Sopon Butcha

Jing Yu

Zikkawas Pasom

Bertrand Goudeau

Chularat Wattanakit

Neso Sojic

Alexander Kuhn

published or not. The documents may come

Electrochemiluminescent enantioselective detection with chiral-imprinted mesoporous metal surfaces

Electrochemiluminescent enantioselective detection with chiral-imprinted mesoporous metal surfaces Sopon Butcha, ‡a,b Jing Yu, ‡a Zikkawas Pasom,a,b Bertrand Goudeau,a Chularat Wattanakit,*b Neso Sojic,*a and Alexander Kuhn*a,b Chiral-imprinted mesoporous Pt-Ir alloy surfaces were combined in a synergetic way with electrochemiluminescence (ECL) to detect the two enantiomers of phenylalanine (PA) as a model compound, acting simultaneously as a chiral target and as a co-reactant to generate significant differences in ECL signals. The chiral features of the metal surfaces are converted into an enantioselective electrogeneration of the excited state of the [Ru(bpy)3]2+ dye, which in fine produces the differentiating light emission with up to 20-fold differences in intensity for the two enantiomers. These findings open up the possibility of developing new ECL-based bioassays and microscopy of chiral environments.

The enantioselective recognition of chiral compounds is crucial for various potential applications, especially in the field of sensing.1 Although multiple techniques have been successfully applied to discriminate chiral molecules, they often suffer from some limitations related to the high cost of advanced instrumentation, the difficulty of sample purification or derivation, and timeconsuming analytical processes.2 Thus, there is still a strong need to develop new and straightforward approaches allowing the discrimination of enantiomers.3

In this context, electrochemical concepts offer several advantages. One of them is electrochemiluminescence (ECL). ECL is a light-emitting phenomenon, which can be decomposed into three sequential main steps: i) the electrochemical trigger for the generation of reactive intermediates, involving a luminophore and a sacrificial co-reactant; ii) their homogeneous reaction leading to the formation of the excited state of the luminophore; iii) its relaxation to the ground state accompanied by photon emission. ECL provides several outstanding advantages, including a controllable electrochemical reaction and ultrasensitive detection of the emitted light.4 Consequently, this allows the investigation of a relevant analyte at very low concentrations (10-11 to 10-15 M), down to imaging at the single-molecule level.5 In addition, ECL is considered a rapid, costeffective, and easy to operate technique.4 Qualitative and quantitative analysis of specific compounds can be carried out based on the luminescence produced by the electrochemical reaction occurring at the electrode surface.6 Up to now, this approach has also been widely associated with other techniques for a large number of practical applications, such as immunosensing, and imaging of (bio)chemical reactions.6-7 However, ECL is a technique that is usually insensitive to chiral information because its intrinsic readout signals, such as the current and the wavelength/intensity of the emitted light, are identical for two enantiomers. To overcome this limitation, we recently reported that resolving the circular polarization of the ECL emission provides valuable information on the excited state of the luminophore. The two individual enantiomers of different luminophores (bispyrene organic macrocycle and the model compound [Ru(bpy)3]2+) were detected selectively by circularly polarizedelectrochemiluminescence (CP-ECL).8 Other groups also reported stereoselective ECL sensors based on various ingredients.9 Nevertheless, in all these cases, a chiral molecular selector had to be integrated into the more or less complex systems.

Herein, we propose a general strategy that is completely different, relying first of all on the fact that specific chiral molecules can play the role of ECL co-reactants, and second, that the enantiodiscrimination occurs on metal surfaces, which are encoded with structural chiral information.

Recently, chiral mesoporous metal structures have been developed.10 They combine the advantages of the mesoporosity, providing a high active surface area and improved mass transport,11 with enantioselective adsorption on surfaces.12 To extend the scope of this strategy, noble and non-noble metals, such as platinum (Pt)10 and nickel (Ni)13 or alloyed platinum-iridium (Pt-Ir),14 have been investigated. These nanostructured surfaces could be used as electrodes for three major aspects of chiral technologies: asymmetric synthesis,10c-e,13,14 chiral separation,15 and enantioselective analysis.10a-b,16 Most importantly, when an alloy is used instead of a monometallic matrix, a significantly improved stability of the chiral features has been observed,14a making these surfaces interesting candidates for the asymmetric synthesis of chiral compounds via both electrocatalysis,14a and heterogeneous chemical catalysis.14b

This structural stability makes an imprinted Pt-Ir alloy also a material of choice for coupling with ECL, because high positive potentials have usually to be applied to the electrode for these experiments. The chiral imprinting should, in this case, allow generating an enantioselective ECL signal when one of the ingredients, either the luminophore or the co-reactant, has chiral features. Thus, we tested an imprinted Pt-Ir alloy as a working electrode for the electrooxidation of the amino acid phenylalanine (PA) as a chiral co-reactant, together with [Ru(bpy)3]2+ as a model luminophore (Scheme 1). In other words, to achieve enantioselective ECL detection, we target in this approach the initial electrochemical triggering step and not the generation of polarized light as in previous studies.8 The amino acid acts simultaneously as the chiral target (Fig. S1, ESI †) and also as the coreactant in the ECL mechanism.14a,17 As illustrated in Scheme 1, when Pt-Ir encoded with D-PA is used as a working electrode, an ECL signal with a significantly higher amplitude is expected for D-PA as an analyte compared to L-PA, due to preferential oxidation at the specific chiral recognition sites. This concept opens up perspectives for a more general use of molecular-imprinted metals in combination with an ECL readout in advanced chiral technologies.

Co-electrodeposition from chloroplatinic acid hydrate (H2PtCl6•xH2O) and dihydrogen hexachloroiridate (IV) hydrate (H2IrCl6•xH2O), in the simultaneous presence of a non-ionic surfactant, polyethylene glycol hexadecyl ether (Brij®C10), and chiral templates, either L-or D-PA, with a molar ratio of PA/(PtCl62-+ IrCl62-)= 1/12, was used to elaborate the chiral encoded mesoporous Pt-Ir electrodes.14a The deposited Pt-Ir alloy, obtained with a deposition charge density of 4 C cm-2, provides outstanding properties such as a perfectly smooth outermost surface (Fig. S2, ESI †) and a uniform thickness of approximately 500 nm (Fig. 1a). The scanning transmission electron microscopy (STEM) image (Fig. 1b) reveals an ordered pore arrangement with an average pore diameter of approximately 5 nm, similar to what has been obtained previously .11,14a,15 The high porosity of the electrode is further emphasized by a significantly larger cyclic voltammetric (CV) signal. Typically, the electrochemically active surface area is estimated in the region of hydrogen adsorption/desorption in a potential window between 0.0 to -0.25 V vs. Ag/AgCl,18 and these measurements allow calculating a roughness factor of up to 100 (Fig. 1c).

PA was selected as a bifunctional model molecule, acting as an analyte and also as a sacrificial co-reactant in combination with the model luminophore [Ru(bpy)3]2+. Both compounds were dissolved in phosphate buffer solution (PBS, pH 7.3). To verify the electrochemical behavior of both PA and [Ru(bpy)3]2+ in PBS solution, a classical Pt electrode was used as a working electrode. Differential pulse voltammetry (DPV) indicates the oxidation of L-PA and [Ru(bpy)3]2+ at approximately 0.75 and 1.1 V vs. Ag/AgCl (sat. KCl), respectively (Fig. 1d).19

The ECL signal starts to be generated at 1 V, where [Ru(bpy)3]2+ oxidation occurs. It reaches a maximum intensity at 1. 15 V.19a The Pt disk electrode does not provide any enantioselectivity, and a specular signal is obtained for both enantiomers (signal ratio of 1.0 ± 0.3 (n=5)). In the case of mesoporous Pt-Ir electrodes, a very pronounced capacitive current (Fig. S3a,ESI †) is observed due to their high active surface area. The ECL signal appears nevertheless at the same oxidation potential as for the classical Pt surface, and its amplitude depends on the PA concentration (Fig. S3b, ESI †). In order to avoid the large capacitive currents, DPV was therefore chosen to further study the chiral discrimination. With a non-imprinted Pt-Ir electrode, the ECL signal was recorded during the DPV scan (DPV-ECL) in a PBS solution containing [Ru(bpy)3]2+ without and with PA (Fig. S3c,ESI †). In a solution containing only the supporting electrolyte there is no ECL signal. A negligible ECL signal was found in the [Ru(bpy)3]2+ solution. However, when adding the PA co-reactant, strong ECL is observed. These observations demonstrate the importance of PA as a co-reactant and that ECL emission is generated at the oxidation potential of [Ru(bpy)3]2+. It further indicates that the mechanism follows an oxidative-reduction route, like in the case of the well-known tri-n-propylamine combined with [Ru(bpy)3]2+.19a-b

Although DPV can be used as a standalone technique for studying chiral recognition,10ab,14a enantioselective ECL emission can lead to an alternative and straightforward readout of analytical information. Fresh L-PA imprinted mesoporous Pt-Ir reveals chiral recognition properties with a higher ECL signal for L-PA than D-PA (Fig. 2a). Similarly, D-PA gives a stronger ECL signal on D-PA imprinted mesoporous Pt-Ir electrodes with respect to L-PA solution (Fig. 2b). In order to check the reproducibility of this discrimination we performed a series of three experiments for every type of electrode. In average the imprinted enantiomer leads to a three times higher ECL signal (Fig. S4) and the recognition ability does not seem to be strongly affected by the scan rate (Fig. S5).

Even though freshly prepared Pt-Ir electrodes can discriminate between PA enantiomers, the sensitivity of this ECL analysis is still limited as the surface can be gradually passivated by the adsorption of molecules. In order to enhance the ECL signal, we therefore employed a potentialinduced surface regeneration concept. The metal surface was activated and cleaned using chronoamperometry, applying a constant negative potential of -0.1 V for 30 s to remove any physisorbed species on the mesoporous metal surface before ECL analysis. 20 The discrimination ability is dramatically improved in this case (Fig. 2c andd). The enantioselectivity, expressed by the ratio of the respective ECL signals, reaches values of over 20. This enhanced enantioselectivity could be ascribed to the fact that the cleaning step equally affects the enantioselective internal surface of the mesopores and the unselective outer surface of the Pt-Ir layer. As the mesopores represent a higher fraction of the total available surface area, this leads to a relative increase in the signal of the imprinted species, which preferentially react inside the pores. This results in a higher probability for the bimolecular reaction between oxidized [Ru(bpy)3]2+ and PA species to occur in a stereoselective way and consequently generates a significantly larger ECL signal for the imprinted enantiomer (Scheme S1).5a,10a-b,19a However, the opposite enantiomers also leads to light emission, even though with much lower intensity. Thus, it is for example not possible to measure enantiomeric excess with a single electrode. However, one could combine in a single experiment two electrodes, one imprinted with the R-enantiomer and the other one with the S-enantiomer. The difference in ECL signal could then be used to estimate the ratio of both enantiomers. From a series of experiments studying the ECL intensity as a function of concentration (Figure S6), one can further estimate that the lower limit of discrimination is around 10 M.

As these ECL experiments require relatively high positive potentials, which might lead to oxidation of the metal surface, and thus to a gradual loss of chiral information, the stability and reusability of the alloy surfaces was investigated. This loss of selectivity relates to the formation of metal oxide while applying highly anodic potentials. The generated metal oxide partially hinders the electrooxidation of PA, resulting in diminished ECL intensities (a loss of 0.4 a.u. of the absolute value of ECL intensity).21 It should be noted that in our previous report,14a the chiral imprinted Pt-Ir alloys demonstrated outstanding discrimination ability of enantiomers, even after several excursions to positive potentials. However, the experiments were carried out in an acidic halide solution (HCl, pH at 1.3), in which the halide species prevent the growth of metal oxides, allowing more efficient oxidation of the reactants. 22 In strong contrast to this, the ECL analysis in this present study was conducted in neutral PBS solution (pH 7.3), in order to maximize ECL intensity which is strongest at neutral pH. Thus, surface passivation leads to a gradual decrease in enantiomeric discrimination efficiency.

