domain: phys nlin

text:
K Veyrinas
email: kevin.veyrinas@u-bordeaux.fr

C Valentin

D Descamps

C Péjot

F Burgy

F Catoire

E Constant

E Mével

Optics-free focusing down to micrometer spot size and spectral filtering of XUV harmonics

Controlling the wavefront of an extreme ultraviolet (XUV) high-order harmonic beam during the generation process offers to focus the beam without resorting to any XUV optics. By characterizing the XUV intensity profile and wavefront, we quantitatively retrieve both the size and the position of the waist of each generated harmonics and show that optics-free focusing leads to focused XUV spot with micrometer size. We use this remarkable coherent effect to demonstrate efficient and adjustable spectral filtering of the XUV light, along with a strong rejection of the fundamental beam, without using any XUV optics.

High-order harmonic generation (HHG) [1,2] is a highly non-linear process producing coherent light in the extreme ultraviolet (XUV) domain and is nowadays commonly used as a table-top source of attosecond pulses [3,4]. Numerous applications rely on these HHG-based sources such as ultrafast molecular dynamics [5][6][7], coherent diffraction imaging [8,9], nonlinear optics with XUV photons [10,11], for instance. A major challenge is now to increase the XUV intensity in order to perform XUV-pump-XUV-probe experiments. This can be done by increasing the energy, shortening the duration of the XUV pulse, or by controlling the focusing properties of the XUV beam which strongly depend on the harmonic order [12][13][START_REF] Wikmark | Proc. Natl. Acad. Sci[END_REF].

For a given laser system, the pulse energy and duration define the typical XUV flux that can be achieved after optimization. The XUV intensity can then be increased by focusing the XUV beam provided the focusing system does not induce significant pulse distortion [START_REF] Bourassin-Bouchet | [END_REF]. Techniques based on reflective mirrors have been used as metallic toroidal mirror [16] or Mo/Si parabolic mirror [17] but they can induce optical aberrations leading to pulse broadening [START_REF] Bourassin-Bouchet | [END_REF]. Multilayer mirrors specifically designed for attosecond pulse-shortening can also be used but suffer from low reflectivity and lack of versatility [18]. Compressors based on XUV gratings have now a good transmission when conical diffraction is used but they require large setup.

Recently, Quintard et al. have demonstrated the possibility of generating harmonics as either diverging or converging beams showing the qualitative ability to control the XUV beam properties [19]. This so-called optics-free focusing results from the possibility of controlling the XUV phase front inside the generating medium. For a thin generating medium where phase matching effects lead to a global prefactor [20], the spatial phase ( , ) q tr  of a given harmonic q can be approximated by:

( , ) ( ) ( ) ( , ) q IR q IR t r q r I t r      (1)
The first term corresponds to q times the spatial phase () IR r  of the driving beam. When only this term is considered, the XUV and IR wavefronts are identical. The second contribution, proportional to the IR intensity ( , ) IR I t r , is an atomic phase intrinsic to the HHG process. The () q  coefficient depends on both q and  , the excursion time of the electronic wavepacket in the continuum [21,22]. The spatial evolution of ( , ) q tr  changes the curvature of the XUV wavefront. From an equivalent optical perspective, the two above-mentioned contributions can be expressed in terms of the radii of curvature IR R and atom R , respectively, and the total radius of curvature R is given by 1 1 1

IR atom R R R 
. Both terms can have similar values. While atom R is always positive, IR R can either be negative (when the IR beam is converging, i.e. before the IR focus) or positive (when the IR beam is diverging, i.e. after the IR focus). It is thus possible to control the curvature of the XUV wavefront by controlling the interaction parameters such as the IR intensity, the value of q  and the gas jet position with respect to the IR focus.

In this Letter, we use a spectrally-resolved wavefront characterization of the XUV radiation to retrieve the waist of each generated harmonic for different positions of the gas jet in the laser focus. This constitutes a direct measurement of coherent focusing and we show that (i) foci as small as 5 µm are achieved by controlling the XUV beam without any XUV optics and (ii) the studied harmonics are not all focused at the same position. This reduces the XUV intensity and makes the attosecond temporal structure space-dependent. We also control harmonic optics-free focusing to achieve efficient and adjustable XUV spectral filtering without using optics.

Experiments have been performed on the terawatt Ti:sapphire Eclipse laser facility at CELIA delivering 800-nm, 100-mJ, 40-fs pulses at a 10-Hz repetition rate. The experimental setup has already been described [19]. Briefly, we used 9mJ of laser energy. The beam is spatially filtered to ensure a near-Gaussian spatial profile ( 21.02

vert M  and 2 1.09 hor M 
) with a transmission of 50 %. Pulses are then compressed under vacuum down to 40 fs and propagate under vacuum after the compressor. Optimization of the IR wavefront is performed by reflecting the beam on a deformable mirror (HIPAO, ISP System) coupled to an HASO wavefront sensor (Imagine Optic) for characterization, allowing for Strehl ratio higher than 0.97. IR pulses are then focused by a 2-m focal length spherical mirror (peak intensity of ~ 2×10 14 W/cm 2 at focus) in a pulsed neon jet mounted on a translation stage able to move over 15 cm around the laser focus. Negative (resp. positive) value of z jet means that HHG occurs upstream (resp. downstream) of the IR focus. Generated harmonics are then transmitted through the 500-µm-wide entrance slit of an XUV spectrometer before being dispersed by a grazing incidence Hitachi spherical grating (1200 gr/mm) onto an XUV detector consisting of a 40-mm-diameter dual microchannel plates (MCPs) and a phosphor screen imaged by a CCD camera (2.9 m after the laser focus). A movable pinhole with diameter Ø = 140 µm can be inserted 37 cm after the IR focal plane to characterize the intensity profile and wavefront of each harmonic. At this position, the pinhole can withstand the IR power without damage.

Characterization of the XUV wavefront has been performed using the SWORD (Spectral Wavefront Optical Reconstruction by Diffraction) technique [23]. It consists of recording the diffraction pattern of the XUV radiation through a slita pinhole in our casewhile scanning the slit across the XUV beam. For each harmonic, the spatial phase of the sampled wavefront slice is extracted from the relative vertical position of the diffraction pattern's centroid with respect to the pinhole position. The corresponding local XUV intensity is obtained by integrating the signal of the diffraction pattern on the MCP. Figure 1 presents the results of the SWORD measurement for harmonics H31, H39 and H47. Fitting the measured XUV intensity profile (a) and reconstructed spatial phase (b) by Gaussian and quadratic functions, respectively, provides the beam size q w and the radius of curvature q R of the wavefront in the pinhole plane. We observe that the beam size and wavefront curvature change with the harmonic order and gas jet position. For z jet = -35 mm, we obtain w = 309, We observe that the spatial profile is close to Gaussian and that the curvature is quadratic. Under these conditions, we can assume that the XUV beam propagates like a Gaussian beam and retrieve, from this intensity profile and wavefront characterization, both the size ,0 q w and the position ,0 q z of the waist of the considered harmonic:

,0 2 2 1 q q q qq w w w R         (2) ,0 2 2 1 q q qq q R z R w         (3)
In Fig. 2, we present the results of this analysis for harmonics H31, H33, H49 and H51, as a function of the gas jet position z jet relative to the IR focus. Strong dependences of ,0 q w (Fig. 2(a)) and ,0 q z (Fig. 2(b)) with q and z jet are observed and the harmonic waists are separated longitudinally. The waist size varies from ~ 5 µm to ~ 15 µm for H31-H33 and from ~ 5 µm to ~ 20 µm for H49-H51. As the harmonic order increases, the maximum waist size is larger and obtained for generation further upstream of the IR focus. Fig. 2(b), the gas jet position is denoted by the dashed line and we observe that there is a full range of parameters where the XUV foci are located after the gas jet and are therefore real. XUV waists below (resp. above) this line are located before (resp. after) the generating medium and thus virtual (resp. real). For z jet > -35 mm, the waists of harmonics H49 and H51 are virtual and the XUV radiations are emitted as diverging beams. For z jet < -35 mm, these waists are real meaning that harmonics H49 and H51 are emitted as converging beams. The waists of harmonics H31 and H33 are real for z jet < -5 mm and virtual for z jet > -5 mm. We observe that these converging XUV beams are focused to spot size of 5-20 µm at distances up to 120 mm (4.4×z R_IR ) after the IR focus without using any XUV optics. Overall, these measurements are in good qualitative agreement with the simulated results reported recently by Quintard et al. [19] where harmonic optics-free focusing was introduced and observed qualitatively by an inversion of the XUV beam spatial chirp. Our results of ,0 q w and ,0 q z for each harmonic constitute a direct and quantitative evidence of coherent focusing in HHG and show that focus size of a few microns can be obtained with real foci. This direct focusing can lead to high XUV intensities. Indeed, even by considering a single harmonic, an 100-nJ energy per harmonic and a 5-fs duration lead to an intensity of 10 13 W/cm 2 . Higher intensities could be obtained by focusing all harmonics at the same position which should be possible by controlling the IR intensity profile and wavefront. FIG. 2. (a) Size ,0 q w and (b) position ,0 q z of the waist of harmonics H31 (blue), H33 (red), H49 (black) and H51 (green) as a function of the gas jet position. In (b), the XUV waists located below (resp. above) the dashed line, that is before (resp. after) the generating medium, are virtual (resp. real).

