Isobel H Norville

Katherine O'shea

Mitali Sarkar-Tyson

Suxin Zheng

Richard W Titball

Gabriele Varani
email: varani@chem.washington.edu

Nicholas J Harmer
email: n.j.harmer@exeter.ac.uk.

The structure of a Burkholderia pseudomallei immunophilin-inhibitor complex reveals new approaches to antimicrobial development

Keywords: Prolyl-peptide isomerase, Burkholderia pseudomallei, X-ray crystallography, NMR, small molecule inhibitor Burkholderia pseudomallei Mip-like protein, BpML1, FK506 binding protein, FKBP, heteronuclear single-quantum coherence, HSQC, Legionella pneumophila Mip, LpMip, Macrophage infectivity potentiator, Mip, Prolyl peptide isomerase, PPIase, threedimensional, 3D

Macrophage infectivity potentiators (Mips) are a subset of immunophilins associated with virulence in a range of micro-organisms. These proteins possess prolyl-peptide isomerase activity and are inhibited by drugs including rapamycin and tacrolimus. We determined the structure of the Mip homologue (BpML1) from the human pathogen and biowarfare threat Burkholderia pseudomallei by nuclear magnetic resonance and X-ray crystallography. The crystal structure suggests that key catalytic residues in the BpML1 active site have unexpected conformational flexibility consistent with a role in catalysis. The structure further revealed BpML1 binding to a helical peptide, in a manner resembling the physiological interaction of human TGF receptor 1 with the human immunophilin FKBP12. Furthermore, the structure of BpML1 bound to the class inhibitor cycloheximide N-ethylethanoate showed that this inhibitor mimics such a helical peptide, in contrast to the extended prolyl peptide mimicking shown by inhibitors such as tacrolimus. We suggest that Mips, and potentially other bacterial immunophilins, participate in protein-protein interactions in addition to their prolyl-peptide isomerase activity, and that some roles of Mip proteins in virulence are independent of their prolyl-peptide isomerase activity.

Introduction

Melioidosis is a community acquired infection frequent in South-East Asia and Northern Australia [START_REF] Wiersinga | Melioidosis: insights into the pathogenicity of Burkholderia pseudomallei[END_REF]. Caused by the Gram-negative bacterium Burkholderia pseudomallei, the disease can range from a localised infection to an acute systemic, chronic or persistent disease. The symptoms of infection are diverse, often resembling tuberculosis or cancer. Consequently, the disease is likely to be often misdiagnosed [START_REF] White | Melioidosis[END_REF]. The disease is especially pernicious as the bacterium is intrinsically resistant to a wide range of antibiotics [START_REF] Cheng | Melioidosis: epidemiology, pathophysiology, and management[END_REF]: even with optimal treatment, mortality in acute cases of disease ranges from 20-40% [START_REF] White | Melioidosis[END_REF]. B. pseudomallei is also a potential bioterror agent [START_REF] Rotz | Public health assessment of potential biological terror agents[END_REF]. Consequently, there has been a recent and concerted effort to develop novel countermeasures against this organism.