In summary, our results demonstrate for the first time that chiral imprinted mesoporous metals can be used for enantio-discrimination by ECL. The selected amino acid, phenylalanine, was efficiently employed as a co-reactant to generate the stereoselective emission of ECL signals. A pronounced selectivity was observed for the target enantiomer. This example illustrates the power of using an ECL-based readout, combined with its usual advantages, such as a low background signal. As the ECL activity is based on the presence of the amino group in the target molecule, it is easy to imagine that the strategy can be extended to other molecules with amino functions and, most notably, to other members of the family of amino acids. Therefore, the findings of these proof-ofprinciple experiments open up interesting perspectives for developing other molecular encoded metal structures that can be used for such an optical detection strategy.

We would like to thank the Frontier Research Center (FRC) at VISTEC for SEM and TEM characterizations. S. B. acknowledges financial support from Campus France, the French Embassy in

Fig. 1

 1 Fig. 1 Characterization of the chiral imprinted mesoporous Pt-Ir electrode. (a) cross-sectional view of the SEM image; (b) STEM image of a thin layer of a porous Pt-Ir sample; (c) Cyclic voltammograms of porous Pt-Ir (red) and commercial flat Pt (black) recorded in 0.5 M H2SO4 solution at a scan rate of 100 mV s -1 ; the Pt-Ir alloy is obtained by electrodeposition with a charge density of 4 C cm -2 . (d) Differential pulse voltammogram and ECL intensity recorded with a commercial Pt disk electrode in a solution composed of 10 mM L-PA and 1 mM [Ru(bpy)3] 2+ dissolved in 0.1 M PBS solution (pH 7.33).

  Fig. S7a (ESI †) shows the ECL response of a D-PA imprinted Pt-Ir surface in the presence of D-and L-PA during the first anodic scan of fresh electrodes, and Fig. S7b-e (ESI †) display the ECL signals recorded during four subsequent anodic scans with the same electrodes. The relative ECL intensity measured in the solution containing L-PA tends to increase compared to D-PA. It shows that the imprinted Pt-Ir surface can be reused for up to four detection cycles, despite a decrease in enantioselectivity.

Fig. 2

 2 Fig. 2 Chiral discrimination by ECL on pristine (upper row) and activated (lower row) encoded Pt-Ir alloy electrodes. ECL signals for discrimination between PA enantiomers of (a), (c) L-PA, and (b), (d) D-PA imprinted Pt-Ir electrodes. The experiments were conducted in both 10 mM L-PA or D-PA/1 mM [Ru(bpy)3]2+

Thailand, and VISTEC for a Ph.D. cotutelle scholarship, and the National Research Council of Thailand (NRCT) for the graduate research fund (N41B650204). J. Y. acknowledges the China Scholarship Council for her Ph.D. fellowship. The project has been financed by the European Research Council (ERC) (grant agreement no. 741251, ERC Advanced Grant ELECTRA) and has also been supported by the CNRS IRP project ChiraChem. C. W. thanks the Mid-Career Research Grant 2020 from NRTC (NRCT5-RSA63025-03) and support from the NSRF via the Program Management Unit for Human Resources & Institutional Development, Research and Innovation (B05F640207).

Conflicts of interest

There are no conflicts to declare. Soc., 2001, 123, 3173-3174;(c) M. Lämmerhofer, J. Chromatogr. A, 2010, 1217, 814-856.


Louis Guillot

Bruno Cochelin

Christophe Vergez

A Taylor series based continuation method for the periodic solutions of a wide range of dynamical systems

This extended abstract is a companion of the one called A Taylor series based continuation method for solutions of Ordinary Differential Equations. It is shown how a quadratic framework allows to solve a large class of dynamical systems. A brief introduction on the continuation method is followed by an explanation on the quadratic rewriting of systems of equations. A few types of dynamical systems that can be treated using this framework are finally given.

Introduction

This paper emphasizes how a quadratic framework can handle a wide range of applications for the continuation of dynamical systems. The continuation method used, the so-called Asymptotic Numerical Method (ANM) that has first been described in [START_REF] Cochelin | A path-following technique via an asymptotic-numerical method[END_REF], relies on a high-order Taylor series representation of the solution-branch. This technique has already proven its efficiency for a lot of applications in enginering, mechanics or acoustics for example. While some implementations relying on automatic differentiation do exist, see [START_REF] Charpentier | The diamant approach for an efficient automatic differentiation of the asymptotic numerical method[END_REF] for example, the choice is made here to work with a quadratic framework. A generic implementation of this latest approach which minimizes problem-dependent implementation has been developed [START_REF] Guillot | A generic and efficient Taylor series based continuation method using a quadratic recast of smooth nonlinear systems[END_REF]. A simplified scheme of it is represented in figure 1. It is based on the numerical continuation of algebraic systems of the form

R(V ) = 0 (1)
where V ∈ R n+1 and R(V ) ∈ R n is an analytic function of its argument. This system is always written in a quadratic format as a prerequisite of the method. Again, this formalism is not a constraint that we suffer but a choice that allows to treat a very wide range of problems as shown in [START_REF] Karkar | A high-order, purely frequency based harmonic balance formulation for continuation of periodic solutions: The case of non-polynomial nonlinearities[END_REF]. The system (1) can then be written

R( Ṽ ) = C + L( Ṽ ) + Q( Ṽ , Ṽ ) = 0 (2)
where Ṽ ∈ R N +1 , N ≥ n and C, L and Q are respectively a constant, a linear and a quadratic operator. This step can be tough and some hints are given in the second section. Again, the key idea of the ANM is to compute a high-order Taylor series development of the solution-branch. Let consider that Ṽ0 is a regular solution of the system (2). Let Ṽ1 be a tangent vector at Ṽ0 . The classical arc-length parameter a = Ṽ t 1 ( Ṽ0 -Ṽ ) is introduced and the solution-branch around Ṽ = Ṽ0 is written as a power series with respect to a

Ṽ (a) = Ṽ0 + a Ṽ1 + a 2 Ṽ2 + a 3 Ṽ3 + • • • (3)
The quadratic framework

The quadratic rewriting of the system allows to compute the terms in the development of the series explicitly with discrete convolution for the non-linear terms (see [START_REF] Guillot | A generic and efficient Taylor series based continuation method using a quadratic recast of smooth nonlinear systems[END_REF] for some details about this section). If the solutions seeked are periodic functions of time expanded in Fourier series as it is the case in this paper, the product of two variables in the frequency domain is similarly represented by an explicit discrete convolution. Another huge advantage of the quadratic rewriting is that the exact Jacobian matrix of the system (2) is known explicitly if the constant, linear and quadratic operators are known, and can be derived automatically from these operators.

Rational variables

The equations with inversions y = 1

x or monomials of degree more than two y = x 3 can be rewritten either implicitly yx = 1 or with the use of auxiliary variables yxr = 0 rx 2 = 0 respectively as detailed in [START_REF] Guillot | A generic and efficient Taylor series based continuation method using a quadratic recast of smooth nonlinear systems[END_REF].

Transcendental functions

Transcendental non-linearities like y = exp(x) require a specific treatment. The idea is to write the differential form of this equation in a quadratic format. For this simple example, the differential form is dy = exp(x)dx. It is easy to recast it using y = exp(x) to obtain dy = ydx. Apart for the term of the series (3) at order 0 that shall still be computed using y = exp(x), all the terms of higher order can be computed with the differentiated form which is quadratic. More details are given about these type of computation in the book [START_REF] Griewank | Evaluating derivatives: principles and techniques of algorithmic differentiation[END_REF].

Classes of systems that can be treated

The quadratic rewriting described in the previous section allows to treat a very wide range of dynamical systems with the same continuation tool. For clarity this section is reduced to periodic solutions of dynamical systems, but in the companion extended abstract, the case of static solutions, quasi-periodic solutions and transients will be treated. For now, there are 5 different types of differential equations that have been treated. The Ordinary Differential Equations (ODE), Differential-Algebraic Equations (DAE) and Implicit Differential Equations (IDE) are illustrated through the same example of the pendulum, using three different formulations of the problem.

Equations (E) Unknowns (X)

Type of the unknowns: Algebraic, Function of the time Quadratic recast of the equations (E q )

If X(t) is periodic, quasi-periodic, or discretized on time samples (E q ) is projected on a suitable basis of representation: by Harmonic Balance Method (HBM), collocation, etc A quadratic algebraic system (E q,alg ) is obtained.

The continuation of this quadratic algebraic system (E q,alg ) is performed using the Asymptotic Numerical Method.

   ẍ + T x = 0 ÿ + T y -1 = 0 x 2 + y 2 -1 = 0 (5)

IDE : conservation of the energy

The pendulum is a conservative mechanical system meaning that its energy h is a constant of the motion. It leads to this implicit differential equation:

1 2 θ2 + 1 -cos(θ) = h (6)
Other types of systems In addition to these three widespread types of systems, some very recent works have shown that equations with fractional derivatives [START_REF] Vigué | Continuation of periodic solutions for systems with fractional derivatives[END_REF] or time-delayed variables could also be treated using the quadratic rewriting presented in this paper.

Conclusion

The full paper corresponding to this extended abstract and to the companion extended abstract will show how to take advantage from a quadratic rewriting to carry out the continuation of solutions of a large class of dynamical systems. The method requires very few effort to switch between the different types of systems. Its implementation is freely available online on a dedicated website http://manlab.lma.cnrs-mrs.fr/.

Figure 1 :

 1 Figure 1: This scheme describes from a general perspective the implementation of the Asymptotic Numerical Method used in this work. ODE : second order polar coordinates This is the very classical equation of motion of the pendulum. θ + sin(θ) = 0 (4) DAE : second order Cartesian coordinates This projection on fixed axis of the equations of the motion leads to an algebraic equation to constrain the length of the pendulum. In addition, the tension of the string T appears in the equations.