Spatially-separated foci can also be useful for spectrally-resolved experiments. Here we demonstrate a direct application of the XUV beam properties control by performing XUV spectral filtering without using any XUV optics. This is realized by comparing the spatiallyresolved HHG spectrum obtained with a 140-µm-diameter pinhole centered on the pathway of the harmonics and the reference obtained without the pinhole, as shown in Fig. 3(a) and (b) for z jet = -58 mm. The transmission factor (T) is then defined as the ratio of the HHG spectra after integrating over the spatial coordinate (Fig. 3(c)). Figure 3(d) displays the measured transmission factors of harmonics H29 to H47 for z jet = -58, 36, -20 mm and clearly shows that the characteristics of this filter are fully tunable by adjusting the gas jet position with respect to the IR focus. For z jet = -20 mm (black curve), we observe a high transmission (~ 80 %) of harmonics H29-H33 and a good attenuation (T < 20 %) of harmonics H41-H47 (low-pass filter). For z jet = -58 mm (blue curve), the situation is reversed with very high (close to 100 %) and poor (T < 30 %) transmissions of harmonics H45-H47 and H29-H37, respectively. In both cases, the high spectral selectivity and contrast ensure a proper selection of a group of harmonics. On the other hand, for z jet = -36 mm (red curve), broadband transmission is also achieved with 60 < T < 90 % over the H29-H47 range, allowing for producing broadband XUV pulses with similar spatial profiles imposed by the spatial filter. When refocused into an experiment, this can lead to intense attosecond pulses. Finally, this spectral filtering is accompanied with a strong rejection of the IR fundamental beam (T < 1 %). Furthermore, the transmitted IR beam will be strongly diffracted [24] and therefore poorly intercepted by any optics that could be located downstream in an experiment. This can avoid the use of metallic filters that usually reduce significantly the XUV signal. We point out here that the present approach does not require the use of any XUV optics and thus does not suffer from limitations inherent to this energy range and encountered with traditional optical techniques: a pair of gratings offers high spectral selectivity but with limited transmission, while multilayer mirrors have good transmission but are designed for a specific wavelength. In conclusion, we investigated the spatial properties of XUV harmonic beams as a function of the position of the generating medium relative to the IR focus, and observed that opticsfree focusing of high-order harmonics can be obtained in specific conditions. By means of the SWORD technique, we characterized the XUV intensity profile and spatial phase of the wavefront to retrieve both the size and position of the waists of the generated harmonics. While diverging harmonics are generated with virtual waists as small as 5 µm, converging XUV beams are also produced with spot size of 5-20 µm and can be focused up to 4.4×z R_IR (120 mm) after the IR focus. We emphasize that no XUV optics are used in this work, thus preserving high photon flux and preventing from optical aberrations that can lead to pulse broadening. This approach is a way to reach higher XUV intensities and should benefit to non-linear XUV-pump-XUV-probe experiments.

Taking advantage of the control of the harmonic focusing properties, we demonstrated adjustable XUV spectral filtering without resorting to any XUV optics. It is characterized by high XUV transmission (60 < T < 100 %), full tunability (low-pass, high-pass or broadband filter) controlled by adjusting the gas jet position with respect to the IR focus, and is accompanied with a strong rejection of the IR fundamental and low-order harmonic beams. This technique allows narrowband transmission with high spectral selectivity and contrast of particular interest, for instance, in studying molecular dynamics with experiments based on photoelectron spectroscopy, in which we may prefer to reduce the number of harmonics contained in the XUV pulse as the complexity of measured photoelectron spectra increases drastically with the number of ionizing photon energies. On the other hand, spectral filtering also allows broadband XUV transmission and thus supporting the production of focused attosecond pulses.

The next step of this work will consist of shaping the spatial intensity profile of the IR fundamental beam to further control the spatial properties of the generated harmonics. It has been shown that using a flat-topor super-Gaussianintensity profile near focus produces very collimated XUV harmonics with a very low divergence associated with a high photon flux [25,26]. In this case, the spatial phase ( , ) q tr  of each harmonic given in Eq. ( 1) would not depend on the harmonic order q leading to a tight focusing over a broad XUV spectral range. This spatial control should directly affect the temporal profile of the XUV beam and produce intense and short attosecond pulses.

FIG. 1 .

 1 FIG. 1. Measured intensity profile (dots) and Gaussian fit (line) (a) and reconstructed spatial phase of the wavefront (dots) and quadratic fit (line) (b) of harmonics H31 (blue), H39 (orange) and H47 (yellow), using the SWORD technique for z jet = -35 mm.

FIG. 3 .

 3 FIG. 3. Far-field spatially-resolved HHG spectrum measured for z jet = -58 mm with (a) and without (b) the pinhole centered on the XUV path. (c) Corresponding HHG spectra after summing over the spatial coordinate. (d) Measured transmission of harmonics H29 to H47 for z jet = -58 (blue), -36 (red) and -20 mm (black).

We acknowledge the financial support from the Agence Nationale de la Recherche (ANR) through the 'CIRCE' project (ANR-16-CE30-0012) and from the Région Nouvelle-Aquitaine through the 'OFIMAX' project.


MD Bianca Magro
email: bianca_magro@hotmail.it

MD Alessandra Mazzola

PhD Mona Munteanu

PhD Claire Goumard

MD Valerie Martinez

PhD Denis Bernard

MD Olivier Scatton

PhD Salvatore Battaglia

MD Calogero Cammà

PhD Filomena Conti

Consequences of Extended Spectrum Beta-Lactamase-Producing Enterobacteriaceae and Methicillin-Resistant Staphylococcus aureus Carriage in Awaiting Liver Transplant Patients Consequences of ESBLE and MRSA carriage in awaiting liver transplant patients

Keywords: ESBLE, fecal carriage, cirrhosis, liver transplantation Extended spectrum beta-lactamase producing Enterobacteriaceae MRSA: Methicillin-resistant Staphylococcus aureus LT : liver transplantation MDROs: Multi-drug resistant organism LRC : liver-related complications sponatneous bacterial peritonitis

Background: Infections in cirrhotic patients are associated with liver-related complications (LRC), especially, in patients awaiting liver transplantation (LT). The aim of this study was to evaluate the impact of Methicillin-Resistant-Staphylococcus-Aureus (MRSA) and Extended-Spectrum-Beta-Lactamase (ESBL) colonization, on infections and LRC on the waitlist and on infections after LT.

Method:

We retrospectively included 250 of 483 cirrhotic placed on waitlist for LT from December 2015 to January 2018, screened for MRSA or ESBLE, at the time of waitlist placement and after LT.

Results: 76% of patients were male with mean age of 57.5  10, the most frequent cause of liver disease was alcohol (39%). Median MELD score was 19 (12-28). Only 1 patient was positive for MRSA, 19% of patients (n = 47) were ESBLE fecal carriage at the moment waitlist placement and 15% (n = 37) after LT. Infection free-survival on the waitlist and after LT, according to fecal carriage status, was not statistically different between two groups. LRC-free-survival at 6 and 12 months was significantly lower in ESBLE fecal carriage [HR 1; p < 0.04)]. MELD score > 19 [HR 3; (p = 0.01)] and occurrence of infection during the first 3 months on the waitlist [HR 4.13; (p<0.001)] were independent risk factors for LRC occurrence at multivariate analysis Conclusions: Our study is the first showing that, in a cohort of cirrhotic patients waiting for LT LRC-free survival is lower in ESBLE fecal carriage, but infection free survival is not different in the two groups.

INTRODUCTION:

Infections are common in cirrhotic patients, about 25-30% for each hospitalization, and they develop as a consequence of the immune dysfunction that occurs progressively during the course of cirrhosis. When infections occur, they were associated with an increased risk of liverrelated complications (LRC), liver failure and mortality. The overall mortality of infected patients was reported to be as high as 38% (odds ratio for death of infected vs. non-infected patients of 3.75). Incidence of infections, could represent a further prognostic stage, defining the "critically ill cirrhotic" and it increases mortality 4-fold: 30% of patients die within 1 month and another 30% die by 1 year. (1,2)(1)(2) Bacterial infections, especially those involving gram-negative bacteria, represent a major complication in liver transplant recipients, the frequency ranging between 20% and 80% of cases.

(3) Moreover, in patients with advanced cirrhosis, prevalence of multi drug resistance organism (MDROs) infections is alarmingly increased, mainly in nosocomial setting. Repeated hospitalizations, invasive procedures and the expositions to antibiotics, as prophylaxis (long-term primary and secondary prophylaxis of spontaneous bacterial peritonitis (SBP) and short-term prophylaxis in upper gastrointestinal bleeding) or as treatment, seem to have contributed to increase the prevalence of MDROs infections across different geographical areas in the cirrhotic population. (4)

In the field of the antibiotic resistance an important role is played by the spread of ESBLproducing Enterobacteriaceae (ESBLE) especially infection related to E. Coli, most represented in Asia, and Klebsiella Pneumonia in Latin America. [START_REF] Arias | Unmet needs and prospects for oritavancin in the management of vancomycin-resistant enterococcal infections[END_REF] Guidelines for infection management to reduce MDROs transmission in hospital, recommended to implement screening culture trough rectal swab at the admission and to realize it weekly until a new infection or colonization but no consensus exists on the optimal timing and interval. [START_REF] Tacconelli | ESCMID guidelines for the management of the infection control measures to reduce transmission of multidrug-resistant Gram-negative bacteria in hospitalized patients[END_REF] Although, there are few data concerning the prevalence of ESBLE fecal carriage in cirrhotic patients. Recent studies suggest that it is similar to the general population prevalence (15.7%). [START_REF] Bert | Risk factors associated with preoperative fecal carriage of extended-spectrum β-lactamase-producing Enterobacteriaceae in liver transplant recipients[END_REF] However, as a consequence of the recurrent hospitalizations, cirrhotic patients could be considered such as "the vehicle" of MDROs between the healthcare environment and the general population. This spread is difficult to control because of the potential huge intestinal reservoir and the international travels in a globalized world. [START_REF] Hawkey | Multidrug-resistant Gram-negative bacteria: A product of globalization[END_REF] Infections caused by MDROs are associated to poor prognosis increasing morbidity and mortality, both in the pre-and post-transplant settings.

The problem of MDR is particularly challenging in patients awaiting LT. The use of inappropriate empiric antimicrobial in septic shock secondary to any type of infection or to SBP is associated

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This article is protected by copyright. All rights reserved with an adjusted odds ratio of death of 1.1 and 1.9, respectively for every hour delay in appropriate antibiotic therapy. (9) [START_REF] Arabi | Antimicrobial therapeutic determinants of outcomes from septic shock among patients with cirrhosis[END_REF] Before LT, colonization with staphylococcus methicillin resistant (MRSA) or ESBLE is an independent risk factor for post-transplant infections. Especially in the early postoperative period, infections are caused by bacteria that have colonized the digestive tract or the oropharynx before. Bert et al. in their retrospective study demonstrated that previous ESBL-producing isolate infection, recent exposure to a b-lactam agent, and an history of SBP are risk factors for preoperative fecal carriage of ESBLE in liver transplant recipients. Moreover, fecal carrier was an independent risk factor for post-transplant infections. [START_REF] Bert | Risk factors associated with preoperative fecal carriage of extended-spectrum β-lactamase-producing Enterobacteriaceae in liver transplant recipients[END_REF] [START_REF] P-B | Pretransplant fecal carriage of extended-spectrum β-lactamaseproducing enterobacteriaceae and infection after liver transplant, France [Internet[END_REF] An important clinical problem is for patients hospitalized for SBP having a risk of ESBLE infection (positive culture at rectal swab), because there isn't any recommendation for the empiric antibiotic treatment. [START_REF] Moreau | Acute-on-chronic liver failure is a distinct syndrome that develops in patients with acute decompensation of cirrhosis[END_REF] All the studies regarding this issue were based on a single rectal swab at the moment of LT, there are no data on clinical impact of ESBLE fecal carriage on the waitlist.