One potential target class for novel antimicrobials is the FK506 (tacrolimus) binding proteins. These ubiquitous enzymes catalyse the isomerisation of pre-proline peptide bonds between the cis and trans configurations (peptidyl-prolyl isomerase, or PPIase, activity [START_REF] Kang | FKBP family proteins: immunophilins with versatile biological functions[END_REF]), and form part of the broader category of PPIase enzymes. Several FKBPs have been found to play a role in virulence in a range of species including the bacteria Legionella pneumophila [START_REF] Cianciotto | A Legionella pneumophila gene encoding a species-specific surface protein potentiates initiation of intracellular infection[END_REF], Chlamydia trachomatis [START_REF] Lundemose | Chlamydia trachomatis Miplike protein has peptidyl-prolyl cis/trans isomerase activity that is inhibited by FK506 and rapamycin and is implicated in initiation of chlamydial infection[END_REF], Neisseria gonorrhoeae [START_REF] Leuzzi | Ng-MIP, a surface-exposed lipoprotein of Neisseria gonorrhoeae, has a peptidyl-prolyl cis/trans isomerase (PPIase) activity and is involved in persistence in macrophages[END_REF] and the protozoan Trypanosoma cruzi [START_REF] Moro | Secretion by Trypanosoma cruzi of a peptidyl-prolyl cis-trans isomerase involved in cell infection[END_REF]. One of the first bacterial FKBPs to be studied in detail is a L. pneumophila protein required for the efficient invasion of macrophages [START_REF] Cianciotto | A Legionella pneumophila gene encoding a species-specific surface protein potentiates initiation of intracellular infection[END_REF]. Consequently, these proteins were labelled Mip (macrophage infectivity potentiator) proteins. The FKBP domain of the Legionella Mip (LpMip) is required for virulence, and both antibodies binding to the active site and specific inhibitors sufficed to reduce the capacity of Legionella to invade macrophages [START_REF] Helbig | The PPIase active site of Legionella pneumophila Mip protein is involved in the infection of eukaryotic host cells[END_REF][START_REF] Wagner | Collagen binding protein Mip enables Legionella pneumophila to transmigrate through a barrier of NCI-H292 lung epithelial cells and extracellular matrix[END_REF]. We recently showed that a Mip-like protein from B. pseudomallei, BPSS1823 (hereafter BpML1), has high PPIase activity; and that it is required for efficient invasion of host cells and virulence in a mouse model of infection. Deletion of this gene significantly attenuates B. pseudomallei (Norville, I., Harmer, N., Harding, S., Fischer, G., Keith, K., Brown, K., Sarkar-Tyson, M., Titball, R., unpublished work).

Despite extensive study of Mips in a range of microorganisms, the true biological targets for these proteins have not been clearly elucidated [START_REF] Leuzzi | Ng-MIP, a surface-exposed lipoprotein of Neisseria gonorrhoeae, has a peptidyl-prolyl cis/trans isomerase (PPIase) activity and is involved in persistence in macrophages[END_REF][START_REF] Wagner | Collagen binding protein Mip enables Legionella pneumophila to transmigrate through a barrier of NCI-H292 lung epithelial cells and extracellular matrix[END_REF][START_REF] Zang | Requirement of a mip-like gene for virulence in the phytopathogenic bacterium Xanthomonas campestris pv. campestris[END_REF]. It has been broadly assumed that they act solely as isomerases [START_REF] Wagner | Collagen binding protein Mip enables Legionella pneumophila to transmigrate through a barrier of NCI-H292 lung epithelial cells and extracellular matrix[END_REF]. Mips have been suggested to be an attractive target class for antimicrobials: there are known high affinity inhibitors, indicating that the proteins are eminently druggable; and their prokaryotic functions are differentiated from those of higher eukaryotic FKBPs.

We have previously shown that the B. pseudomallei Mip-like protein plays a key role in disease progression. In this study, we report the molecular structure of BpML1, identify common features with other Mips, and highlight differences from mammalian FKBPs that could be exploited for drug design. We also identify new approaches to the design of compounds able to block this target. These compounds could be developed as antimicrobials to treat melioidosis, but could also have a much wider utility to treat other diseases where Mips play a key role in infection.