Emile Dupuy

Egbert Sander

Jiusheng Van Der Verren

Mark Alan Lin

Alix Vincent Wilson

Felipe Dachsbeck

Emmanuelle Viela

Alexandra Latour

Didier Gennaris

Yves Frédéric Vertommen

Dufrêne

Van Der Verren

Jiusheng Lin

Mark Alan Wilson

Alix Vincent Dachsbeck

Felipe Viela

Emmanuelle Latour

Alexandra Gennaris

Didier Vertommen

Yves Frédéric Dufrêne

Bogdan Iuliu Iorga

Camille Véronique Goemans
email: camille.goemans@embl.de

Han Remaut
email: han.remaut@vub.be

Jean-François Collet
email: jfcollet@uclouvain.be

A molecular device for the redox quality control of GroEL/ES substrates

Keywords: Protein folding, chaperones, redox, proteostasis, TPR, thioredoxin, chaperonin

établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Following synthesis as linear amino-acid chains, proteins must fold to unique threedimensional (3D) structures to become functional. Seminal work from Anfinsen demonstrated that the information required for a polypeptide to reach its native conformation is contained in its primary sequence. [START_REF] Anfinsen | Principles that govern the folding of protein chains[END_REF] For most small proteins, folding to the native state is a spontaneous process that takes less than a few milliseconds. [START_REF] Jahn | The Yin and Yang of protein folding[END_REF] For larger proteins with multiple domains, however, the path to the native conformation is more tortuous and potentially hazardous. For these proteins, stable intermediates can form, slowing the folding process and potentially leading to aggregation and/or degradation. [START_REF] Ellis | Macromolecular crowding: obvious but underappreciated[END_REF] To deal with this problem, living cells express a network of chaperones that help complex proteins to fold efficiently. [START_REF] Hartl | Molecular chaperones in protein folding and proteostasis[END_REF] The Hsp60 chaperonins are a unique class of chaperones that are essential in all domains of life and prevent unproductive interactions within and between polypeptides using adenosine triphosphate (ATP)-regulated cycles. [START_REF] Goloubinoff | Reconstitution of active dimeric ribulose bisphosphate carboxylase from an unfolded state depends on two chaperonin proteins and Mg-ATP[END_REF][START_REF] Horwich | Chaperonin-assisted protein folding: a chronologue[END_REF][START_REF] Hayer-Hartl | The GroEL-GroES chaperonin machine: a nano-cage for protein folding[END_REF] Chaperonins stand out in the proteostasis network, as they form a complex tetradecameric structure encompassing a large cylindrical cage consisting of two seven-membered rings stacked back to back (Figure S1A). 8,[START_REF] Hendrix | Purification and properties of groE, a host protein involved in bacteriophage assembly[END_REF] Each Hsp60 subunit consists of an ATP-binding equatorial domain, an intermediate domain, and an apical substrate-binding domain (Figure S1A). [START_REF] Braig | The crystal structure of the bacterial chaperonln GroEL at 2.8 Å[END_REF] Hsp60 cooperates with Hsp10 [START_REF] Chandrasekhar | Purification and properties of the groES morphogenetic protein of Escherichia coli[END_REF] , which forms a heptameric dome-like structure (Figure S1A). [START_REF] Hunt | The crystal structure of the GroES co-chaperonin at 2.8 Å resolution[END_REF] In the presence of nucleotides, Hsp10 associates with the apical domain of Hsp60, binding as a lid covering the ends of the ring and forming a folding chamber [START_REF] Xu | The crystal structure of the asymmetric GroEL-GroES-(ADP) 7 chaperonin complex[END_REF] referred to as the "Anfinsen cage." Binding of Hsp10 to a substrate-loaded Hsp60 results in displacement of the substrate into the chamber, where it can fold protected from outside interactions. [START_REF] Langer | Chaperoninmediated protein folding: GroES binds to one end of the GroEL cylinder, which accommodates the protein substrate within its central cavity[END_REF][START_REF] Martin | The reaction cycle of GroEL and GroES in chaperonin-assisted protein folding[END_REF] The mechanism by which chaperonins assist substrate proteins in navigating the folding landscape to their native state is relatively well understood. Although this is particularly true for the Escherichia coli Hsp60 chaperonin GroEL and GroES, its Hsp10 cofactor, several crucial questions remain unsolved. For instance, whether the GroEL-GroES nanomachine actively promotes folding or serves only as a passive folding cage remains controversial. [START_REF] Hayer-Hartl | The GroEL-GroES chaperonin machine: a nano-cage for protein folding[END_REF] It is also unknown why some polypeptides are highly dependent on GroEL-GroES for folding whereas homologous proteins with a similar structure fold independently of the chaperonin. [START_REF] Hayer-Hartl | The GroEL-GroES chaperonin machine: a nano-cage for protein folding[END_REF] Thus, further investigation is required to elucidate the sorting signals that recruit substrate proteins to the Hsp60 folding cage. Moreover, recent results have indicated that the integration of GroEL-GroES in the cellular proteostasis network also needs further exploration. Indeed, the identification of CnoX as the first chaperone capable of transferring its substrates to GroEL-GroES for active refolding [START_REF] Goemans | CnoX is a chaperedoxin: a holdase that protects its substrates from irreversible oxidation[END_REF][START_REF] Goemans | The chaperone and redox properties of CnoX chaperedoxins are tailored to the proteostatic needs of bacterial species[END_REF] suggests that functional links between GroEL-GroES and accessory folding factors remain to be discovered. The extreme complexity of the GroEL-GroES molecular machine, its essential role in cell survival, and redundancy in the bacterial proteostasis system have slowed progress in the field, highlighting the need for new investigation approaches and experimental strategies.

Here, we sought to explore the details of the newly reported CnoX-GroEL functional relationship [START_REF] Goemans | CnoX is a chaperedoxin: a holdase that protects its substrates from irreversible oxidation[END_REF][START_REF] Goemans | The chaperone and redox properties of CnoX chaperedoxins are tailored to the proteostatic needs of bacterial species[END_REF] , with the aim of revealing unsuspected features of the GroEL-GroES system.

CnoX consists of a redox-active N-terminal thioredoxin domain and a C-terminal tetratricopeptide (TPR) domain (Figure S1B) [START_REF] Lin | Escherichia coli thioredoxin-like protein YbbN contains an atypical tetratricopeptide repeat motif and is a negative regulator of GroEL[END_REF] , a fold often involved in protein-protein interactions. CnoX is a "chaperedoxin," meaning that it combines a redox-protective function, by which it prevents irreversible oxidation of its substrates, and a holdase chaperone activity, by which it maintains its substrates in a folding-competent state before transferring them to either GroEL-GroES or the DnaK-DnaJ-GrpE system for ATP-dependent refolding [START_REF] Goemans | CnoX is a chaperedoxin: a holdase that protects its substrates from irreversible oxidation[END_REF] . We reasoned that identifying the molecular attributes that uniquely allow CnoX to work in concert with GroEL-GroES would lead to new insights into the properties of the GroEL-GroES system.

RESULTS

CnoX and GroEL form a stable complex

To start our investigation, we pulled down CnoX from E. coli cellular extracts using specific -CnoX antibodies. We found that CnoX co-eluted with only one partner (Figure 1A), a ~60-kDa protein identified as GroEL by mass spectrometry (MS), confirming previous results suggesting a direct interaction between the two proteins. [START_REF] Lin | Escherichia coli thioredoxin-like protein YbbN contains an atypical tetratricopeptide repeat motif and is a negative regulator of GroEL[END_REF] In this experiment, CnoX and GroEL were expressed from their native locus in cells grown under normal conditions. Noteworthy, intracellular protein abundance estimates [START_REF] Li | Quantifying absolute protein synthesis rates reveals principles underlying allocation of cellular resources[END_REF] indicate that CnoX and GroEL tetradecamers (GroEL14) are expressed at similar levels in rich defined medium at 37°C; thus, there seems to be enough CnoX to satisfy GroEL at a 1:1 CnoX:GroEL14 ratio. Exposing the cells to heat shock (42°C) did not lead to an increase in the amount of GroEL that co-eluted with CnoX (Figure S1C).

We then examined whether the CnoX-GroEL interaction could be reconstituted in vitro using purified proteins. E. coli CnoX and GroEL were independently overexpressed and purified to near homogeneity (Figure S1D). We mixed GroEL and CnoX in a 1:1 molar ratio (1[GroEL subunit]:1 [CnoX]) and found that they co-eluted from both a streptavidin affinity column (Figure 1B; a Strep-tag was fused to the N-terminus of CnoX) and a size-exclusion chromatography column (Figure 1C). The latter showed the co-eluting GroEL-CnoX complex in an approximately 14:1 molar ratio compared with the 1:1 input ratio, corresponding to a ratio of 1 CnoX per GroEL double ring. Notably, we also observed that CnoX formed a complex with a GroEL mutant (GroELR452A/E461A/S463A/V464A) known to form a single heptameric ring (GroEL7; Figure S1E). [START_REF] Weissman | Mechanism of GroEL action: productive release of polypeptide from a sequestered position under GroES[END_REF] Finally, we determined the affinity between the two proteins using fluorescence spectroscopy and fluorescence anisotropy and found that fluorescein (fluorescein-5-maleimide [FM])-labeled CnoX (FM-CnoX) binds GroEL with a dissociation constant (Kd) of 310 ± 10 nM (Figures 1D andS1F). Using atomic force microscopy (AFM), we measured a specific binding force of 175 ± 75 pN between the two proteins (Figures S1G andS1H). Thus, we conclude that CnoX physically interacts with GroEL and that the two proteins form a stable complex both in vitro and in vivo.

GroES binding triggers the release of CnoX from GroEL

We next aimed to unravel the interrelationship among CnoX, GroEL, and GroES. GroES reversibly binds GroEL in the presence of nucleotides. [START_REF] Hayer-Hartl | The GroEL-GroES chaperonin machine: a nano-cage for protein folding[END_REF][START_REF] Chandrasekhar | Purification and properties of the groES morphogenetic protein of Escherichia coli[END_REF] The addition of adenosine diphosphate (ADP), which triggers conformational changes in GroEL and primes the ring for GroES binding, had no impact on the GroEL-CnoX complex (purified proteins were mixed in a 14:1 molar ratio) (Figure 1E), although the affinity of CnoX for GroEL decreased slightly (Kd of ~350 nM) (Figure S2A). Strikingly, however, the subsequent addition of GroES (14[GroEL]:14[GroES]:1[CnoX] molar ratio) triggered the release of CnoX from GroEL (Figure 1E), indicating a direct or allosteric competition between CnoX and GroES for GroEL binding.

We obtained similar results with a non-hydrolysable ATP analogue (Figure S2B). Next, titration of a complex between GroEL and FM-CnoX with increasing amounts of GroES resulted in a dose-dependent loss of FM-CnoX, confirming that GroES dissociates CnoX from GroEL (Figure S2C). Using a single-site competitive binding model, we calculated a fitted inhibitory constant (Ki) of 47 nM. Finally, under conditions in which GroES was overexpressed, the GroELR452A/E461A/S463A/V464A variant (GroEL7) did not co-elute with CnoX in pulldown experiments (Figure S2D). Here, we employed GroEL7 for unambiguous data interpretation. Altogether, these results clearly distinguish CnoX from typical GroEL substrates and indicate that CnoX is a bona fide GroEL partner. Indeed, GroEL does not release substrate proteins such as unfolded citrate synthase (CS) upon GroES addition (Figure 1E); rather, these proteins become encapsulated inside the GroEL-GroES folding chamber for refolding. [START_REF] Horwich | Chaperonin-assisted protein folding: a chronologue[END_REF][START_REF] Hayer-Hartl | The GroEL-GroES chaperonin machine: a nano-cage for protein folding[END_REF] Along the same line, we found that the presence of CnoX does not prevent GroEL from recruiting unfolded CS (Figure S2E). Thus, CnoX does not restrict access to the substrate-binding site of GroEL.

The C-terminal -helix of CnoX binds GroEL near the site of substrate entry into the cage

Intrigued by these results, we sought to obtain structural information on the CnoX-GroEL interaction using cryo-electron microscopy (cryoEM). We reconstituted the CnoX-GroEL complex by mixing purified GroEL and CnoXN-Strep (10[GroEL]:1[CnoX] molar ratio, which corresponds to a ratio of 1[GroEL14]:1.4 [CnoX]) in the absence of nucleotides. The complex was then affinity-purified (Figure S3A) and imaged for single-particle cryoEM analysis (Figures S3B andS3C and Table S1). Analysis of the two-dimensional (2D) class averages showed the two rings of GroEL stacked back-to-back and revealed the presence of a protruding density on top of the two GroEL rings (Figures 2A,2B, and S3D). A c7-symmetrical 3D reconstruction resulted in a 3.4 Å electron potential map (Figure S3E) with a density on the GroEL apical domain corresponding to at least five α-helices and allowing an unambiguous rigid body docking with the TPR domain of CnoX (Figures 2C, 2D, S3F, andS3G). The absence of a clearly resolved thioredoxin domain in the CnoX-GroEL complex is consistent with the prior observation of extensive mobility of this domain in the X-ray crystal structure of CnoX alone. [START_REF] Lin | Escherichia coli thioredoxin-like protein YbbN contains an atypical tetratricopeptide repeat motif and is a negative regulator of GroEL[END_REF] This finding suggests that the thioredoxin domain is highly dynamic, which may be relevant for our proposed model (see below).