The aim of this study is to evaluate impact of MRSA and ESBLE colonization at 6 and 12 months on infections and liver-related complications (LRC) during time on the waitlist and infections after LT.

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METHODS

Study population:

We retrospectively included 250 of 483 cirrhotic placed on waitlist for LT from December 2015 to January 2018 in our liver transplant center at Pitié-Salpêtrière hospital, that correspond to the population systematically screened for MRSA or ESBL carriage with nasal and rectal swab at the time of waitlist placement and in the next six months after LT (Figure 1). 216 patients were transplanted and 34 died before LT. We analyzed consecutively all data. Inclusion criteria were: i) cirrhotic patients, according to histological, clinical, biochemical, ultrasound, and/or endoscopic findings; ii) patients on the waitlist for LT; iii) screened for MRSA or ESBLE carriage with nasal/rectal swab at the moment of waitlist placement and after six months within LT. The exclusion criteria were: i) non-cirrhotic patients; ii) patients not screened at nasal or rectal swab at the moment of waitlist inscription; iii) patients waiting for re-transplantation.

All medical major health events including LRC, infections, re-hospitalisation or deaths were collected.

Outcomes:

The primary outcome was to evaluate the clinical impact of ESBLE fecal carriage on infectionsfree-survival before and after LT, and on LRC-free-survival during time on the waitlist respectively at 6 and 12 months. The secondary outcome was to evaluate the overall survival rate on the waitlist according to ESBLE fecal carriage and also to individuate risk factors of LRC.

Variables:

-Infections before LT: SBP, pneumonia, urinary tract infection (UTI), cholangitis, meningitis, colitis, skin and soft tissues infections, sepsis. Infection-free-survival was considered from the time of waitlist placement to the time of infections occurrence or the time of LT/death. [START_REF] Horan | CDC/NHSN surveillance definition of health careassociated infection and criteria for specific types of infections in the acute care setting[END_REF] -Infections after LT: pneumonia, UTI, biliary infections, bacteremia, cutaneous infections. The infection-free-survival was considered from LT to the time of occurrence of the infections or last follow-up/death. [START_REF] Horan | CDC/NHSN surveillance definition of health careassociated infection and criteria for specific types of infections in the acute care setting[END_REF] -LRC: upper gastrointestinal bleeding (UGB), hepatorenal syndrome (HRS), SBP, hepatic encephalopathy (HE) that occur on waitlist period. The LRC-free-survival was defined from the time of placement on waitlist to the time of LRC occurrence or the time of LT/death. ( 14) -Waitlist mortality: time from waitlist placement to time of death or LT, no matter the cause, at any time, during all the follow-up period.

Data collection:

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This article is protected by copyright. All rights reserved Data sources to complete the prospective database were outpatient or inpatient reports, radiology reports, operative reports, and laboratory or pathology reports, on digital or paper support.

Moreover, a complementary source of data was represented by the CRISTAL database of

Agence National de la Biomedicine ( https://www.sipg.sante.fr/portail/). The quality of data management was compliant to a reference methodology on personal data processing and protection (database hosted on secured hospital servers -CNIL n°2208386 v 0).

For this reason a dedicated patient consensus was not required.

Microbiological assessment:

Rectal and nasal swabs were systematically performed once at the time of waitlist placement for LT and within six months after LT, regardless of the indication to be hospitalized in election or in urgency.

A rectal swab specimen was obtained and plated into a selective commercial agar plate (ESBL agar, bioMerieux, Marcy l'Etoile, France). Identification of ESBL isolates was done by the doubledisk synergy test on Mueller-Hinton agar and, when appropriate, Mueller-Hinton agar containing cloxacillin. Identification to the species level was done by the API20E method. Antimicrobial drug susceptibility was tested by the Mueller-Hinton agar diffusion method and interpreted according to the recommendations of the Committee of the French Society for Microbiology. ( 15)

Statistical analysis:

Data were analyzed with the NCSS-12.0 statistical package. Data for continuous variables are presented as mean and standard deviation (SD) or as median and range, and data for categorical variables as frequency and percentage. Differences between continuous data were analyzed by Student t test or Chi square test was used for dichotomous or categorical variables. Survival probabilities were estimated according to the Kaplan Meier method. The Log Rank test was used to compare patient survival groups. The stepwise Cox proportional hazard regression model was used to assess the independent factors of LRC on the waitlist. Variables with a p value < 0.1 at univariate analysis were inserted in a multivariate model. The multivariate analysis was adjusted for LRC at baseline A p value < 0.05 was considered statistically significant. A non parametric competing risks analysis was performed in order to estimate separately the sub distribution cumulative incidence functions (CIFs) of transplant and death, according to Fine and Gray approach (16) The Gray's

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This article is protected by copyright. All rights reserved test was used to evaluate hypotheses of equality of sub distribution CIFs according to the fecal carriage status (positive versus negative) [START_REF] Gray | A Class of $K$-Sample Tests for Comparing the Cumulative Incidence of a[END_REF]

RESULTS:

Patients:

Baseline characteristics of the 250 cirrhotic patients are shown in Table 1. Seventy-six percent of patients were male with a mean age of 57.510, the most frequent cause of liver disease was alcohol (39%). Median MELD score was 19 (12-28) and 21%of patients had hepatocellular carcinoma (HCC). One patient had a positive nasal swab for Staphylococcus aureus at waitlist placement.

Prevalence rate of LRC on the waitlist was: 17% (n=50) HE, 6% (n=19) SBP, 13% (n=37) HRS and 12% (n=34) UGB. Nineteen percent of patients (n=47) were ESBLE fecal carriage at the time of place on waitlist and 15% (n=37) after LT.

The prevalence rates of organism isolated from rectal swab at the time of waitlist were: 53%

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This article is protected by copyright. All rights reserved wasn't any significant difference between the two groups regarding previous treatment with norfloxacin and rifaximin (p=0.9 and p=0.7, respectively). No significant difference was found for antibiotic treatments, hospitalization rate in intensive or semi-intensive care-unit (ICU or SICU) in the last 3 months (p=0.3 and p=0.6, respectively). Median time on the waitlist was 4 (3-4) months, and median time of follow-up after LT was 17 (4-55) months.

Primary outcomes:

-

Impact of ESBLE fecal carriage on infection

Median time of infection occurrence before LT was significantly different in ESBLE fecal carriage:

2 (1-3) months vs. 4 (2-5) months in Non-fecal carriage (p=0.06).

Infection free-survival rates at 6 and 12 months on the waitlist according to EBLSE fecal-carriage was not statistically different between two groups [HR 1.2; 95% CI (0.5-3.29); p=0.63]; [HR: 1.5, 95% CI (0.6-3.7); p=0.28)] (Figure 2). Among infections on the waitlist, 51% (n=33) percent was related to ESBLE and the most frequent were pneumonia (30%, n=20) and SBP (30%, n=20). In 50% (n= 32) of cases a germ was isolated and the most common was E. Coli (36%, n=12) (Table S).

Infection free-survival rates at 6 and 12 months after LT according to ESBLE Fecal carriage was not significantly different between the two groups [HR 0.99; 95% CI (0.46-2.15); p=0.9] (Figure 2).

After LT, 42%(n=23) of infections were due to ESBLE, the most common were: pneumonia (34%, n=26). In 73 % (n=55) of cases a germ was isolated, the most common germs were K.

Pneumoniae (25%, n=14) and E. Coli (14%, n=8) (Table S). Among 47 fecal carriers, 13 persist to be positive also after LT. The infection rate after LT in patients who had a positive swab was 45,9% (N=17/37) -

Impact of ESBLE fecal carriers on LRC

In our study we evaluate ESBLE fecal carriage impact on LCR occurrence during time on the waitlist. The median time of LRC occurrence was 2 (1-3) and 3 (3-4) months (p=0.2), in ESBLE fecal carriage vs. non ESBLE fecal carriage, respectively. LRC according to fecal carrier status were shown in Figure 3. HRS was significantly higher in the ESBLE fecal carriage compared to not one [30% (n=12) vs. 14% (n=25), (p=0.02)] LRC-free-survival at 6 and 12 months was significantly lower in ESBLE fecal carriage vs. Nonfecal carriage: [HR 1.6; (95% CI 0.9-3; p<0.04)] (Figure 4).

Secondary outcomes:

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Waitlist survival

Table 2 shows the outcomes of patients stratified according to the fecal carriage status. By competing risks analysis, the probability of death at 6 months was 12.7% (95% CI 5.1-24.1%) in patients with positive fecal carriage, and 10.4% (95% CI 6.7-15.1%) in patients with negative fecal carriage, respectively. The probability of death at 12 months was 14.9% (95% CI 6.3-26.7%) in patients with positive fecal carriage, and 11.9% (95% CI 7.9-16.9%) in patients with negative fecal carriage, respectively. No significant differences were observed between patients with positive and negative fecal carriage (p=0.721). (Figure S1)

In patients with HCC, no differences were found in waitlist mortality between patients with positive and negative fecal carriage, by competing risks analysis (p=0.608). (Figure S2)

In patients who developed LRC and who had not HCC, no death competing events were observed. In these patients, the probabilities of transplant according to the fecal carriage status are showed in Figure S3.

The most common causes of death were: infection (53%, n=18) such as pneumonia and SBP, and liver-related (44%, n=15) such as hematemesis.

-Predictors of liver related complications:

Patients characteristics included in univariate and multivariate analysis were reported in Table 3.