Methods

Preparation of BpML1

Full length BpML1 from B. pseudomallei strain K96243 was cloned into pET15b using the NdeI and HindIII sites. The vector was transformed into BL21 (DE3) cells. For enzymology, cells were grown in LB media at 37ºC with agitation until the OD 600 reached 0.4-0.6. Isopropyl β-D-1-thiogalactopyranoside was added to 1 mM and growth continued at 20ºC for 4 hours. Harvested cells were resuspended in 10 mM PBS supplemented with 100 mg/mL DNase I and Complete EDTA-free protease inhibitors (Roche), and disrupted by sonication. Clarified lysate was loaded onto a 1 ml Histrap FF column (GE Healthcare) and the recombinant protein eluted in 10 mM PBS supplemented with 100 mM imidazole. The purified protein was dialysed against 10 mM PBS and samples frozen at -80ºC until use. Protein concentration was determined using a bicinchoninic acid assay (Pierce Biotechnology). For crystallisation, cells were grown in ZYM-5052 media [13] supplemented with 100 g/mL ampicillin at 37°C until the OD 600 was 0.5, and then at 20°C for 16 hours. Harvested cells were resuspended in 20 mM Tris-HCl pH 8.0, 0.5 M NaCl (Buffer A), and lysed using a Soniprep 150 sonicator (MSE). Clarified lysate was purified using a nickel-agarose column (Bioline). Briefly, the loaded protein was washed with Buffer A supplemented with 20 mM imidazole-HCl pH 8.0, and eluted with Buffer A supplemented with 250 mM imidazole-HCl pH 8.0. BpML1 was then loaded onto a Superdex 200 16/60 hr column (GE Healthcare), and eluted isocratically with 10 mM Hepes pH 7.0, 0.5 M NaCl. For NMR studies, 15 N labelled and 13 C 15 N labelled protein was made by substituting [ 15 N]NH 4 Cl and/or [ 13 C]glucose in the cell growth media. 15 N labelled and 13 C 15 N double labelled proteins were dialysed into 20 mM potassium phosphate pH 7.0, 100 mM KCl, 2 mM DTT, to a final protein concentration of approximately 1 mM. Cycloheximide Nethylethanoate (> 95%) was purchased from Life Chemicals (www.lifechemicals.com) without further purification. Samples of the complex were prepared by titrating unlabeled cycloheximide N-ethylethanoate into labelled protein samples while monitoring 15 N heteronuclear single-quantum coherence (HSQC) chemical shift change until a previously established "saturated" spectrum was obtained.

Crystallisation and structure solution

All crystals were grown using the microbatch method, and were prepared using an Oryx6 crystallisation robot (Douglas Instruments). BpML1 at 14 mg/mL was mixed with an equal volume of 2.2 M (NH 4 ) 2 SO 4 , 0.1 M Bis-Tris pH 5.5, and grown at 20°C. Prior to flashfreezing in liquid nitrogen, crystals were soaked for 30-60 seconds in a cryoprotectant solution of 1.1 M (NH 4 ) 2 SO 4 , 0.1 M Bis-Tris pH 5.5, 30% (v/v) glycerol. Single wavelength X-ray diffraction data were collected at a wavelength of 0.861 Å at 100 K at beamline I03 of the Diamond synchrotron (Table 1). Data were processed using iMOSFLM version 1.0.3 [START_REF] Leslie | Recent changes to the MOSFLM package for processing film and image plate data[END_REF] and SCALA [START_REF] Evans | Scaling and assessment of data quality[END_REF]. Initial phases were determined by molecular replacement using the structures of human FKBP12 (PDB ID: 1FKB) and the N-terminal domain of rabbit FKBP59 (PDB ID: 1ROT) as a model. These structures were modified to remove non-homologous side chains using CHAINSAW [16]. Molecular replacement was performed using PHASER [START_REF] Mccoy | Phaser crystallographic software[END_REF]. One protomer was observed in the asymmetric unit. Model building and refinement of the structures was performed using Coot version 0.6.1 [START_REF] Emsley | Features and development of Coot[END_REF] and PHENIX version 1.6. 1 [19]. The structure was solved using anisotropic B-factors for all heavy atoms, riding hydrogen atoms, and optimisation of stereochemical and ADP restraints. Structures were validated using PHENIX, Coot and MOLPROBITY [START_REF] Davis | MolProbity: all-atom contacts and structure validation for proteins and nucleic acids[END_REF]. The Ramachandran plot for the final model showed 100% of residues in the favoured region. Structural images were prepared using the PyMOL molecular graphics system, version 1.3 (Schrödinger). A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