Although the N-terminal thioredoxin domain of CnoX is not visible, the structure provides crucial molecular details regarding the CnoX-GroEL interaction. First, the structure reveals that CnoX binds GroEL via its C-terminal α-helix (Figure 3A, 3B, 3C); accordingly, a CnoX mutant lacking the last 10 C-terminal residues (CnoX∆Cter) is unable to bind GroEL, both in vivo (Figure 3D) and in vitro (Figure S4A). Furthermore, addition of a His-tag to the Cterminus of CnoX (CnoXC-His) or mutation of conserved residues in the C-terminal α-helix (CnoXR277L, CnoXY284L) prevented CnoX binding to GroEL (Figures 3D andS4A). Using AFM, we found that the C-terminal α-helix on its own binds to GroEL; we measured a specific binding force of 135 ± 53 pN between GroEL and the 10 C-terminal residues of CnoX (Figure S1I), which is similar to the binding force measured between full-length CnoX and the chaperonin.

We previously reported that CnoX is able to transfer unfolded CS to GroEL-GroES for refolding. [START_REF] Goemans | CnoX is a chaperedoxin: a holdase that protects its substrates from irreversible oxidation[END_REF] As shown in Figure S4B, we found that deleting the last 10 residues of CnoX significantly decreases its ability to cooperate with GroEL for CS refolding. Thus, the Cterminal α-helix of the TPR domain of CnoX functions as a specific GroEL affinity tag that is required and sufficient for GroEL binding and is important for function.

Using cells lacking trigger factor (∆tig) and a functional DnaK-DnaJ-GrpE system (∆dnaKdnaJ), we obtained further in vivo evidence that the C-terminal α-helix controls the CnoX-GroEL interaction. ∆tig∆dnaKdnaJ cells exhibit a heat-sensitive phenotype that is rescued by overexpression of GroEL-GroES [START_REF] Genevaux | In vivo analysis of the overlapping functions of DnaK and trigger factor[END_REF] , presumably because the chaperonin, when present at high levels, folds proteins that normally depend on trigger factor and/or DnaK-DnaJ-GrpE. We found that overexpressing CnoX, but not CnoX∆Cter, together with GroEL-GroES prevents the chaperonin from suppressing the growth defect of ∆tig∆dnaKdnaJ cells at 37°C (Figure S4C). This result suggests that CnoX, when bound to GroEL, limits the ability of the chaperonin to fold non-substrate proteins. This finding further demonstrates the importance of the Cterminal -helix and provides additional evidence that the GroEL-CnoX interaction occurs in vivo. Finally, we noted that while the sequence of the TPR domain of CnoX is diverse among species, the last C-terminal helix is highly conserved (Figure S4D) and is structurally and electrostatically distinct from the remainder of the TPR domain [START_REF] Lin | Escherichia coli thioredoxin-like protein YbbN contains an atypical tetratricopeptide repeat motif and is a negative regulator of GroEL[END_REF] , suggesting that the ability to bind GroEL is widespread and central to CnoX activity.

The structure also reveals where CnoX binds to GroEL; the interaction zone, which has a buried surface area of 472 Å 2 (-4.6 kcal/mol; PDBePISA [START_REF] Brinker | Ligand discrimination by TPR domains. Relevance and selectivity of EEVD-recognition in Hsp70 x Hop x Hsp90 complexes[END_REF] ) and encompasses residues D224, K286-M307, K311, D316, R345, and Q348 (Figures 3B and3C), corresponds to a shallow surface cleft formed by helices J and K in the apical domain of GroEL. This region does not overlap with the substrate-binding site of GroEL in helices H and I [START_REF] Horwich | Chaperonin-assisted protein folding: a chronologue[END_REF][START_REF] Hayer-Hartl | The GroEL-GroES chaperonin machine: a nano-cage for protein folding[END_REF] , as also corroborated by the above results (Figure S2E). At least five potential hydrogen bonds or electrostatic interactions stabilize the contacts between CnoX and GroEL (R255-E304, R277-G298, R277-T299, Y284-E304, and Y284-R345, listed as CnoX-GroEL), as well as a hydrophobic interaction between CnoX residues L279, Y280, and L283 and GroEL residues V300, I305, and M307 (Figures 3B and3C). Accordingly, introducing a set of mutations in the interaction interface of GroEL (GroEL § ) with CnoX disrupted the GroEL-CnoX interaction (Figure 3E), without affecting the chaperone function of GroEL as the mutant was still able to rescue the temperature sensitivity of ∆tig∆dnaKdnaJ cells (Figure S4C).

GroEL is a highly dynamic protein that undergoes substantial conformational rearrangements depending on the binding of a nucleotide, its position in the folding pathway, or the binding of GroES [START_REF] Clare | ATP-triggered conformational changes delineate substrate-binding and-folding mechanics of the GroEL chaperonin[END_REF] . Comparison of our structure with the different conformational states of GroEL shows that the GroEL rings are in a conformation corresponding to that of the nucleotide-free protein (Figure S4E), as expected. Our findings also indicate that the CnoX-binding paratope remains fully accessible in all conformations, except when GroES is bound (Figure S4E). The persistence of the CnoX-binding site in various conformations of GroEL is consistent with the ability of CnoX to bind to GroEL irrespective of the presence of a nucleotide (Figures 1B, 1C, 1E, andS2B). Available structures also show a large conformational rotation of the GroEL apical domain in the GroEL-GroES complex.

Although the GroES-binding site does not directly overlap with that of CnoX, the conformation of the apical domain results in a steric occlusion of the CnoX-binding paratope (Figure S4E), providing a molecular explanation to our finding that GroES docking onto GroEL is incompatible with CnoX binding (Figures 1E andS2B).

CnoX forms mixed disulfides with obligate GroEL substrates when bound to GroEL

We next aimed to gain insight into the physiological relevance of the CnoX-GroEL complex in vivo. GroEL-GroES substrates often need minutes to fold after leaving the ribosome [START_REF] Ewalt | In vivo observation of polypeptide flux through the bacterial chaperonin system[END_REF] , which raises a question regarding how their amino acids are protected from oxidative damage before reaching their native state. This question is particularly relevant for cysteine residues, which are highly sensitive to oxidation by the molecular oxidants present in cells even in the absence of stress. [START_REF] Imlay | Cellular defenses against superoxide and hydrogen peroxide[END_REF][START_REF] Ezraty | Oxidative stress, protein damage and repair in bacteria[END_REF] Indeed, the thiol side chain of a cysteine is readily oxidized to a sulfenic acid (-SOH), an unstable derivative that can react with another cysteine in the vicinity to form a disulfide or that can be irreversibly oxidized to sulfinic and sulfonic acids.

Similar to Anfinsen's experiments showing that noncanonical disulfide pairing thwarts in vitro protein folding, one can expect the GroEL chaperonin to require its substrates' cysteines to be reduced for proper folding. CnoX stands out in the proteostasis network in that it combines a chaperone and a redox-protective function; [START_REF] Goemans | CnoX is a chaperedoxin: a holdase that protects its substrates from irreversible oxidation[END_REF] therefore, CnoX may bind GroEL to function as a redox rescue mechanism for slow-folding GroEL-GroES substrates.

By performing additional pull-down experiments, we obtained a crucial result shedding light onto the function of CnoX. When GroEL is pulled down from cellular extracts, it co-elutes with CnoX, as expected. Intriguingly, we found that high-molecular-weight complexes involving CnoX are also pulled down (Figure 4A). When a reducing agent was added, these complexes disappeared, indicating that they correspond to mixed disulfides comprising CnoX and unknown proteins. Accordingly, we did not detect high-molecular-weight complexes when the experiment was repeated with a CnoX mutant lacking the two cysteine residues (CnoXno_cys; Figure 4A), confirming that tripartite complexes of GroEL, CnoX, and unidentified proteins exist in the cell. We identified the proteins involved in the mixed disulfides using MS (Table S2); remarkably, the identified proteins exhibited strong enrichment in GroEL obligate substrates (9 of the 57 obligate GroEL substrates were detected [START_REF] Fujiwara | A systematic survey of in vivo obligate chaperonin-dependent substrates[END_REF] ), including two lowabundance proteins, acetylornithine deacetylase, and nicotinamide adenine dinucleotide phosphate (NADP)-specific glutamate dehydrogenase (<400 copies per cell; [START_REF] Li | Quantifying absolute protein synthesis rates reveals principles underlying allocation of cellular resources[END_REF] ) (Figure 4B and Table S2). We did not detect these proteins when the experiment was performed using lysates prepared from cells expressing CnoXno cys. Thus, we conclude that CnoX forms mixed disulfides with obligate GroEL substrates when bound to GroEL in the cell.

CnoX functions as a molecular plugin providing redox quality-control for GroEL substrates

Altogether, our results suggest the following model (Figure 4C). Regardless of stress, CnoX binds GroEL via its highly conserved C-terminal α-helix in a nucleotide-independent manner.

The CnoX-binding interface on GroEL does not overlap with the substrate-binding site. If the substrate that reaches GroEL for folding presents oxidized cysteine residues (to a sulfenic acid or in a disulfide bond), CnoX reacts with the substrate via the cysteines of its thioredoxin domain, resulting in the formation of a mixed disulfide. Cytoplasmic reducing pathways then reduce the mixed disulfide, releasing the substrate in a reduced, foldingcompetent state. The binding of GroES to GroEL induces conformational changes in the chaperonin and occludes the CnoX-binding site, triggering CnoX release from GroEL and encapsulation of the substrate within the folding cage for folding. This model implies that segments of unfolded substrates are already present in the cavity of GroEL when the substrates form a mixed disulfide with GroEL-bound CnoX. We confirmed this implication by purifying a tripartite complex of CnoXN-Strep, CS, and GroEL (Figure 5A) and subjecting it to crosslinking MS analysis: we detected crosslinked peptides between several GroEL residues, including four facing the inside of the cavity, and CS (Figures 5B and5C).

Thus, we propose that CnoX functions as a molecular plugin that provides redox quality control for GroEL substrates. Our model is compatible with both the binding of CnoX to unfolded oxidized client proteins in solution followed by delivery to the GroEL chaperonin and the surveillance performed by CnoX to identify erroneously oxidized client proteins that may become stuck at the substrate entrance to the Anfinsen cage of GroEL. This function of CnoX as a quality control plugin for GroEL is constitutive and not inducible by stress. It is not incompatible with the previously reported role of CnoX in the protection of cellular proteins, including GroEL substrates, from HOCl-induced aggregation and oxidation. [START_REF] Goemans | CnoX is a chaperedoxin: a holdase that protects its substrates from irreversible oxidation[END_REF]

DISCUSSION

Investigations of Hsp60 chaperonins started in the 1970s 6 , when researchers described mutations that blocked phage head assembly in groE and discovered the tetradecameric structure of GroEL, the archetypical member of the Hsp60 family, using EM. Since then, a large body of studies has examined the mechanistic and structural properties of Hsp60 proteins and their Hsp10 co-chaperones, not only in bacteria but also in chloroplasts and mitochondria. [START_REF] Horwich | Chaperonin-assisted protein folding: a chronologue[END_REF] This impressive amount of work has rendered chaperonins a textbook example of folding systems. In the current study, the identification of CnoX as a qualitycontrol protein that physically interacts with GroEL-GroES for optimal folding has further widened this field of investigation by uncovering an unsuspected feature of Hsp60.