In our study, at multivariate analysis, MELD score > 19 [HR 3; 95% CI (1.8-5); p= 0.01] and infection during the first three months on the waitlist [HR 4.13; 95% CI (2.13-7.9); p<0.001] were independent risk factors for LRC occurrence on the waitlist. (Table 3) SBP was censored as infection.

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DISCUSSION

In our retrospective study prevalence rate of ESBLE fecal carriage among cirrhotic patients is higher (19%) than reported in literature (15,7%). [START_REF] Bert | Risk factors associated with preoperative fecal carriage of extended-spectrum β-lactamase-producing Enterobacteriaceae in liver transplant recipients[END_REF] This could mean that the spread of ESBLE colonization is increased in the last years, and it represents an important source of morbidity and mortality, especially for patients waiting for LT.

The recent multicentric study, conducted on 1302 cirrhotic patients, has shown that MDR bacterial infections are associated with a higher incidence of septic shock and higher in-hospital mortality at 28 days (29% vs 20%; P= 0.014). Moreover, this study showed that MDR prevalence depends on the geographic area (73% in India; 16% in United States and 24% in Canada) [START_REF] Piano | Epidemiology and Effects of Bacterial Infections in Patients With Cirrhosis Worldwide[END_REF] Previous studies have investigated the role of the ESBLE fecal carriage on infections in a cohort of cirrhotic patients and especially after LT. placement on the waitlist and after LT.

After LT many variables can impact on infection occurrence such as ICU hospitalization, surgery and their related complications, and a single rectal swab, probably, cannot determinate infections occurrence.(

While during the time on the waitlist it would be interesting to repeat rectal swab routinely to assess patient colonization and to minimize antibiotic use. (4) The presence of a single specimen is one of the limits of our study, because we don't know if the fecal carriage status changes and how it changes during pre-transplant period. The retrospective design of the study cannot allow to explore all the fecal carriage variations.

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Regarding the overall survival on the waitlist, there is no difference between the two groups [HR 1.57; 95% CI 95% (0.7-3.56); p=0.17]. The most common cause of death is infection (53%) and among the hepatic ones the most common was SBP (40%).

To our knowledge our study is the first that evaluates impact of ESBLE colonization on LCR on the waitlist. Our results show a significant difference on LRC-free-survival on waitlist between the two groups at univariate analysis [HR 1.6; (95% CI 0.9-3; p<0.04)], even if this result is not confirmed in multivariate analysis. We want to underline the importance to identify colonized patients because of their higher risk of complication, ant it would be supposed that this kind of patients could be decolonized or it would be necessary an antibiotic treatment at the moment of the complication to reduce over infection or clinical worsening.

It is known that in cirrhosis the gut liver axis and the homeostasis of the immune system are A recent multicentric randomized trial evaluated, in a cohort of patients' carriers for ESBLE or CPE at stool culture, the possible decolonization by an antibiotic oral treatment followed by microbiota fecal transplantation (MFT). Sample size was small (n=39), but 50% of MFT and 3% of control patients achieved decolonization (OR 3.3, 95% 0.7e16.8), in this study non-cirrhotic patients were enrolled.( 26) It seems to be necessary to validate the strength of rectal swab

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screening, decolonization strategies and also spread prevention, especially in the setting of LT recipients.

Our study presents different limits: the retrospective and monocentric design of the study, the small sample size, and the single rectal swab taken before and after LT. Moreover, data from patients placed on waitlist not screened for ESBLE or MRSA are not available. and we did not have the second swab for all patients swabbed at the moment of waitlist placement (n=187/250).

In conclusion, our study shows an increase of ESBLE colonization among cirrhotic patients in the last years (19% vs 15%). This is the first study that reported a significant association of fecal carriage status and the occurrence of LRC in LT recipients. The implications of this study for us are significant: indeed, even if all the results are not significant, which is probably due to the small number of patients and the retrospective nature of the evaluation, sheds light on us by showing that ESBLE colonized patients have more LRC, and are therefore more likely to decompensate.

We are convinced that this screening will help in the prevention of pre LT complications, whether infectious or not.

Future prospective investigations with a large simple size could be performed to clarify this data.

It is important to underline that MELD score >19 and earliness of infections occurrence (< 3 months) were the independent predictors of LRC. Moreover, our results didn't show a correlation between ESBLE fecal carriage status end infection occurrence before and after LT, probably because we have not done rectal swab routinely on waitlist and because after LT there are many variables that can impact on infections occurrence. It is necessary to optimize prevention strategies, and in this context the role and the validity of the rectal swab needs to be more explored by a prospective and multicentric studies, especially in cirrhotic patients.

BIBLIOGRAPHY

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  Giannella et al. investigated in a large prospective cohort study (553 patients) the impact of colonization with carbapenemase-producing Enterobacteriaceae (CPE) on the CPE infection risk after LT, and they showed that CPE colonization at LT or acquired after LT were the strongest predictors of CPE infection after LT. In this study rectal swab was performed at the time of LT.[START_REF] Giannella | The impact of carbapenemase-producing Enterobacteriaceae colonization on infection risk after liver transplantation: a prospective observational cohort study[END_REF]

  altered. The increased intestinal permeability allows the bacterial translocation, bacterial products spread through the portal circulation, triggering an inflammatory and systemic response. This dysbiosis could lead to the development of LRC. (22) Moreover, bacterial translocation in cirrhotic patients is a crucial step leading to LRC, but little is known about the mechanism that could explain it. (1) We explored the possible risk factors of LRC occurrence on the waitlist, in multivariate analysis and we identified: MELD score >19 [HR 3; 95% CI (1.8-5); p= 0.01] and time of infection during the first 3 months on the waitlist [HR 4.13; 95% CI (2.13-7.9); p<0.001] as independent risk factors, but ESBLE fecal carriage is not confirmed [HR:1.37; 95%CI (-0.4-1.04) p=0.3]. Probably we need a bigger sample size to validate its role in this context. Dionigi et al. in their retrospective study showed how infection occurrence in cirrhotic patients defines another stage of disease, associated with higher morbidity and mortality despite of infection resolution. (23)In a recent study, Kimman et al., reported that in cirrhotic patients with a MELD score between 15 and 25, SBP incidence is the most frequent infection and it increased mortality (p = 0.02; HR = 2.10), and patients included in our study have a similar MELD score.[START_REF] Kimmann | Sustained impact of nosocomial-acquired spontaneous bacterial peritonitis in different stages of decompensated liver cirrhosis[END_REF] Infections in this setting of patients, waiting for LT, represent a big issue especially for public health and it is fundamental to have solid prevention strategies. Different studies, in non-cirrhotic population, tried different strategies of decolonization for ESBLE carriers, but they showed a moderate efficacy.[START_REF] Rieg | Intestinal decolonization of Enterobacteriaceae producing extended-spectrum β-lactamases (ESBL): A retrospective observational study in patients at risk for infection and a brief review of the literature[END_REF]

  Bert et al, in a cohort of 317 cirrhotic patients, demonstrated that pre-transplant ESBLE fecal carriage confers a high risk for infection (48% vs. 6,7%, p>0.001) with the same organism after LT. (7) A recent study of Logre et al. showed that about 40% of ESBLE carriers before transplantation develop infection with ESBLE after LT. But also, in this case authors used the rectal swab obtained before LT to analyze infections after LT.

Our study is the first that evaluate ESBLE fecal carriage impact before and after LT on relevant clinical outcomes. Contrary to published data, we reported any difference on infection-freesurvival before and after LT at 6 and 12 months between ESBLE fecal carriage and Non ESBLE fecal carriage [HR: 1.5, 95% CI (0.6-3.7); p=0.28)]; [HR 0.99; 95% CI (0.46-2.15); p=0.9]. But in our study, we considered for the survival analysis two rectal swab at different moments:

  1. Jalan R, Fernandez J, Wiest R, et al. Bacterial infections in cirrhosis: A position statement based on the EASL Special Conference 2013. J. Hepatol. 2014;60:1310-1324. 2. Arvaniti V, D'Amico G, Fede G, et al. Infections in patients with cirrhosis increase mortality four-fold and should be used in determining prognosis. Gastroenterology 2010;139 3. Rubin RH. The direct and indirect effects of infection in liver transplantation: pathogenesis, impact, and clinical management. [Internet]. Curr. Clin. Top. Infect. Dis. 2002;22:125-54.[cited 2019 Oct 28] Available from: http://www.ncbi.nlm.nih.gov/pubmed/12520651 4. Fernández J, Bert F, Nicolas-Chanoine MH. The challenges of multi-drug-resistance in

Table 3 . Predictors of LRC in univariate and multivariate analysis

 3 The multivariate analysis was adjusted for LRC at baseline. To take into account the competing risk of LT on LRC rate, the model was adjusted for the number of events that occurs during the waitlist time lapse.