NMR structure solution

All NMR spectra were collected at 298 K. 15 N HSQC, triple-resonance spectroscopy, HCCH TOCSY [START_REF] Kay | A Gradient-Enhanced Hcch Tocsy Experiment for Recording Side-Chain H-1 and C-13 Correlations in H 2 O Samples of Proteins[END_REF], three-dimensional (3D) 15 N-edited NOESY and 3D 13 C-edited NOESY [START_REF] Muhandiram | An Enhanced-Sensitivity Pure Absorption Gradient 4d N-15, C-13-Edited Noesy Experiment[END_REF] were recorded on a Bruker Avance 500-MHz spectrometer with cryoprobe. The 2D 1 H-1 H NOESY and 1 H-1 H TOCSY spectra were recorded on a Bruker Avance 750-MHz spectrometer. Backbone resonance assignments of H, N, C α , C′, and side-chain C β were performed using triple-resonance HNCA [START_REF] Yamazaki | An Hnca Pulse Scheme for the Backbone Assignment of N-15,C-13,H-2-Labeled Proteins -Application to a 37-Kda Trp Repressor DNA Complex[END_REF], CBCA(CO)NH [START_REF] Muhandiram | Gradient-Enhanced Triple-Resonance 3-Dimensional Nmr Experiments with Improved Sensitivity[END_REF], HNCACB, and HN(CO)CA. The remaining side-chain resonance assignments were obtained from the analysis of 15 N-edited NOESY, 13 C-edited NOESY, and HCCH-TOCSY spectra. Aromatic ring protons and protected amides were identified from two-dimensional 1 H-1 H NOESY, 1 H-1 H TOCSY, and 15 N HSQC, respectively, collected from a sample exchanged into a 100% 2 H 2 O NMR buffer. The assignments of the ligand were obtained based on the 2D 13 C-filtered NOESY and total correlation spectroscopy (TOCSY) spectra of the "F1fF2f" type [START_REF] Peterson | New applications of 2D filtered/edited NOESY for assignment and structure elucidation of RNA and RNA-protein complexes[END_REF]. The 2D 13 C- 12 C NOESY spectra were collected to facilitate the assignments of protein-ligand intermolecular NOEs [26].

Spectral data were processed using NMRPipe [START_REF] Delaglio | Nmrpipe -a Multidimensional Spectral Processing System Based on Unix Pipes[END_REF] and analyzed using CcpNmr Analysis1.0. [START_REF] Vranken | The CCPN data model for NMR spectroscopy: Development of a software pipeline[END_REF]. The structure calculations were conducted automatically by CYANA2.1 [START_REF] Güntert | Automated NMR protein structure calculation[END_REF][START_REF] Herrmann | Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA[END_REF] based on nuclear Overhauser enhancements (NOEs) selected from 15 N-edited NOE spectroscopy (NOESY), 13 C-edited NOESY, 1 H-1 H NOESY and 13 C- 12 C NOESY, as well as additional dihedral-angle and hydrogen-bond constraints generated from the NMR data.

Inhibitor assay

PPIase activity of BpML1 protein was determined by a protease coupled assay as described previously [START_REF] Fischer | Detection of Enzyme Catalysis for Cis-Trans-Isomerization of Peptide-Bonds Using Proline-Containing Peptides as Substrates[END_REF]. Briefly, 10 nM BpML1 protein was incubated for 6 min at 6ºC in 1.2 mL 35 mM Hepes buffer, pH 7.8 with succinyl-Ala-Phe-Pro-Phe-p-nitroanilide (10 mg/mL; Bachem). α-chymotrypsin (Sigma) was added to the cuvette to a final concentration of 1.25 mg/mL and mixed. Hydrolysis of the substrate was measured at 390 nm using a Shimadzu 1800 UV/Vis spectrophotometer at 1 sec intervals until there was no further change in absorbance. For inhibition measurements, BpML1 protein was pre-incubated with varying concentrations of cycloheximide-N-ethylethanoate from 1 -500 M for 6 min prior to addition of chymotrypsin. At least three independent readings were taken at each data point. All data fitting and statistical analyses were performed using SPSS v16.0 (IBM). The pseudo first order rate constant was calculated using equation 1 [START_REF] Kullertz | Semiautomated microtiter plate assay for monitoring peptidylprolyl cis/trans isomerase activity in normal and pathological human sera[END_REF]; data from 10-50 sec (following the lag phase, and before substrate became limiting) were taken, and k obs calculated by linear regression.