Additional questions remain unsolved and will be the subject of future research. For instance, the biologically active stoichiometry of the CnoX-GroEL complex warrants careful investigation, as well as the specific role of the cytoplasmic reducing pathways in the reduction and release of mixed disulfides. Future work must also establish the location of the N-terminal thioredoxin domain when CnoX is bound to GroEL. Our results show that CnoX forms mixed disulfides with GroEL substrates while being bound to GroEL, but future research will elucidate whether CnoX also functions as a tugboat to locate endangered GroEL substrates in the cytoplasm and escort them to the chaperonin. Finally, it will be important to determine whether similar proteins with a redox quality-control function exist in other organisms, including eukaryotes. The facts that E. coli CnoX stably interacts with human mitochondrial Hsp60 (mHsp60; Figure S5A) and that proteins sharing structural features with CnoX exist in eukaryotes (Figures S5B, S5C, andS5D) support this idea. Along the same line, it is tempting to speculate that living cells could also contain Hsp60 molecular "plugins" with specific, redox-independent functions yet to be discovered.

Limitations of the study

Our study focused on the interaction between CnoX and GroEL. We did not study the ability of CnoX to cooperate with the DnaK-DnaJ-GrpE system. Moreover, although we identified several eukaryotic proteins with structural features similar to those of CnoX -which suggest that these proteins could function as molecular plugins for eukaryotic Hsp60 proteins-we did not investigate this attractive hypothesis. Our model implies that cytoplasmic reducing systems catalyze the reduction of mixed disulfides between CnoX and its substrates. The respective roles of the thioredoxin and glutaredoxin pathways in this process remain to be determined. Further studies are also required to obtain additional structural information on the ternary complexes between GroEL, CnoX, and their substrates.

STAR METHODS

Detailed methods are provided in the online version of this paper and include the following:

• Key resources table

• Resource availability

Lead contact

Materials availability

Proteins were pulled down with the GroEL-CnoX complexes using a-GroEL antibodies and identified using LC-MS/MS. These proteins were not detected when the experiments were repeated in cells expressing CnoXno_cys. spectrometry: DV. Data analysis and interpretation: ED, SEVdV, CVG, HR, JL, MAW, YFD, FV, and JFC. All authors discussed the results and commented on the manuscript. (C) Model: 1. CnoX forms a stable complex with GroEL via its C-terminal α-helix in a nucleotide-independent manner. Positioned on the apical domain of GroEL, CnoX interacts with incoming substrates for GroEL, acting as a redox quality-control plugin. 2. If the substrate that reaches GroEL for folding presents oxidized cysteine residues (to a sulfenic acid or in a disulfide bond), CnoX reacts with the substrate via the cysteines of its thioredoxin domain, and a mixed disulfide is formed. Interactions between the substrate and the GroEL cavity occur. 3. Cytoplasmic reducing pathways reduce the mixed disulfide, releasing the substrate in a reduced, folding-competent state. 4. GroES binding triggers CnoX release from GroEL and encapsulation of the substrate within the folding cage for folding. (H) AFM measurement of the specific adhesion force between CnoX and GroEL. Two typical adhesion force curves are shown in the inset. We measured a specific binding force of 175 ± 75 pN (mean and standard deviation for a total of 5000 curves from three independent experiments). In these experiments, we used a GroEL mutant (GroELD490C) with a cysteine residue exposed on its equatorial domain to allow binding to the gold surface and a CnoX variant (CnoXN-link/C38A/C63A) lacking the two cysteines of the thioredoxin domain to prevent disulfide bond formation between CnoX and GroEL.

Main Figure Titles and Legends

(I) AFM measurement of the specific adhesion force between the C-terminal 10 residues of CnoX and GroEL. We measured a specific binding force of 135 ± 53 pN (mean and standard deviation for a total of 5000 curves from three independent experiments). In these experiments, we used a GroEL mutant (GroELD490C) with a cysteine residue exposed on its equatorial domain to allow binding to the gold surface.

STAR*Methods

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Jean-François Collet (jfcollet@uclouvain.be).

Materials availability

Strains and plasmids generated in this study are available upon request to the lead contact.  Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

The E. coli strains used in this study are listed in Table S3. We prepared the cnoX deletion mutant by transferring the corresponding allele (ybbN::kan R ) from the Keio collection 29 into MG1655 by P1 phage transduction, with verification by polymerase chain reaction (PCR). To excise the kanamycin-resistance cassette, we used pCP20 [START_REF] Cherepanov | Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibioticresistance determinant[END_REF] . Unless otherwise indicated, we grew the cells in lysogeny broth (LB) at 37°C, and when necessary, we supplemented the growth media with ampicillin (100-200 µg/mL), chloramphenicol (100 µg/mL), or kanamycin (50 µg/mL).

METHOD DETAILS

Plasmid construction

Table S4 shows the plasmids used in this study. The primers used for their construction are shown in Table S5. To obtain high expression levels of wild-type CnoX, CnoXC-His, GroEL, and GroES, we cloned the corresponding genes into the high copy vector pET22b(+) (Novagen).

DNA encoding CnoXN-Strep was cloned into the medium copy vector pACYCDuet-1. To prepare an expression vector for cnoX without the last 10 C-terminal residues (CnoX∆Cter), we amplified the corresponding sequence from E. coli genomic DNA and ligated the sequence into pET22b(+). The CnoX and GroEL substitution variants used in this study were generated by site-directed mutagenesis, except for GroELG298A/T299L/V300K/E304L/I305K/M307K/R345L, which was generated using Gibson assembly. We generated the CnoXN-link/C38A/C63A variant using sitedirected mutagenesis by inserting the coding sequences of Met, Ala, Cys, Ala, and Gly residues at the N-terminal extremity. To prepare an expression vector for mHsp60, we amplified the corresponding sequence from the MAC_C_CH60 vector (Addgene) without the mitochondrial targeting sequence, which was replaced by the coding sequences of Met, Gly, and Ser residues, and cloned the sequence into pET22b(+) using Gibson assembly.

Expression and purification of CnoX, CnoX∆Cter, CnoXN-Strep, CnoXC-His, and CnoXNlink/C38A/C63A

We utilized E. coli BL21 (DE3) carrying pET22b_cnoX, pET22b_cnoX∆Cter, or pET22b_cnoXNlink/C38A/C63A to overexpress wild-type CnoX, CnoX∆cter, or CnoXN-link/C38A/C63A, respectively. Cells grew at 37°C until the optical density at 600 nm (OD600) reached 0.5, and then, we added 1 mM IPTG for 3 h to induce protein expression. Cells were pelleted, resuspended in 50 mM For all proteins, we verified the purity via SDS-PAGE with Coomassie staining and concentrated samples using a Vivaspin Turbo (Sartorius) with a 5-kDa molecular-weight cutoff.

We assessed protein concentrations by measuring the absorbance at 280 nm using a Varian Cary 50 UV-Vis spectrophotometer.

Reconstitution of the GroEL-CnoXN-Strep complex

To reconstitute GroEL-CnoXN-Strep, we mixed GroEL (150 µM) in excess (10:1) or with equimolar amounts of CnoXN-Strep in 500 µL of buffer A. After 15 min at room temperature, the mixture was loaded onto a 1-mL Strep-Tactin column (IBA Lifesciences) previously equilibrated in buffer A. After applying a washing step with buffer A, we performed elution with buffer A supplemented with 2.5 mM D-desthiobiotin.

Expression and purification of the GroEL-CS-CnoXN-Strep complex

We used E. coli BL21 (DE3) cells carrying pACYCDuet-1-cnoXN-Strep _gltA_groL to overexpress CnoXN-Strep, CS, and GroEL. Cells were grown at 37°C. When an OD600 of 0.5 was reached, we added 1 mM IPTG for 4 h to induce protein expression. Cells were pelleted, resuspended in buffer B (50 mM Tris-HCl pH 8, 150 mM NaCl) supplemented with 1 mM EDTA, and lysed with a French press. After centrifugation for 20 min at 17,418g at 4°C, the supernatant was filtered through 0.45-μm filters and loaded onto a 2-mL Strep-Tactin column (IBA Lifesciences) previously equilibrated in buffer B. After applying a washing step with buffer B, we performed elution with buffer B supplemented with 2.5 mM D-desthiobiotin. We then purified the sample using size-exclusion chromatography on a HiLoad 10/300 Superdex 200 column (GE Healthcare) using buffer B supplemented with 1 mM EDTA.

In vivo pull-down of CnoX

E. coli MG1655 wild-type, E. coli MG1655 ∆cnoX, and E. coli MG1655 cells carrying pET22b-cnoX, pET22b-cnoX∆Cter, pET22b-cnoXR277L, pET22b-cnoXY284L, or pET22b-cnoXC-His grew in LB (25 mL) at 37°C in a shaking incubator until the mid-log phase was reached. The cells were then harvested, resuspended in 1 mL of buffer E (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA), and sonicated. After a 5-min centrifugation at 16,000g at 4°C, we added 5 µL of undiluted rabbit anti-CnoX antibody and 50 µL of protein A/G magnetic beads (Pierce) to the supernatant to immunoprecipitate CnoX; then, we incubated the samples for 30 min on a wheel at room temperature. After three washes with 500 µL of buffer E, CnoX was eluted with 20 µL of 100 mM glycine pH 2.5. We neutralized the samples with 2 µL of 1.5 M Tris-HCl pH 8 before SDS-PAGE analysis and Coomassie staining.

In vitro pull-down of GroEL

We mixed GroEL (5 µM) with equimolar amounts of either CnoX, CnoX∆Cter, CnoXC-His, or CnoX and unfolded CS in 100 µL of buffer E. After 30 min at room temperature, we immunoprecipitated GroEL by adding 5 µL of undiluted rabbit anti-GroEL antibody and 50 µL of protein A/G magnetic beads (Pierce); samples were incubated for 30 min on a wheel at room temperature. After three washes with buffer E, GroEL was eluted with 20 µL of 100 mM glycine pH 2.5. We then neutralized the samples with 2 µL of 1.5 M Tris-HCl pH 8 before SDS-PAGE analysis and Coomassie staining.

In vitro pull-down of CnoX

CnoX, CnoX∆Cter, or CnoXC-His (5 µM) was mixed with equimolar amounts of GroEL in 100 µL of buffer E. After 30 min at room temperature, we immunoprecipitated CnoX by adding 5 µL of undiluted rabbit anti-CnoX antibody and 50 µL of protein A/G magnetic beads (Pierce); then, we incubated the samples for 30 min on a wheel at room temperature. After three washes with buffer E, CnoX was eluted with 20 µL of 100 mM glycine pH 2.5. We neutralized the samples with 2 µL of 1.5 M Tris-HCl pH 8 before SDS-PAGE analysis and Coomassie staining.

Size-exclusion chromatography analysis

To reconstitute GroEL-CnoX, we mixed both proteins using the ratios indicated in the manuscript. After 15 min at room temperature, the sample was loaded onto a Superdex S200 10/300 column equilibrated with buffer E. The fractions corresponding to absorbance peaks at 280 nm were collected and analyzed by gradient (4%-12%) SDS-PAGE.

To determine the impact of GroES addition on the GroEL-CnoX complex, we mixed GroEL (45 µM) with CnoX using the ratios indicated in the manuscript in 1 mL of buffer F (50 mM Tris-HCl pH 8, 150 mM KCl, 10 mM MgCl2, 1 mM DTT, 1 mM EDTA) supplemented with either ADP (1 mM) or AMP-PNP (1 mM). After 15 min at room temperature, GroES was added at a molar ratio of 14:1:14 (GroEL:CnoX:GroES). After 15 min, we loaded the sample onto a Superdex S200 10/300 column equilibrated with buffer A supplemented with ADP (50 µM) or AMP-PNP (50 µM). The column ran at a flow rate of 0.5 mL/min with 1-mL fractions using an AKTA Purifier (Cytiva). Fractions corresponding to absorbance peaks at 280 nm were collected and analyzed by gradient (4%-12%) SDS-PAGE.