	VARIABLES	p	HR 95% CI	p	HR 95% CI
	Sex male	0.3	1.33(0.75-2.38)	NS
	Age mean SD	0.01	1(1-1.002)	NS
	MELD-score > 19 (median)	0.01	3.1(1.98-5)	0.01	3.0 (1.79-5.17)
	HCC	0.03	0.5(0.2-0.9)	NS
	Faecal carriers	0.04	1.7(1.01.-2.8)	NS
	Etiology	0.15	1.12(0.96-1.32)	NS
	Invasive procedures	0.65	1.2(0.5-2.7)	NS
	Infections before LT wo SBP	0.05	0.6(0.3-1)	NS
	Time on waitlist > 4 months
	(median)	<0.001	3.4 (1.92-6.21)	NS
	Time of Infection
	on waitlist < 3 months (median)	<0.001	0.9 (0.8-0.9)	< 0.001	4.13 (2

LRC: Liver related complications; ICU: intensive care unit; SICU: semi-intensive care unit; wo: without; HCC: hepatocellular carcinoma. *

.13-7.9) Antibiotic treatment in the last 3 months before LT 0.01 2.2(1.2-3.9) NS Hospitalization in ICU or SICU in the past 3 months before LT 0.01 2.196(1.26-3.8) 0.07

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Competing Risk [Internet]. Ann. Stat. 1988;16:1141-1154.[cited 2020 Jul 3] Available from:

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domain: chim sdv

text:
Marta Bonaccorsi

Michael J Knight

Tanguy Le Marchand

Hugh R W Dannatt

Tobias Schubeis

Loïc Salmon

Isabella C Felli

Lyndon Emsley

Roberta Pierattelli

Guido Pintacuda

Gui- Do Pintacuda

Multimodal Response to Copper Binding in Superoxide Dismutase Dynamics

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

Introduction

Metal ions have a key role in the cell machinery as cofactors of almost one third of all the known proteins. [START_REF] Gray | Biological Inorganic Chemistry at the Beginning of the 21st Century[END_REF] They perform a variety of functions such as catalysis, electron transfer and oxygen storage. [START_REF] Bertini | Handbook on Metalloproteins[END_REF] Often, metal ion binding also has a structural effect, modulating the stability and the folding pathways of metalloproteins. It is commonly thought that metal binding promotes a rigidification of the protein scaffold due to changes in side chain structure and dynamics in the metal binding site upon metal uptake. [START_REF] Arnold | Metal-Mediated Protein Stabilization[END_REF][START_REF] Babor | Flexibility of Metal Binding Sites in Proteins on a Database Scale[END_REF][START_REF] Li | Molecular Simulations of Metal-Coupled Protein Folding[END_REF][START_REF] Matsuo | Global Structural Flexibility of Metalloproteins Regulates Reactivity of Transition Metal Ion in the Protein Core: An Experimental Study Using Thiol-Subtilisin as a Model Protein[END_REF] However, a number of intriguing exceptions to this paradigm have been reported, [START_REF] Deng | Molecular Basis for the Cu2+ Binding-Induced Destabilization of Β2-Microglobulin Revealed by Molecular Dynamics Simulation[END_REF][START_REF] Sedlák | In Vitro Unfolding of Yeast Multicopper Oxidase Fet3p Variants Reveals Unique Role of Each Metal Site[END_REF][START_REF] Morales | Dynamic Response of the C2 Domain of Protein Kinase Cα to Ca2+ Binding[END_REF][START_REF] Ariöz | Folding of Copper Proteins: Role of the Metal?[END_REF][START_REF] Yadav | Differences in the Mechanical Unfolding Pathways of Apoand Copper-Bound Azurins[END_REF] suggesting that the repercussions of metal binding on stability, oligomerization and catalytic activity cannot be reconciled simply in terms of an overall reduction in flexibility. A more complete description of the modulation of motions on different timescales (from fluctuations at amino acid level to collective motions involving larger structural elements) [START_REF] Henzler-Wildman | Dynamic Personalities of Proteins[END_REF] is indeed required to disentangle all the effects. One of the unique features of nuclear magnetic resonance (NMR) toward this goal is its capability to probe internal molecular motions at atomic resolution over a broad dynamic range in physiological environments. The continuous development of solid-state NMR has allowed to extend these investigations to complex targets of different aggregation states, to insoluble oligomers, sediments, and proteoliposomes. [START_REF] Lewandowski | Advances in Solid-State Relaxation Methodology for Probing Site-Specific Protein Dynamics[END_REF][START_REF] Lewandowski | Direct Observation of Hierarchical Protein Dynamics[END_REF][START_REF] Schanda | Studying Dynamics by Magic-Angle Spinning Solid-State NMR Spectroscopy: Principles and Applications to Biomolecules[END_REF] Solid-state NMR additionally provides the intriguing possibility to probe dynamical parameters of soluble proteins inside hydrated crystals or microcrystals [START_REF] Giraud | Quantitative Analysis of Backbone Dynamics in a Crystalline Protein from Nitrogen-15 Spin-Lattice Relaxation[END_REF][START_REF] Ma | Observing the Overall Rocking Motion of a Protein in a Crystal[END_REF][START_REF] Kurauskas | Slow Conformational Exchange and Overall Rocking Motion in Ubiquitin Protein Crystals[END_REF][START_REF] Marchand | Conformational Dynamics in Crystals Reveal the Molecular Bases for D76N Beta-2 Microglobulin Aggregation Propensity[END_REF][START_REF] Rovó | Mechanistic Insights into Microsecond Time-Scale Motion of Solid Proteins Using Complementary 15 N and 1 H Relaxation Dispersion Techniques[END_REF][START_REF] Singh | Fast Microsecond Dynamics of the Protein-Water Network in the Active Site of Human Carbonic Anhydrase II Studied by Solid-State NMR Spectroscopy[END_REF] and can therefore expand solution NMR methods. Pioneering studies have shown that the local flexibility of backbone and side-chains in crystals is reminiscent, if not identical, to that occurring in solution, [START_REF] Agarwal | Protein Side-Chain Dynamics As Observed by Solution-and Solid-State NMR Spectroscopy: A Similarity Revealed[END_REF][START_REF] Mollica | Atomic-Resolution Structural Dynamics in Crystalline Proteins from NMR and Molecular Simulation[END_REF] and that relaxation parameters under MAS can even reveal residual collective motions that are associated with large-amplitude conformational changes in solution. [START_REF] Lewandowski | Anisotropic Collective Motion Contributes to Nuclear Spin Relaxation in Crystalline Proteins[END_REF][START_REF] Ma | Probing Transient Conformational States of Proteins by Solid-State R 1ρ Relaxation-Dispersion NMR Spectroscopy[END_REF] In addition, crystal contacts abolish the overall tumbling of the molecules, which dominates spin relaxation in solution, and allow observation of motions over an expanded time window, necessary to obtain a comprehensive description of the functional dynamics of the proteins.

14,26,27

Here, we use state-of-the-art solid-state NMR to characterize motions in a metalloenzyme, human copper/zinc superoxide dismutase (SOD), and to uncover the links between internal dynamics over various timescales and metal ions coordination, which are central to the processes of catalytic efficiency, maturation and stability of the dimeric enzyme.

SOD is a well-known metalloenzyme found in almost all eukaryotic cells and in a few prokaryotes, which catalyzes the dismutation of the superoxide radical to hydrogen peroxide and oxygen. This system has been widely studied over the years as a benchmark for structure-function relationships in metalloproteins. [START_REF] Ferraroni | The Crystal Structure of the Monomeric Human SOD Mutant F50E/G51E/E133Q at Atomic Resolution. The Enzyme Mechanism Revisited[END_REF][START_REF] Tainer | Determination and Analysis of the 2 Å Structure of Copper, Zinc Superoxide Dismutase[END_REF] Human Cu,Zn-SOD is a remarkably stable protein, [START_REF] Valentine | Copper-Zinc Superoxide Dismutase and Amyotrophic Lateral Sclerosis[END_REF] with two identical subunits (153 aa) featuring a very well conserved β-barrel structure composed of eight antiparallel β strands (Figure 1a), held together by hydrophobic interactions involving mainly two loops (namely 4 and 6). [START_REF] Banci | Backbone Dynamics of Human Cu,Zn Superoxide Dismutase and of Its Monomeric F50E/G51E/E133Q Mutant: The Influence of Dimerization on Mobility and Function[END_REF][START_REF] Hörnberg | The Coupling between Disulphide Status, Metallation and Dimer Interface Strength in Cu/Zn Superoxide Dismutase[END_REF] Each subunit contains in the active site a Zn II ion, buried inside the protein (Figure 1b) and known to have a primarily structural role. A solvent-accessible Cu ion (with oxidation state I or II during dismutation reaction) lies at the bottom of a wide active channel (Figure 1c) which is involved in efficiently driving the superoxide to the active site. Dismutation occurs with high catalytic efficiency, which is diffusion limited (kCAT~10 9 M -1 s -1 ). [START_REF] Banci | Backbone Dynamics of Human Cu,Zn Superoxide Dismutase and of Its Monomeric F50E/G51E/E133Q Mutant: The Influence of Dimerization on Mobility and Function[END_REF][START_REF] Banci | Structure and Dynamics of Copper-Free SOD: The Protein before Binding Copper[END_REF][START_REF] Fisher | The Role of Arginine 143 in the Electrostatics and Mechanism of Cu,Zn Superoxide Dismutase: Computational and Experimental Evaluation by Mutational Analysis[END_REF] The coordination of copper and zinc in the active site is mainly mediated by the side chains of histidine residues (Figure 1d): Cu I is coordinated by His 46 (strand β4), His 48 (strand β4) and His 120 (strand β7). Zn II is coordinated by His 63, His 71, His 80 and Asp 83, all located in loop 4. In addition, His 63 is responsible of coordinating copper when it adopts an oxidation state II. Systematic comparative studies have been performed on different forms of SOD, carrying or lacking Cu, by solution NMR. [START_REF] Ferraroni | The Crystal Structure of the Monomeric Human SOD Mutant F50E/G51E/E133Q at Atomic Resolution. The Enzyme Mechanism Revisited[END_REF][START_REF] Banci | Backbone Dynamics of Human Cu,Zn Superoxide Dismutase and of Its Monomeric F50E/G51E/E133Q Mutant: The Influence of Dimerization on Mobility and Function[END_REF][START_REF] Banci | Structure and Dynamics of Copper-Free SOD: The Protein before Binding Copper[END_REF][START_REF] Banci | The Solution Structure of Reduced Dimeric Copper Zinc Superoxide Dismutase: The Structural Effects of Dimerization[END_REF][START_REF] Banci | Solution Structure of Apo Cu,Zn Superoxide Dismutase: Role of Metal Ions in Protein Folding[END_REF][START_REF] Shipp | Dynamic Properties of the G93A Mutant of Copper-Zinc Superoxide Dismutase as Detected by NMR Spectroscopy: Implications for the Pathology of Familial Amyotrophic Lateral Sclerosis[END_REF][START_REF] Banci | Human SOD1 before Harboring the Catalytic Metal: Solution Structure of Copper-Depleted, Disulfide-Reduced Form[END_REF][START_REF] Luchinat | In-Cell NMR in Human Cells: Direct Protein Expression Allows Structural Studies of Protein Folding and Maturation[END_REF] These studies have contributed a picture showing that Cu uptake induces rigidification of both the active site and of the backbone of the protein overall. Nevertheless, in all these studies the effect of Cu uptake could not be separated from the effect of punctual mutations, which were designed specifically to produce an (inactive) monomeric variant suitable for solution studies. Moreover, motional processes occurring on different timescales, such as slow conformational changes and fast fluctuations, could not be connected in a single frame of reference.