To investigate the binding of CS on GroEL, CS (82 µM, Sigma-Aldrich) was unfolded by dilution in 4 M guanidinium chloride and incubated at room temperature for 2 h. To assay binding on GroEL, we mixed GroEL (45 µM) with unfolded CS (CSdenat) at a molar ratio of 14:1 GroEL:CSdenat in 1 mL of buffer F supplemented with 1 mM ADP. After 15 min at room temperature, GroES was added at a molar ratio of 14:1:14 GroEL:CSdenat:GroES; after another 15 min, we loaded the mixture onto a Superdex S200 10/300 column equilibrated with buffer A supplemented with 50 µM ADP. The column ran at a flow rate of 0.5 mL/min with an AKTA Purifier, and 1-mL fractions were collected. The fractions corresponding to absorbance peaks at 280 nm were collected and analyzed by gradient (4%-12%) SDS-PAGE.

Purification of GroEL, GroES, and CnoX for quantitative binding studies

CnoX and GroES were expressed and purified as previously described [START_REF] Lin | Escherichia coli thioredoxin-like protein YbbN contains an atypical tetratricopeptide repeat motif and is a negative regulator of GroEL[END_REF] . Purified proteins had their N-terminal hexahistidine tags removed by thrombin cleavage and were passed again over Ni 2+ -NTA resin. These proteins were then collected in the flow-through, dialyzed into storage buffer (25 mM HEPES pH 7.5, 100 mM KCl), snap-frozen in 50-to 100-μL aliquots in liquid nitrogen, and stored at -80°C. We verified tag removal by electrospray MS. We induced GroEL expression from pET15b as an untagged protein by removing the sequence between the "CC" of the NcoI site and the "ATG" of the NdeI site using mutagenic PCR primers (see Table S5). GroEL lacking any tag-or vector-derived amino acids was expressed by 0.5 mM IPTG induction, and cells were harvested by centrifugation as described. After sonication and centrifugation at 12000g, clarified lysate was loaded onto a Macro-Prep High Q resin (Bio-Rad) column, and proteins were eluted over a 0.1-1.1 M NaCl gradient in 25 mM HEPES pH 7.5.

The fractions ran on SDS-PAGE gels, and those enriched in GroEL were pooled. We precipitated the crude GroEL fraction by gradually adding finely ground (NH4)2SO4 powder to 75% saturation at room temperature and centrifuging the fraction at 20,000g for 45 min. The pellet was then dissolved in storage buffer (25 mM HEPES pH 7.5, 100 mM KCl) and dialyzed against storage buffer overnight. GroEL was applied to a Sephacryl S-300 HR gel filtration column and eluted near the void volume due to the large size of the GroEL oligomer. We identified the fractions containing GroEL by SDS-PAGE and then pooled and dialyzed the fractions against 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 30% methanol (Tris-methanol buffer).

The pooled GroEL fractions were loaded onto a UNO Q12 anion exchange column (Bio-Rad) and eluted with a gradient of 0-1 M NaCl in Tris-methanol buffer. We identified GroELcontaining fractions by SDS-PAGE, and those lacking any visible contaminants were pooled, dialyzed into storage buffer, concentrated by centrifugation with a 10-kDa molecular-weight cutoff concentrator, snap-frozen in 50-to 100-μL aliquots in liquid nitrogen, and stored at -80°C.

Fluorescent labeling of CnoX

We fluorescently labeled CnoX using FM (Thermo Fisher Scientific) for binding studies. The maleimide moiety of FM is cysteine-reactive and is therefore expected to label the N-terminal thioredoxin-like domain of CnoX, which contains the only two cysteine residues in the protein, Cys38 and Cys63. A 25-fold molar excess of FM to CnoX in 20 mM sodium phosphate buffer pH 7.2, 150 mM NaCl, and 5 mM EDTA was incubated for 4 h at room temperature in the dark.

We then dialyzed the sample using 3.5-kDa-cutoff regenerated cellulose tubing against storage buffer overnight at 4°C to remove any unreacted FM.

We determined the stoichiometry of FM-CnoX labeling via the ratio of absorbance at 495 nm and 280 nm. The calculated ε280 for CnoX is 23,000 M -1 cm -1 (ExPASY), the ε495 of FM is 75,000 M -1 cm -1 , and the ratio of FM extinction coefficients at 280 and 495 nm (

Measurement of the CnoX-GroEL binding affinity

We performed fluorescence measurements on a Cary Eclipse fluorescence spectrophotometer (Varian) at room temperature (~22°C). We measured fluorescence emission spectra between 500 and 560 nm with an excitation wavelength of 495 nm. The excitation and emission slits were set to 5 nm, and spectra were recorded at a scan rate of 10 nm/s. To measure the binding of GroEL to FM-CnoX, we titrated 119 μM GroEL into 0.5 μM FM-CnoX in a total volume of 0.5 mL storage buffer. Final GroEL concentrations ranged from 0.035 to 1 μM. We calculated the normalized fluorescence ratio (D) as:

𝐷 = 𝐹 𝑖 𝐹 0 -1
where Fi is the fluorescence emission of FM-CnoX at peak (typically 520 nm) measured after the addition of GroEL and F0 is the fluorescence emission at peak measured for FM-CnoX alone.

To complement the fluorescence emission measurements, we performed a fluorescence anisotropy binding assay using a Jasco J-815 CD spectrophotometer at 20°C. We employed an excitation wavelength of 495 nm and a 540-nm emission filter. Measurements were collected using a cell with a 1-cm path length at a scan rate of 0.833 nm/s from 450 to 550 nm.

In a total volume of 2 mL of storage buffer, we titrated 0.5 μM FM-CnoX with 119 μM GroEL to final concentrations ranging from 0.036 to 1 μM. The final volume change after GroEL titration was less than 1%. We measured the fluorescence anisotropy for each titration point 2 min after the addition of GroEL. The data were averaged from 3-4 measurements. Binding data were plotted and fitted in Prism (GraphPad) using nonlinear least-squares minimization. We utilized a single-site binding model with positive cooperativity for the CnoX-GroEL interactions and a single-site competition model with a logarithmic competitor concentration for the GroES competition experiment. No data linearization was performed.

Influence of nucleotides on the CnoX-GroEL and GroES interaction

To examine the effect of ATP or ADP on GroEL-CnoX binding, we repeated the experiment described in the previous section using 0.5 μM FM-CnoX and GroEL in storage buffer with 2 mM ATP (Alfa Aesar) or 2 mM ADP (Sigma) and 10 mM MgCl2. For the GroES competition experiment, 0.5 μM FM-CnoX was incubated with 0.5 μM GroEL, with or without 2 mM ATP in storage buffer, and then titrated with GroES to final concentrations ranging from 0.035 to 2 μM.

We measured the fluorescence emission at peak 2 min after adding titrated protein at room temperature. In all titrations, the final volume change was less than 1%; hence, no dilution correction was performed. Data were averaged from 3-4 measurements.

In vivo trapping of GroEL-CnoX-substrate tripartite complexes

To identify proteins trapped in a mixed disulfide with CnoX when CnoX is bound to GroEL, we immunoprecipitated GroEL from extracts prepared from E. coli BL21 (DE3) cells carrying pET22b-cnoX and pET22b-cnoXC38A/C63A as follows. After an overnight preculture, cells were diluted in 500 mL of LB containing 100 mg/mL ampicillin and grown at 37°C. At the mid-log phase, we induced protein expression for 1 h with 1 mM IPTG. Cultures were cooled on ice for 10 min, and iodoacetamide (25 mM) was added. After 30 min, the cells were harvested, resuspended in 20 mL of buffer E, and sonicated. After performing centrifugation at 12,000g at 4°C, we added 150 µL of undiluted rabbit anti-GroEL antibody and 1 mL of protein A/G magnetic beads (Pierce) to the supernatant. After 30 min on a wheel at room temperature, magnetic beads were collected and resuspended with 1 mL of buffer E. After applying three washes with 1 mL of buffer E, we eluted GroEL with 100 µL of 0.1 M glycine pH 2.5.

Chaperone-mediated reactivation of chemically unfolded CS

We adapted our protocol from Haslbeck and Buchner (2015 mM, respectively) or the GroEL14/GroES7 refolding system (0.15 mM and 0.5 mM, respectively) was added to the refolding solution and incubated for 2 h at 25°C. We added 4 mL of this mixture to 200 mL of reaction buffer (100 mM oxaloacetic acid, 100 mM 5,5'dithiobis(2-nitrobenzoic acid), and 160 mM acetylCoA in Tris-EDTA buffer) in a 96-well plate.

)
We monitored the absorbance at 412 nm for 1 min with a Biotek Synergy H1 Hybrid microplate reader and used the initial slope of the absorbance values to calculate the CS activity.

Bacterial viability assay

E. coli MC4100 ∆tig∆dnaKdnaJ cells carrying p29SEN-EV, p29SEN-groSgroL, p29SEN-groSgroLheptuple, p29SEN-groSgroLcnoX, or p29SEN-groSgroLcnoXΔCter grew in LB supplemented with ampicillin at 20°C until the mid-log phase was reached. Cells were serially diluted and spotted on LB agar plates containing ampicillin, with or without 100 μM IPTG, overnight at 37°C.

AFM

Gold-coated glass coverslips and cantilevers (OMCL-TR4, Olympus Ltd.; nominal spring constant of ∼0.02 N m -1 ) were incubated with 0.1 mg mL -1 of CnoXN-link/C38A/C63A, the last Cterminal 10 amino acids of CnoX fused to a linker (CGGGSGGGYRRQLYALLY), or GroELD490C solution for 1 h, rinsed with buffer D, and then immediately used without dewetting. We performed measurements at room temperature in 50 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA with a Force Robot 300 AFM (JPK Instruments). We recorded multiple (32 × 32) force-distance curves over an area of 500 x 500 nm 2 with an applied force of 250 pN, a constant approach, and a retraction speed of 1,000 nm s -1 . A histogram was generated based on the force of the last rupture event for each curve. We measured the spring constants of the cantilevers by the thermal noise method and analyzed these data with data-processing software from JPK Instruments.

Native PAGE

We performed Blue native electrophoresis analysis of the concentrated complex on a 3%-12%

Bis-Tris gel (Life Technologies) following the manufacturer's instructions. We identified the protein complex bands separated in the native electrophoresis via SDS-PAGE. Briefly, bands of interest were excised, boiled in SDS-PAGE sample buffer, and applied to the top of a polyacrylamide gel.

Immunoblotting

Samples were boiled before being loaded onto a precast NuPAGE Bis-Tris 12% gel (Life Technologies). We performed immunoblotting according to standard procedures using 1:5000 anti-CnoX antibody (rabbit serum, CER group, Belgium) followed by a horseradish peroxidaseconjugated anti-rabbit antibody (Sigma). We conducted chemiluminescence imaging (ECL Prime Western Blotting Detection Reagents; GE Healthcare) with an ImageQuant LAS 500

Camera (GE Healthcare Life Sciences).

Mammalian homologs of CnoX

The (Thioredoxin), 10,056 (TPR_19), and 44 (TPR_20) eukaryotic sequences. We downloaded all 44 eukaryotic sequences from UniProt 31 and built corresponding AlphaFold 3D models using ColabFold [START_REF] Mirdita | ColabFold: making protein folding accessible to all[END_REF] . Most of these models contained all three domains (Thioredoxin, TPR_19, and TPR_20). Three representative structures are shown in Figure S5.

Protein identification by MS

After in-gel digestion with trypsin, peptides were dissolved in solvent A (0.1% trifluoroacetic acid in 2% acetonitrile), directly loaded onto a reversed-phase precolumn (Acclaim PepMap 100, Thermo Fisher Scientific), and eluted in backflush mode. We performed peptide separation using a reversed-phase analytical column (Acclaim PepMap RSLC, 0.075 x 250 mm, Thermo Fisher Scientific) with a linear gradient of 4%-27.5% solvent B (0.1% formic acid in 98% acetonitrile) for 40 min, 27.5%-50% solvent B for 20 min, 50%-95% solvent B for 10 min, and holding at 95% for the last 10 min at a constant flow rate of 300 nL/min on an Ultimate 3000 RSLC system. We analyzed the peptides by an Orbitrap Fusion Lumos tribrid MS (Thermo Fisher Scientific). The peptides were subjected to a nanospray ionization source followed by MS/MS in a Fusion Lumos coupled online to a nano-LC. Intact peptides were detected in the Orbitrap at a resolution of 120,000. We selected peptides for MS/MS using a higher-energy collision dissociation setting of 30 and detected ion fragments in the Orbitrap at a resolution of 30,000. A data-dependent procedure that alternated between one MS scan and one MS/MS scan was applied for 3 s for ions above a threshold ion count of 2.0×10 4 in the MS survey scan with 40.0-s dynamic exclusion. An electrospray voltage of 2.1 kV was applied. We obtained MS1 spectra with an automatic gain control target of 4×10 5 ions and a maximum injection time of 50 ms and MS2 spectra with an automatic gain control target of 5×10 4 ions and a maximum injection set to dynamic. For MS scans, the m/z scan range was 375-1800.

We processed the resulting MS/MS data using the Sequest HT search engine within Proteome Discoverer 2.4 SP1 against an E. coli K12 protein database obtained from Uniprot (4,349 entries). Trypsin was specified as a cleavage enzyme allowing up to two missed cleavages, four modifications per peptide, and up to five charges. We set the mass error to 10 ppm for precursor ions and 0.1 Da for fragment ions. Oxidation on Met (+15.995 Da) and conversion of Gln (-17.027 Da) or Glu (-18.011 Da) to pyro-Glu at the peptide N-terminal were considered as variable modifications. We assessed the false discovery rate using Percolator, with the thresholds for proteins, peptides, and modification sites specified at 1%. For abundance comparison, we calculated abundance ratios by label-free quantification of the precursor intensities within Proteome Discoverer 2.4 SP1.

Crosslinking identification by MS

We applied 100 µL of the purified ternary complex between CS, CnoX, and GroEL onto a NAP-5 column for buffer exchange into 500 µL of 20 mM HEPES pH 8. Then, DSSO was not added or added to the protein sample at a final concentration of 2 mM. After 60 min at room temperature, we ended the reaction by adding 40 mM of Tris pH 8. Protein extraction and digestion with trypsin were performed as in [START_REF] Luo | Thiol-disulphide independent in-cell trapping for the identification of peroxiredoxin 2 interactors[END_REF] . Briefly, protein samples were precipitated by adding three volumes of methanol, one volume of chloroform, and three volumes of water, vortexed, and centrifuged at 15,000g for 2 min at room temperature. The supernatant was removed carefully and the precipitated protein layers at the interface were further washed and pelleted by adding three volumes of methanol and centrifuging at 15,000g for 2 min.

Subsequently, the protein pellets were air-dried for 10 min after removing the methanol. The Vivaspin membrane with a 5-kDa cutoff (Sartorius). Peptide separation was performed on an Ultimate 3000-nLC RSLC essentially as described in [START_REF] Luo | Thiol-disulphide independent in-cell trapping for the identification of peroxiredoxin 2 interactors[END_REF] except that the analytical column was a C18 reversed-phase nano-analytical column (BioZen Polar-C18, 250 x 0.075 mm, Phenomenex). We subjected the peptides to a nanospray ionization source followed by tandem MS/MS in a tribrid Fusion Lumos Orbitrap analyzer coupled online to a nano-LC.

Spectra were acquired by an XLMS Clevable MS2-MS3 scan routine specific for DSSO Xlinks with MS1 and MS2 detection in the Orbitrap and MS3 in the Ion Trap. In summary, we acquired full MS1 spectra at a resolution of 120,000 and MS2 scans at a resolution of 30,000. The first MS2 scan was performed in data-dependent acquisition mode with collision-induced dissociation (CID) fragmentation and analysis of the daughter ions in the Orbitrap. If a targeted mass difference specific for DSSO (31.9721 Da) was detected in an ion pair, this finding would trigger a supplemental MS2 fragmentation of the same parent ion by EThcD and several MS3 scans for both ions in the pair by CID fragmentation. We processed the resulting MS/MS data using Sequest HT and the Xlink 2.5 search engine within Proteome Discoverer 2.5 against an E. Coli protein database obtained from Uniprot (4,353 entries) and a specific homemade library containing six proteins (groEL, cnoX, tufB, ompA, gltA, lpp) for Xlink identification. Trypsin was specified as a cleavage enzyme allowing up to two missed cleavages, four modifications per peptide, and up to seven charges. We set the mass error to 10 ppm for precursor ions and 0.02 Da for fragment ions. Oxidation on methionine, carbamidomethyl on cysteine, and DSSO monolinks were considered as variable modifications. We assessed the false discovery rate using Percolator and set specified thresholds for proteins, peptides, and modification sites at 1%.

Single-particle cryoEM imaging and data processing

Prior to vitrification, we assessed the sample quality using negative-stain EM. For this step, 3

μL GroEL-CnoX at 0.4 mg/mL was applied to a glow-discharged formvar Cu400 grid (Electron Microscopy Sciences) and screened using in-house 120-kV JEOL JEM 1400 and 1400+ microscopes equipped with an LaB6 filament at the VIB-VUB BECM facility. We vitrified the GroEL-CnoX sample in liquid ethane using a CP3 Cryoplunge (Gatan) set to 100% humidity and room temperature. R2/1 (Quantifoil) grids were coated with graphene oxide (Sigma), and a 3-μL sample at 0.4 mg/mL was applied and manually back-blotted for 4 s before plunging.

We collected data on an in-house (BECM) 300-kV JEOL CRYOARM 300 equipped with a K3 direct electron detector (Gatan). We collected 3,015 movies in counting mode, with a dose of 68.27 e/Å 2 spread over 61 frames at a nominal magnification of 60.000X, corresponding to a pixel size of 0.784 Å. Images were motion-corrected via MotionCor2.1 34 using 5 x 5 patches with dose-weighting, and the contrast transfer function was estimated using ctffind4.1 [START_REF] Rohou | CTFFIND4: Fast and accurate defocus estimation from electron micrographs[END_REF] . We picked particles using crYOLO 1.5 [START_REF] Wagner | SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM[END_REF] with a dataset-specific trained crYOLO network on six micrographs (709 picked particles). The picking quality and CnoX occupancy were assessed using ISAC2 [START_REF] Yang | Iterative stable alignment and clustering of 2D transmission electron microscope images[END_REF] as implemented in the SPHIRE package [START_REF] Moriya | High-resolution single particle analysis from electron cryo-microscopy images using SPHIRE[END_REF] .

We performed all subsequent processing using RELION3.0 [START_REF] Zivanov | New tools for automated high-resolution cryo-EM structure determination in RELION-3[END_REF] . A total of 670,080 picked particles were extracted with a box size of 400 and were binned twice for processing. A c1 initial 3D model was generated ab initio. In all subsequent steps, we applied c7 symmetry throughout. After extensive rounds of 3D classification to optimize the density for CnoX on GroEL, we utilized 170,458 particles for the final reconstruction at 3.4 Å.

Final maps were density-modified using phenix.resolve [START_REF] Terwilliger | Improvement of cryo-EM maps by density modification[END_REF] , and the model was fitted and symmetry-expanded in UCSF Chimera 41 using the second TPR domain of CnoX (helices 2A-2A'-2B-2B'-2C [197-284] from PDB: 3QOU) and GroEL (pdb:1XCK) as starting coordinates. We conducted further docking and refinement using phenix.real.space [START_REF] Afonine | Real-space refinement in PHENIX for cryo-EM and crystallography[END_REF] with manual curation in COOT. 43 After the first refinement, we generated a second local resolution-sharpened map using LocScale [START_REF] Jakobi | Model-based local density sharpening of cryo-EM maps[END_REF] , as implemented in the CCPEM project package [START_REF] Burnley | Recent developments in the CCP-EM software suite[END_REF] . We used this map together with the density-modified map to aid further map building and refinement in Phenix [START_REF] Liebschner | Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix[END_REF] .

The reported FSC-model curve, resolution, and local-resolution maps were calculated using the Phenix validation output and the Phenix implementation of local-resolution assessment.

QUANTIFICATION AND STATISTICAL ANALYSIS

We performed statistical analysis using GraphPad Prism 6. Results are represented as the mean ± standard error of the mean. We conducted comparisons of two groups using unpaired two-tailed Student's t-tests with an assumed Gaussian distribution and equal variances.

Experiments were performed in triplicate (n = 3) unless otherwise indicated.

Figure 1 .

 1 Figure 1. CnoX interacts stably with GroEL.

Figure 2 .

 2 Figure 2. CryoEM shows that the TPR domain of CnoX binds GroEL.

Figure 3 .

 3 Figure 3. The C-terminal -helix of CnoX binds a shallow cleft in the apical domain of

Figure 4 .

 4 Figure 4. CnoX functions as a molecular plugin to rescue GroEL substrates from oxidative

Figure 5 .

 5 Figure 5. Unfolded CS is present in the cavity of GroEL when in a mixed disulfide with

Figure S2 .

 S2 Figure S2. GroES binding to GroEL displaces CnoX from the chaperonin. CnoX binding to

Figure S3 .Figure 2 (

 S32 Figure S3. Single-particle cryoEM analysis of the purified GroEL-CnoX complex, related to

Figure S4 .

 S4 Figure S4. CnoX binds GroEL via its conserved C-terminus, related to Figure3

Figure S5 . 4 (

 S54 Figure S5. The C-terminus of CnoX interacts with human mitochondrial Hsp60 and is

Data and code availability 

 availability Coordinates and electron potential maps for the GroEL:CnoX cryoEM structure have been deposited in the PDB and EMDB under accession codes 7YWY and EMD-14352, respectively. Raw data from Figures 1, 2, 3, 4, 5 and Supplemental Figures 1, 2, 3, 4, 5, 6 were deposited on Mendeley at http://dx.doi.org/10.17632/9pt4v7hc93.1.

  called the correction factor) is 0.3. The labeling stoichiometry is 2.2 FM per CnoX molecule, as expected for the two cysteine residues in the N-terminal thioredoxin domain. FM-CnoX was snap-frozen in liquid nitrogen and stored at -80°C.

  and Hoffmann et al. (2004). Briefly, 15 mM CS (Sigma-Aldrich) was unfolded by dilution in 4 M guanidinium chloride in Tris-EDTA buffer (50 mM Tris-HCl [pH 8], 2 mM EDTA) and incubated at 16°C for 2 h. We diluted the unfolded CS by 1:160 (final concentration of 75 nM) in 40 mM HEPES-KOH (pH 8), 10 mM KCl, and 2 mM Mg-ATP (unless otherwise specified) containing 0 or 0.45 mM YbbN. After a 20-min incubation at 25°C, the DnaK/DnaJ/GrpE refolding system (0.4 mM, 0.16 mM, and 0.4

  protein pellets were then resuspended in 200 μL of a buffer containing 4 M urea, 50 mM NH4HCO3 and sonicated at 80 % amplitude (20 kHz) for 30 s. The protein suspension was reduced by 5 mM tris(2-carboxyethyl)-phosphine (TCEP) at 55 °C for 20 min and then alkylated by 10 mM iodoacetamide in the dark at 25 °C for 20 min with vigorous shaking using a Thermomixer Eppendorf Comfort at 1000 rpm. Afterwards, the proteins were digested by adding 250 μL 50 mM NH4HCO3, 2.5 μL 1 % ProteaseMAX™ Surfactant dissolved in 50 mM NH4HCO3, and 1:100 (enzyme/protein, w/w) Sequencing Grade Modified Trypsin to reach a final reaction volume of 500 μL. The digestion reactions were incubated overnight at 37 °C with vigorous shaking using a Thermomixer Eppendorf Comfort. The next day, the protein digestion reactions were stopped by adding trifluoroacetic acid (TFA) to 0.1 % final concentration. We then conducted size-exclusion enrichment of crosslinked peptides on a

  Figure 1

  CnoX structure (http://pfam.xfam.org/protein/P77395) is organized into three domains, corresponding to the Pfam entries Thioredoxin (http://pfam.xfam.org/family/Thioredoxin),

	TPR_19 (http://pfam.xfam.org/family/TPR_20). As of March 9, 2022, these Pfams contain 40,361 (http://pfam.xfam.org/family/TPR_19), and TPR_20

Table S3 : Bacterial strains used in this study, related to STAR Methods.