In the following, we show that the previous picture of Cuinduced rigidification was incomplete, and that in fact Cu-binding increases local dynamics on fast timescales and reduces dynamics on slower timescales. We do this through the observation of dynamical parameters by solid-state NMR on hydrated microcrystalline preparations that conserve the enzymatic activity (SI text and figure S1). We study here an intermediate that contains all the features of the dimeric mature form, differing only by the absence of Cu (E,Zn-SOD hereafter). The dynamics of this intermediate are compared with those of the mature Cu Ibound enzyme (Cu,Zn-SOD). The structures of both forms have been determined, [START_REF] Banci | Structure and Dynamics of Copper-Free SOD: The Protein before Binding Copper[END_REF][START_REF] Strange | The Structure of Holo and Metal-Deficient Wild-Type Human Cu, Zn Superoxide Dismutase and Its Relevance to Familial Amyotrophic Lateral Sclerosis[END_REF][START_REF] Banci | The Solution Structure of Reduced Dimeric Copper Zinc Superoxide Dismutase: The Structural Effects of Dimerization[END_REF][START_REF] Knight | Fast Resonance Assignment and Fold Determination of Human Superoxide Dismutase by High-Resolution Proton-Detected Solid-State MAS NMR Spectroscopy[END_REF] and no evident structural changes are induced in the protein backbone by the uptake of Cu. Nonetheless, significant variations are observed in the backbone mobility of relevant regions of SOD. By virtue of the definition of the target systems, such variations are not associated with point mutation nor with dimerization, and are uniquely ascribed to the copper binding, clarifying the key determinants relating copper uptake, functional protein backbone and sidechain motions and dimer stability.

Materials and Methods

Sample preparation. The C6A/C111S thermostable mutant of SOD, U-[ 2 H, 13 C, [START_REF] Schanda | Studying Dynamics by Magic-Angle Spinning Solid-State NMR Spectroscopy: Principles and Applications to Biomolecules[END_REF] N] labelled and completely reprotonated at exchangeable positions, was prepared as described previously. [START_REF] Knight | Fast Resonance Assignment and Fold Determination of Human Superoxide Dismutase by High-Resolution Proton-Detected Solid-State MAS NMR Spectroscopy[END_REF] Selective metallation was achieved by treating the purified and back-exchanged protein sample with EDTA followed by dialysis, stoichiometric addition of Zn II as a 10 mM ZnCl2 solution, followed, in the case the Cu,Zn-SOD sample, by slow titration with a 10 mM solution of CuCl2 . The sample was then concentrated to 20 mg/ml and 1.5 molar equivalents of iso-ascorbic acid under nitrogen atmosphere were added to obtain the Cu Iform. Both samples were then crystallized using the same procedure and timing by the sitting drop method, yielding microcrystals comparable in shape and dimension at visual inspection, and packed into 1.3 mm rotors by centrifugation. The Cu,Zn-SOD sample was determined to be functionally active. Details of all these procedures are described in the Supporting Information.

NMR spectroscopy. NMR spectra were carried out on a 23.5 T ( 1 H Larmor frequency 1000 MHz) and an 18.8 T (800 MHz) narrow-bore Bruker Avance III spectrometers, with 60 kHz MAS and a sample temperature of 27 °C. A proton-detected 3D (H)CNH experiment was employed to resolve resonances from histidine sidechains in three dimensions and correlate side-chain NH protons with sidechain heavy atoms. All site-specific [START_REF] Schanda | Studying Dynamics by Magic-Angle Spinning Solid-State NMR Spectroscopy: Principles and Applications to Biomolecules[END_REF] N relaxation measurements for backbone and histidine sidechains were performed with the pulse sequences reported in [START_REF] Knight | Paramagnetic Systems in Biochemistry: Solid-State NMR Studies[END_REF] . A full description of the NMR experiments is provided in the Supporting Information.

Results

Histidine side-chain structure. The metal ion coordination pattern in the active site is readily accessible to NMR through [START_REF] Schanda | Studying Dynamics by Magic-Angle Spinning Solid-State NMR Spectroscopy: Principles and Applications to Biomolecules[END_REF] N-1 H cross-polarization-based 2D spectra, CP-(H)NH, which contain resonances from HN pairs in δ and/or ε positions of the imidazole groups of histidines. Such spectra were acquired for microcrystalline preparations of the two metallated forms of SOD and are shown in Figure 2a. Their site-specific assignment, performed using 1 H-detected triple-resonance 3D experiments, is detailed in the SI text. In both forms, a single correlation peak was observed for each histidine sidechain in the active site, associated to a singly protonated form. In contrast, two correlations were observed for His43, indicating a doubly protonated form for this non-coordinating sidechain at the pH used for crystallization. The spectra of the two molecules are very similar, with 15 N chemical shift perturbation upon copper binding smaller than 1.5 ppm. A noticeable difference is represented by the signal of His 120 NHδ1, which is absent in E,Zn-SOD, while it appears in the case of Cu,Zn-SOD. These first observations indicate an overall conservation of structure, with the conservation of the tautomeric state of each histidine, but also suggest different dynamics between the two forms, with higher flexibility of His 120, and consequent inefficient dipolar transfer, in the absence of Cu.

Histidine side-chain dynamics. Interconversion between structurally similar states (separated by low energy barriers) occurs over a broad ps to μs timescale, and nuclear spin relaxation parameters determined by MAS-NMR are informative of such motions. [START_REF] Giraud | Quantitative Analysis of Backbone Dynamics in a Crystalline Protein from Nitrogen-15 Spin-Lattice Relaxation[END_REF][START_REF] Krushelnitsky | Microsecond Time Scale Mobility in a Solid Protein as Studied by the 15N R1ρ Site-Specific NMR Relaxation Rates[END_REF][START_REF] Lewandowski | Site-Specific Measurement of Slow Motions in Proteins[END_REF][START_REF] Smith | Because the Light Is Better Here: Correlation-Time Analysis by NMR Spectroscopy[END_REF] Here, the 15 N-1 H correlation spectra provide a window for the direct measurement of such parameters at the catalytic center with atomic resolution. A set of 15 N R1 and R1ρ relaxation rates was measured for all the observed His side-chain signals, in both Cu,Zn-SOD and E,Zn-SOD. These parameters monitor differences in dynamics between metallation states covering the aforementioned time window, specifically from ps-ns ( 15 N R1) to ns-μs ( 15 N R1ρ) processes, respectively (SI text and figure S2,S3). The relaxation rates measured for the two forms are compared in Figure 2b and listed in Table S1. Longitudinal relaxation rates ( 15 N R1) are very well conserved between the two forms, with noticeable difference for the 15 Nε2 of His 63, showing considerably larger R1 in the absence of Cu. The residues in the Cu pocket (His 46, His 48 and His 63) showed increased R1ρ in E,Zn-SOD. Conversely, His 43, which is not exposed to the active site, showed conserved R1 and R1ρ rates between metallation states.

In addition, we explored even slower conformational dynamics (µs-ms) by measuring 15 N R1ρ at variable spin-lock field, the commonly named relaxation dispersion (RD) experiment. The high magnetic field strengths and the high MAS rates used here, together with proton dilution obtained in perdeuterated samples, reduce the coherent effect on the measured relaxation rates which are therefore dependent on dynamic processes only. [START_REF] Kurauskas | Slow Conformational Exchange and Overall Rocking Motion in Ubiquitin Protein Crystals[END_REF][START_REF] Lewandowski | Site-Specific Measurement of Slow Motions in Proteins[END_REF] A sloped profile in the relaxation dispersion is the signature of the presence of exchange between states with different chemical shifts on a μs-ms timescale which translate into the existence of structural fluctuations. [START_REF] Schanda | Studying Dynamics by Magic-Angle Spinning Solid-State NMR Spectroscopy: Principles and Applications to Biomolecules[END_REF] N R1ρ relaxation dispersion profiles were measured for the His-sidechains of both SOD samples (Figure 2c-d). While a flat dependence of R1ρ with respect to the spin lock field was observed for most of the histidine side-chains, a large dispersion was observed for His 63 Nε2 in the absence of Cu. This spinlock dependence is abolished upon Cu binding. Protein backbone structure. The observations above can be complemented by recording the analogous experiments on the 1 H, [START_REF] Schanda | Studying Dynamics by Magic-Angle Spinning Solid-State NMR Spectroscopy: Principles and Applications to Biomolecules[END_REF] N pairs of the backbone amide groups, extending the investigation from the metal binding core to the whole protein scaffold. Fingerprint 1 H, [START_REF] Schanda | Studying Dynamics by Magic-Angle Spinning Solid-State NMR Spectroscopy: Principles and Applications to Biomolecules[END_REF] N dipolar correlation spectra CP-(H)NH acquired on both Cu,Zn-SOD and E,Zn-SOD are reported in Figure 3a. The two spectra are almost superimposable, evidencing backbone structure and crystal packing conservation in the two forms with the exception of small variations in chemical shifts for backbone NH pairs close to the Cu pocket. For both samples, a single set of resolved peaks was detected, indicating the absence of exchange processes with other substantially populated conformers on timescales slower than ms. S1. While R1 relaxation rates are rather similar for the two forms for most of the backbone amide groups, three regions with globally larger values in Cu,Zn-SOD than in E,Zn-SOD were observed: around the active channel in loops 4 (residues 56,58,61) and 7 (residues 133,137,141,142), and in loop 6 (residues 107-111). These regions are highlighted in Figure 3c and visible in Figure S4, in which the values of the relaxation rates are projected on the crystal structure of dimeric SOD. In these regions, metal binding clearly results in an increase of motions on fast timescales. In parallel, an overall decrease of 15 N R1ρ was observed for Cu,Zn-SOD with respect to E,Zn-SOD, indicating a reduction of motions in the intermediate timescales all along the sequence.

As for relaxation dispersion profiles (a representative example is shown in Figure 3b), backbone residues can be sorted into three categories (Figure S5). For a first set of residues the RD-profiles are flat for both samples, with little or no dependence of R1ρ with the spin lock field, indicating the absence of exchange. In a second set, the same profile is observed for both samples, showing chem-ical exchange unrelated to copper binding. Lastly, for a third set of residues R1ρ values at low spin-lock field are increased in E,Zn-SOD translating into an increase of motion on μs-ms timescales in the absence of copper. Intriguingly, for these residues the features of the dispersion profile (slope and plateau) mirror those observed for His 63 side-chain Nε2. Slow dynamics is assessed here qualitatively, without imposing any particular model of motion, by simply using ΔR1ρ, the difference of R1ρ between the lowest and the highest spin lock fields (Figure 3c). The projection of ΔR1ρ values onto the crystal structure of SOD (Figure S4) reveals that the portions of the loops with higher values and the major differences between the two metallated forms cluster in loops 4 (residues 51,53,57-59,67,68), 6 (residues 106-111) and 7 (residues 126-129,138) close to the metal binding site but also around the dimer interface.

Discussion

The structures of many differently metallated intermediates, with various permutations of post-translation metallations, have been solved by NMR and X-ray crystallography, elucidating the maturation pathway of SOD. [START_REF] Ferraroni | The Crystal Structure of the Monomeric Human SOD Mutant F50E/G51E/E133Q at Atomic Resolution. The Enzyme Mechanism Revisited[END_REF][START_REF] Banci | The Solution Structure of Reduced Dimeric Copper Zinc Superoxide Dismutase: The Structural Effects of Dimerization[END_REF][START_REF] Banci | Solution Structure of Apo Cu,Zn Superoxide Dismutase: Role of Metal Ions in Protein Folding[END_REF][START_REF] Strange | Variable Metallation of Human Superoxide Dismutase: Atomic Resolution Crystal Structures of Cu-Zn, Zn-Zn and as-Isolated Wild-Type Enzymes[END_REF] It is known that, after protein expression, Zn is taken up first and induces folding of the protein. [START_REF] Banci | Structure and Dynamics of Copper-Free SOD: The Protein before Binding Copper[END_REF] In particular, loop 7, which is randomly oriented in the apo form, [START_REF] Banci | Solution Structure of Apo Cu,Zn Superoxide Dismutase: Role of Metal Ions in Protein Folding[END_REF][START_REF] Strange | The Structure of Holo and Metal-Deficient Wild-Type Human Cu, Zn Superoxide Dismutase and Its Relevance to Familial Amyotrophic Lateral Sclerosis[END_REF] adopts a defined orientation in the presence of Zn. Additionally the copper binding site adopts a geometry suitable for coordination. [START_REF] Sheng | Exposure of Solvent-Inaccessible Regions in the Amyloidogenic Protein Human SOD1 Determined by Hydroxyl Radical Footprinting[END_REF] The uptake of Cu occurs as the final step in maturation of SOD and, while not inducing sensible changes in the overall structure, [START_REF] Banci | Human SOD1 before Harboring the Catalytic Metal: Solution Structure of Copper-Depleted, Disulfide-Reduced Form[END_REF][START_REF] Strange | Variable Metallation of Human Superoxide Dismutase: Atomic Resolution Crystal Structures of Cu-Zn, Zn-Zn and as-Isolated Wild-Type Enzymes[END_REF] it affects the flexibility and the stability of the enzyme. [START_REF] Ding | Dynamical Roles of Metal Ions and the Disulfide Bond in Cu, Zn Superoxide Dismutase Folding and Aggregation[END_REF] The effect of copper coordination on the dynamics of the enzyme has been investigated by solution NMR on a monomeric mutant, showing a rigidification of the protein backbone upon metal binding on the sub-ns and ms-μs timescales. [START_REF] Banci | Structure and Dynamics of Copper-Free SOD: The Protein before Binding Copper[END_REF] Similarly, the crystal structure of a Cu depleted mutant showed that in the absence of copper the functional loops 4 and 7 are poorly resolved, likely due to dynamics. [START_REF] Antonyuk | Structural Consequences of the Familial Amyotrophic Lateral Sclerosis SOD1 Mutant His46Arg[END_REF] Nevertheless, with this approach the effect of metal binding could not be disentangled from the effect of dimerization and of punctual mutations. More importantly, the effect of metal uptake and its implication in the functionality of the enzyme cannot be ascribed to a simple linear response. [START_REF] Henzler-Wildman | A Hierarchy of Timescales in Protein Dynamics Is Linked to Enzyme Catalysis[END_REF] Culik et al. recently expanded this in a multi-time scale analysis of dynamics, [START_REF] Culik | Effects of Maturation on the Conformational Free-Energy Landscape of SOD1[END_REF] investigating both the presence of conformational exchange at slow timescale and fast dynamics with measured nuclear Overhauser effect. However, the focus of the study being the investigation of the maturation pathway, motions in the fully mature form were not characterized.

We show here that the function of SOD seems to require fine-tuned dynamics, and does it in an articulated way. Cu binding, investigated here in absence of any other structural modifications, is indeed responsible of modulation of internal dynamics on multiple timescale. Making use of the site-specific precision and high sensitivity of solid-state NMR investigation on a large window of timescales, a richer picture of the effects induced by Cu uptake could be obtained. More specifically, the data permit us to associate copper binding with several interconnected layers of dynamics, each linked to a specific aspect of activity or stability, as detailed below and sketched in Figure 4. Cu binds to a pre-structured rigid pocket. Absence of chemical shift changes in the 1 H, [START_REF] Schanda | Studying Dynamics by Magic-Angle Spinning Solid-State NMR Spectroscopy: Principles and Applications to Biomolecules[END_REF] N correlations for the His side-chains indicates no change in the geometry of the metal binding pocket even upon copper binding. We can confirm that this pocket is extremely rigid on the fastest timescale sampled here (ps-ns), and its dynamical properties closely resemble those of the backbone residues in well-structured portions of the protein. With the notable exception of His 63 (see below), the presence of copper has little or no impact on such dynamics. The picture is dramatically different for slower timescales. In the ns-μs window, the relaxation rates of side-chains reflect their degree of coordination, with smaller 15 N R1ρ for histidines coordinated to Zn in E,Zn-SOD and twice larger values for those facing the empty site. These values become uniformly small over the whole metal binding pocket in Cu,Zn-SOD, which can be interpreted in terms of a further rigidification of the metal binding pocket on a slower timescale, consistently with previous work from Banci et al. [START_REF] Banci | Backbone Dynamics of Human Cu,Zn Superoxide Dismutase and of Its Monomeric F50E/G51E/E133Q Mutant: The Influence of Dimerization on Mobility and Function[END_REF] by solution NMR. The fact that copper binding abrogates the protonation equilibria between cationic and neutral forms of the His imidazole rings within the metal binding pocket, [START_REF] Li | Tautomerization, and Rotameric Structure of Histidine: A Comprehensive Study by Magic-Angle-Spinning Solid-State NMR[END_REF] may produce an additional reduction in [START_REF] Schanda | Studying Dynamics by Magic-Angle Spinning Solid-State NMR Spectroscopy: Principles and Applications to Biomolecules[END_REF] N R1ρ of His 46, 48 and 120 in Cu,Zn-SOD.

Cu binding activates fast dynamics in the active channel.

Dynamics on the fastest timescale probed in this study were conserved for most of the backbone residues between the two forms. A local increase of longitudinal relaxation rates was however selectively observed for residues located around the active channel, indicating that Cu uptake acts as a "positive switch", triggering an increase of motions in the fast regime for this portion of the protein. This behavior is contrary to the general idea that metal binding is associated with a stiffening of the protein scaffold in metalloproteins. Interestingly, these results also counter the interpretation of previous solution NMR data which postulated a global rigidification of the backbone following Cu binding based on the change of the average S 2 along the sequence. [START_REF] Banci | Structure and Dynamics of Copper-Free SOD: The Protein before Binding Copper[END_REF] However, solid-state NMR is able to sample internal dynamics in the absence of overall molecular tumbling, allowing here for a clear separation of fast and slow timescale motions. The high catalytic efficiency in Cu,Zn-SOD (kcat ~ 2 x 10 9 M -1 s -1 55 ) is connected not only to the correct conformation of the metal coordinating residues, but also to an efficient diffusion of the superoxide towards the active site. [START_REF] Fisher | The Role of Arginine 143 in the Electrostatics and Mechanism of Cu,Zn Superoxide Dismutase: Computational and Experimental Evaluation by Mutational Analysis[END_REF] The fast motions detected here and activated by Cu binding match the estimated timescale for the diffusion of the superoxide anion through the active channel, suggesting a specific role in driving the superoxide ion. While the requirement of this kind of motions for SOD activity had been speculated on in the past, [START_REF] Banci | Backbone Dynamics of Human Cu,Zn Superoxide Dismutase and of Its Monomeric F50E/G51E/E133Q Mutant: The Influence of Dimerization on Mobility and Function[END_REF] here the observation of increased fast motions compellingly connects to the presence of the catalytic ion.

Cu binding clogs intermediate dynamics overall. The effect of Cu binding is opposite if we move the observation win-dow to an intermediate timescale (ns-μs). Here a uniform decrease of [START_REF] Schanda | Studying Dynamics by Magic-Angle Spinning Solid-State NMR Spectroscopy: Principles and Applications to Biomolecules[END_REF] N R1ρ in both the Cu-coordinating sidechains and in the backbone suggests a Cu-driven "negative switch" of global motion. Such global motion could in principle be associated to a residual collective movement of the protein in the crystalline lattice, and evidence of such phenomena has recently been reported. [START_REF] Ma | Observing the Overall Rocking Motion of a Protein in a Crystal[END_REF][START_REF] Kurauskas | Slow Conformational Exchange and Overall Rocking Motion in Ubiquitin Protein Crystals[END_REF] The occurrence of this "rocking" motion cannot, however, account for the different 15 N R1ρ rates observed between the two SOD forms. Indeed, the properties of such motion are determined by the global protein shape (its tensor of inertia) and the lattice parameters (the crystal packing), [START_REF] Lewandowski | Site-Specific Measurement of Slow Motions in Proteins[END_REF] which are both conserved in our two differently metallated samples. A more likely model which explains the differences between the two forms in our experimental data would involve segmental motions [START_REF] Good | Conformational Dynamics of a Seven Transmembrane Helical Protein Anabaena Sensory Rhodopsin Probed by Solid-State NMR[END_REF] displacing the secondary structure elements with respect to each other. The inhibition of collective motions of the protein in this regime by Cu binding that we observe here is in line with the common framework of ligand-induced rigidification of metalloproteins communicated from the metal-binding site to distant regions.