 S3

	Strain	Genotype	Plasmid	Source and notes
				From Jim
				Bardwell
				(University of
	JF179	MG1655 wt		Michigan)
				Goemans et al.,
	ED002	MG1655 ΔcnoX		2018
				Goemans et al.,
	ED004	MG1655 ΔcnoX pET22b-cnoX	2018
	ED207	MG1655 ΔcnoX pET22b-cnoXR277L	This study
	ED217	MG1655 ΔcnoX pET22b-cnoXY284L	This study
	ED006	MG1655 ΔcnoX pET22b-cnoXC-His	This study
	ED005	MG1655 ΔcnoX pET22b-cnoXΔCter	This study
				Goemans et al.,
	CG233	BL21 (DE3)	pET22b-groL	2018
	ED035	BL21 (DE3)	pET22b-groLD490C	This study
				This study;
				Weissman et al.,
	ED126	BL21 (DE3)	pET22b-groLR452A/E461A/S463A/V464A	1995, 1996
	ED187	BL21 (DE3)	pET22b-groLG298A/T299L/V300K/E304L/I305K/M307K/R345L	This study
	CG183	BL21 (DE3)	pET22b-groS	This study
				Goemans et al.,
	CG232	BL21 (DE3)	pET22b-cnoX	2018
	CG229	BL21 (DE3)	pET22b-cnoXΔCter	This study
	CG191	BL21 (DE3)	pET22b-cnoXC-His	This study
				Goemans et al.,
	CG260	BL21 (DE3)	pET22b-cnoXC38A/C63A	2018
	ED042	BL21 (DE3)	pET22b-cnoXN-link/C38A/C63A	This study
	ED145	BL21 (DE3)	pET22b-mhsp60	This study
	ED049	BL21 (DE3)	pACYCDuet-1-cnoXN-Strep	This study

Table S5 : Primers used in this study, related to STAR Methods.

 S5

	EDo324_AD_groEL_G298A-
	T299L-E304L-	AGATCACAAGCGCGCCAGTCAG	cloning (pET22b-
	R345L_Gibson_frag2_R	GGTTGCGATATC	groLG298A/T299L/V300K/E304L/I305K/M307K/R345L)
	Primer EDo325_AD_groEL_G298A-	Sequence (5' -> 3')	Comments
	T299L-V300K-E304L-I305K-	AAAACCATGGCAAGCTGGAGCC
	M307K-	ACCCGCAGTTCGAAAAGTCCGTA AATCCAGGGCCTTGTTGCTCAGA	cloning (pET22b-
	AGo230_cnoX_Strep_NcoI_F R345L_Gibson_frag1_F	GAAAATATTGTCAAC TCCGTCAGCAG	cloning (pACYCDuet-1-cnoXN-Strep) groLG298A/T299L/V300K/E304L/I305K/M307K/R345L)
	AGo231_cnoX_Strep_BamHI EDo326_AD_groEL_G298A-	AAAAGGATCCTCAATACAACAAT
	_R T299L-V300K-E304L-I305K-	GCATACAGC	cloning (pACYCDuet-1-cnoXNterStrep)
	CGo174_GroES_NdeI_F M307K-CGo185_GroES_stop_XhoI_ R R345L_Gibson_frag1_R EDo327_AD_groEL_G298A-CGo178_cnoX_H6_NdeI_F T299L-V300K-E304L-I305K-M307K-CGo71_cnoX_H6-stop_XhoI_R R345L_Gibson_frag2_F EDo328_AD_groEL_G298A-T299L-V300K-E304L-I305K-CGo57_cnoX_NdeI_F M307K-R345L_Gibson_frag2_R CGo234_cnoXdel10_HindIII_ stop_R EDo34_22b_MACAG_cnoX_ F EDo35_22b_MACAG_cnoX_	GAGCATATGAAGTTTCGTCCC CTTTACCTTTAAGTTCAGAGATTT TAAGCGCGCCAGTCAGGGT GAGCTCGAGTTAGGCTTCGACCA CGCC GAGCATATGCACCACCACCACCA CCACTCCCTGATCGGC AAAATCTCTGAACTTAAAGGTAA AGAGCTGGAAAAAGC CTCCTCGAGTCAGTGGTGGTGGT GGTGGTGGCTGAAGAGGATCGA GG GAGCAACAAGGCCCTGGATTGC GAGCATATGATGTCCCTGATCGG C AGCTTC ATGGCTTGTGCTGGTATGTCCGT CTCAAGCTTTCACTTCGACGCCA GTGCATCACC AGAA ACCAGCACAAGCCATCATATGTA	cloning (pET22b-cloning (pET22b-groS) groLG298A/T299L/V300K/E304L/I305K/M307K/R345L) cloning (pET22b-groS) cloning (pET22b-cloning (pET22b-cnoXC-His) groLG298A/T299L/V300K/E304L/I305K/M307K/R345L) cloning (pET22b-cnoXC-His) cloning (pET22b-groLG298A/T299L/V300K/E304L/I305K/M307K/R345L) cloning (pET22b-cnoXΔCter) cloning (pET22b-cnoXN-link/C38A/C63A) cloning (pET22b-cnoXΔCter)
	EDo227_22b(mHSP60)_F R	TGAGATCCGGCTGCTAAC TAATC	cloning (pET22b-mhsp60) cloning (pET22b-cnoXN-link/C38A/C63A)
		ATGTATATCTCCTTCTTAAAGTTA AGCTCCGCTGGCTCAGATCGTAT	cloning (pET22b-
	EDo228_22b(mHSP60)_R EDo92_22b_groEL_R452A_F	AACAAAATTATTTC TGAACTGC	cloning (pET22b-mhsp60) groLR452A/E461A/S463A/V464A)
		TTTAAGAAGGAGATATACATATG	cloning (pET22b-
	GGAAGTGCCAAAGATGTAAAATT EDo93_22b_groEL_R452A_R TCCATTGCACGCAGTGCA	groLR452A/E461A/S463A/V464A)
	EDo229_(22b)mHSP60_F EDo94_22b_groEl_E461A/S4	TGGTGC GCTGCTGTTGCTAACACCGTTAA	cloning (pET22b-mhsp60) cloning (pET22b-
	63A/V464A_F	TTGTTAGCAGCCGGATCTCAGAA AG	groLR452A/E461A/S463A/V464A)
	EDo230_(22b)mHSP60_R EDo95_22b_groEl_E461A/S4	CATGCCACCTCCCATAC CGGAGCTTCGCCGCAGTTCAATA	cloning (pET22b-mhsp60) cloning
	63A/V464A_R	TGGATCCAGGATACCCATGCAGA C	(pET22b_groLR452A/E461A/S463A/V464A)
	EDo31_GroEL-D490C_F CnoX_NdeI_for	TCATGTTGCCGTATTCT GCCGACGCCCCTTGCATATGTCC	cloning (pET22b-groELD490C)
		AGAATACGGCAACATGATCTGCA GTAGAAAATATTGTC	cloning (pET15b-cnoX)
	EDo32_GroEL-D490C_R CnoX_XhoI_rev	TGGGTATCCTGGATCCA CCGTGCTTTCTTGCTCGAGTCAAT	cloning (pET22b-groELD490C)
	EDo321_AD_groEL_G298A-	ACAACAATGCATACAGC	cloning (pET15b-cnoX)
	T299L-E304L-GroES_NdeI_for	GACTGGCGCGCTTGTGATCTCTG CCTGAGAAGCGTTCCATATGAAT	cloning (pET22b-
	R345L_Gibson_frag1_F	AACTTATCG ATTCGTCCATTGCATGATCG	groLG298A/T299L/V300K/E304L/I305K/M307K/R345L) cloning (pET15b -groS)
	EDo322_AD_groEL_G298A-GroES_XhoI_rev	CCGTGCTGCCTTGCTCGAGTTAC
	T299L-E304L-	GTCACGCTCGTCGTTTGGTATGG GCTTCAACAATTGCCAGAATG	cloning (pET22b-cloning (pET15b -groS)
	R345L_Gibson_frag1_R GroEL_mHis_for	CTTCATTC CTTTAAGAAGGAGATATACCCAT	groLG298A/T299L/V300K/E304L/I305K/M307K/R345L)
	EDo323_AD_groEL_G298A-	ATGGCAGCTAAAGACGTAAAATT
	T299L-E304L-	TACCAAACGACGAGCGTGACACC C	cloning (pET22b-cloning (pET15b-groL)
	R345L_Gibson_frag2_F GroEL_mHis_rev	ACGATG GAATTTTACGTCTTTAGCTGCCAT	groLG298A/T299L/V300K/E304L/I305K/M307K/R345L)
		ATGGGTATATCTCCTTCTTAAAG cloning (pET15b-groL)

ACKNOWLEDGEMENTS

We thank Asma Boujtat and Gaetan Herinckx for technical help. We are indebted to Dr. Michael Deghelt, Dr. Seung-Hyun Cho, and Dr. Pauline Leverrier for helpful suggestions and discussions and for providing comments on the manuscript. M.A.W. received support from National Institutes of Health grant R01GM139978. We thank Dr. Tommi White and Dr. Javier Seravalli for assistance with the GroEL-CnoX quantitative interaction studies and Dr. Aron Fenton for discussions about cooperative protein binding. We thank the staff at the VIB-VUB facility for Bio Electron Cryogenic Microscopy (BECM) for assistance in data collection. This work was funded by the Fonds de la Recherche Scientifique (FNRS) grant agreements WELBIO-CR-2015A-03 and WELBIO-CR-2019C-03, the Excellence of Science in Research Program of the FWO and FRS-FNRS (G0G0818N), the Fédération Wallonie-Bruxelles (ARC 17/22-087), GENCI-IDRIS (2021-A0100711524), the Flanders Research Foundation Hercules grant (G0H5916N), the Flanders Research Foundation PhD fellowship program, and the Flanders Institute for Biotechnology -VIB.

Data availability

• Method details • Quantification and statistical analysis

SUPPLEMENTAL INFORMATION

Table S2: Proteins involved in the mixed disulfides with CnoX, related to Figure 4.

AUTHOR CONTRIBUTIONS

Writing: JFC, SEVdV, ED, HR, and CVG. Conceptualization: CVG, ED, HR, and JFC.

Investigation, strain construction, construct cloning: ED, CVG, AVD, AG, SEVdV, JL, MAW, EL, YFD, and FV. Identification and AlphaFold modeling of CnoX mammalian homologs: BII. Mass

DECLARATION OF INTERESTS

The authors declare no competing interests.

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