Cu binding reduces slow dynamics at the dimer interface.

The presence of large [START_REF] Schanda | Studying Dynamics by Magic-Angle Spinning Solid-State NMR Spectroscopy: Principles and Applications to Biomolecules[END_REF] N relaxation dispersions is a signature of the occurrence of slow conformational exchanges between a ground state and thermally accessible states which are invisible both in X-ray data and in NMR spectra. These slow-timescale fluctuations affect residues close to the dimer interface in E,Zn-SOD, and are abrogated by copper binding. The absence of Cu therefore introduces the possibility of transient restructuring of the interface. With the regions concerned lying far from crystal contacts, 18,57 these conformational exchange processes represent an intrinsic property of the molecule, and are likely to persist in solution. As dimerization is known to be a key feature for overall stability, the presence of transient states localized at the interface represents a mechanical explanation to interpret the large difference in the thermal stability measured in solution for E,Zn-SOD (melting temperature Tm of 75°C) and Cu,Zn-SOD (Tm of 92°C). [START_REF] Valentine | Copper-Zinc Superoxide Dismutase and Amyotrophic Lateral Sclerosis[END_REF][START_REF] Furukawa | Amyotrophic Lateral Sclerosis Mutations Have the Greatest Destabilizing Effect on the Apo-and Reduced Form of SOD1, Leading to Unfolding and Oxidative Aggregation[END_REF] This may also provide a link to interpret the increased aggregation propensity observed in mutants designed to abrogate Cu-binding. [START_REF] Rodriguez | Familial Amyotrophic Lateral Sclerosis-Associated Mutations Decrease the Thermal Stability of Distinctly Metallated Species of Human Copper/Zinc Superoxide Dismutase[END_REF][START_REF] Wang | Disease-Associated Mutations at Copper Ligand Histidine Residues of Superoxide Dismutase 1 Diminish the Binding of Copper and Compromise Dimer Stability[END_REF] It is fascinating that such conformational transitions can be captured in a crystalline sample, in line with a growing body of evidence connecting slow dynamics in hydrated crystals with the corresponding phenomena in solution. [START_REF] Marchand | Conformational Dynamics in Crystals Reveal the Molecular Bases for D76N Beta-2 Microglobulin Aggregation Propensity[END_REF][START_REF] Rovó | Mechanistic Insights into Microsecond Time-Scale Motion of Solid Proteins Using Complementary 15 N and 1 H Relaxation Dispersion Techniques[END_REF][START_REF] Tollinger | Site-Resolved Measurement of Microsecond-to-Millisecond Conformational-Exchange Processes in Proteins by Solid-State NMR Spectroscopy[END_REF] It is also interesting to note that these conformational exchange processes present in E,Zn-SOD cannot be captured by solution NMR Carr-Purcell-Meiboom-Gill (CPMG) experiments, [START_REF] Culik | Effects of Maturation on the Conformational Free-Energy Landscape of SOD1[END_REF] as their investigation requires high RF fields available only to solid-state NMR. [START_REF] Schanda | Studying Dynamics by Magic-Angle Spinning Solid-State NMR Spectroscopy: Principles and Applications to Biomolecules[END_REF][START_REF] Ban | Exceeding the Limit of Dynamics Studies on Biomolecules Using High Spin-Lock Field Strengths with a Cryogenically Cooled Probehead[END_REF] These findings interestingly mirror the above analysis of fast dynamics ( 15 N R1). The very same regions (loops 4, 6 and 7) undergoing increased ns motions in Cu,Zn-SOD experience an opposite effect on the μs-ms timescales sampled by relaxation dispersion. This suggests Cu binding couples the structural stabilization of the dimer interface and the activation of motions in the superoxide diffusion channel.

Histidine side-chains couple metal binding to dynamics in remote protein regions. In the above picture of the effect of Cu binding on the histidine side-chains, outstanding is the behavior of Zn-coordinating His 63, halfway between the Zn and Cu pockets, and His 120. The side-chain of His 63 experiences abnormally large dynamics over all timescales in E,Zn-SOD. The presence of copper quenches most of these motions and levels its behavior to that of the other histidine residues. A similar behavior explains the appearance of a signal in the HN correlation from His 120 only in the presence of Cu. Importantly, the consistency between the general features of the relaxation dispersion profiles for backbone amides in loops 4 and 7 and that of His63 Nε2 suggests a comparable timescale for the slow conformational exchanges occurring in the absence of Cu, both in close proximity of the metal site and far from it. This observation highlights a pivotal role of His 63 in propagating the effect of Cu-binding to spatially remote regions of the enzyme.

It is interesting to speculate on the mechanism of this transfer. Although not directly coordinated to Cu I , His 63 faces the Cu-binding pocket and it is sterically affected by the presence of the metal. Different from the three other His in the same pocket (46, 48 and 120), which all belong to β-strands, His 63 is located in the middle of loop 4, which provides a broader conformational space compatible with larger changes of dynamics. Remarkably, the very same loop 4 anchors the metal center with the active channel and the dimer interface, so that dynamical changes for His 63 can easily be transferred away from the Cu I site. [START_REF] Khare | Common Dynamical Signatures of Familial Amyotrophic Lateral Sclerosis-Associated Structurally Diverse Cu, Zn Superoxide Dismutase Mutants[END_REF]

Conclusions

In this work we have applied state-of-the-art solid-state NMR methods with fast MAS at the highest magnetic fields to disentangle the interplay between Cu uptake and the internal dynamics in human SOD. This was performed here regardless of any other post-translational events occurring in the maturation pathway (disulfide bond formation, dimerization, Zn-binding), by the comparison of two crystalline preparations differing uniquely for the presence of Cu I in the active site.

The measurement of solid-state NMR observables expands the window of timescales of motion accessible for atomic-scale investigation with respect to earlier analyses in solution, unraveling a purely dynamical, multimodal response of the skeleton of dimeric SOD upon binding of Cu I in its active site. In SOD, we find that metal ion uptake does not simply rigidify the enzyme but acts as a switch simultaneously both activating and deactivating motional processes on different timescales, with additionally coupling of the dynamics of histidine sidechains and those of remote key backbone elements of the protein. The multi-timescale dynamic mechanisms unveiled here for SOD are difficult to observe using other methods, and so they may be general and our observations could be pertinent for the description of metal ion binding in a variety of metalloproteins.

Figure 1 .

 1 Figure 1. (a) Structure of the homodimer of Cu,Zn-SOD (PDB code:1SOS). The Cu and Zn metal ions are represented by a gold and a blue sphere respectively. (b) Solvent excluded surface of Cu,Zn-SOD, where the active channel is shown in orange (T58 in loop 4) and green (E133, T137, R143 in loop 7). This has a role in attracting superoxide anions and guide them into the channel to the copper ion. The binding sites of Cu and Zn ions are shown in yellow and purple respectively. Cu is solvent accessible at the end of the active channel, while Zn shielded at the protein core. (c) Zoom of the structure in the region of the active channel. The residues involved in metal coordination are shown with sticks. Loops 4 and 7 are colored in orange and green respectively. The fully conserved disulfide bridge between C57 and C146 is shown as a yellow stick. (d) Structure of the active site. Side chains of the residues involved in the metal coordination are shown as sticks and the residue numbering is indicated. Cu-

Figure 2 . 15 N

 215 Figure 2. (a) Superposition of solid-state 15 N-1 H CP-HSQC spectra of E,Zn-SOD (blue) and Cu,Zn-SOD (red) in the region of the side chains of histidine residues. The assignment

Figure 3 . 15 N- 1 H 15 N 15 N 15 N

 3151151515 Figure 3. (a) Superposition of 15 N-1 H CP-HSQC spectra of E,Zn-SOD (blue) and Cu,Zn-SOD (red), recorded at 1 GHz 1 H Larmor frequency and 60 kHz MAS. The assignment of the most shifted residues and of the residues exposed in the active site (underlined) is reported according to Knight et al. (32, 33). (b) Relaxation profiles of representative residues in E,Zn-SOD (blue) and Cu,Zn-SOD (red). Top: 15 N R1 experimental decay curves and fit curves of residue T137 (loop 7, active channel). Middle: 15 N R1ρ experimental decay curves and fit curve of residue D101 (in β-strand). Bottom: 15 N relaxation dispersion profiles of residue T58 (loop 4, active channel and close to dimer interface). (c) 15 N backbone relaxation rates measured for E,Zn-SOD (blue triangles) and Cu,Zn-SOD (red circles). Top: longitudinal 15 N relaxation rates R1 (ps-ns dynamics). Middle: spin-lattice 15 N relaxation rates in the rotating frame R1ρ with a spin-lock RF amplitude of 20 kHz (ns-μs dynamics). Bottom:15 N ΔR1ρ (μs-ms dynamics) for E,Zn-SOD (top, blue) and Cu,Zn-SOD (bottom, red). The secondary structure is indicated above the plots. Orange and green boxes highlight the residues located in loop 4 and 7 respectively, where noticeable differences between the two metallated states were revealed.

Figure 4 .

 4 Figure 4. Schematic view of the dynamical modes activated by Cu binding for the backbone and the metal-binding histidine residues of SOD over the three timescales investigated. For sake of clarity, SOD is represented by its monomer. Por-

Funding Sources

The work was co-funded by the European Research Council (ERC-2015-CoG GA 648974), the Agence Nationale de la Recherche (10-BLAN-713-01), Fondazione CR Firenze, Egide (programme Galilée 22397RJ), the Università Italo-francese (programma Galileo 11/12), CNRS (IR-RMN FR3050) as well as by the EC-project iNext (infrastructure for NMR, EM, and Xrays for Translational Research, GA 653706).

Supporting Information

The Supporting Information contains a full description of the NMR experimental details, formulas and figures illustrating the sensitivity of the NMR to different timescales of motion, details on the activity assays, relaxation dispersion profiles as well as tables with site-specific relaxation data. This material is available free of charge on the ACS Publications website.

AUTHOR INFORMATION

Corresponding Author

* Email addresses: lyndon.emsley@epfl.ch, roberta.pierattelli@unifi.it, guido.pintacuda@ens-lyon.fr

Author Contributions

All authors have given approval to the final version of the manuscript.