(

1) ln [A ∞ -A t ] = -k obs t + ln [A ∞ -A 0 ]
The enzymatic rate was determined by comparing the observed rate to the uncatalysed rate (equation 2).

(2) k enz = k obs -k uncat Data for inhibitor assays were fit to equation 3 [START_REF] Williams | The kinetics of reversible tight-binding inhibition[END_REF] using least squares non-linear fitting. [E] was treated as a constant (10 nM); v 0 and K I app were fit, using initial estimates based on the raw data.

( A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

) ] [ 2 ] [ 4 ) ] [ ] ([ ] [ ] [ 2 0 E K E K I E K I E v v 3

Results and Discussion

The BpML1 fold and active site residue configuration are highly similar to LpMip We solved the X-ray structure of BpML1 to 0.91 Å (Table 1). The structure confirmed that BpML1 adopts a classical FKBP fold (Figure 1A). Closer inspection of the active site in the crystal revealed that key side chains adopt a conformation that closely resembles that of the ligand free L. pneumophila Mip FKBP domain (Figure 1B). NMR structures of BpML1 that were determined independently show an excellent similarity in overall fold with the crystal structure. They also demonstrate that there is considerable flexibility in both peripheral loops and side chains (Figure 1C) in the well defined active site (see Supplementary Figure 1 online [START_REF] Ceymann | Solution structure of the Legionella pneumophila Mip-rapamycin complex[END_REF]35]). In particular, the 4-5 loop, which contributes the key Tyr 89 side chain to the active site, shows two distinct conformations in solution; and several of the key conserved active site side chains have considerable flexibility. These observations are in contrast to Mip from L. pneumophila and T. cruzi, which show a remarkable commonality in the conformations adopted by these regions (see Supplementary Figure 2 online [START_REF] Ceymann | Solution structure of the Legionella pneumophila Mip-rapamycin complex[END_REF][START_REF] Pereira | Trypanosoma cruzi macrophage infectivity potentiator has a rotamase core and a highly exposed alpha-helix[END_REF]37]). These observations suggest that previous structures (especially crystal structures) might be trapping a preferred, low energy conformation that may be catalytically relevant, but which does not capture the full range of dynamics available to the enzyme in solution.

BpML1 forms protein-protein interactions reminiscent of other FKBPs

The protein sample used for crystallisation of BpML1 had been cloned with a 6*His tag, linked to the protein by a thrombin cleavage site (sequence MSSHHHHHHSSGLVPRGSHM…, thrombin site underlined). The thrombin cleavage peptide was clearly resolved in the electron density, and forms an interaction with a neighbouring molecule in the crystal. This interaction placed the peptide directly in the active site of the neighbouring molecule, forming an apparently tight interaction, burying 890 Å 2 of surface (Figure 2A). Intriguingly, although BpML1 is a very active PPIase (Norville, I., Harmer, N., Harding, S., Fischer, G., Keith, K., Brown, K., Sarkar-Tyson, M., Titball, R., unpublished work), the conformation of the peptide is inconsistent with the expected binding for a proline-containing peptide. The proline side chain does not sit in the deep pocket of the enzyme: complexes with the inhibitor FK506 (see Supplementary Figure 1B online [START_REF] Wilson | Comparative X-ray structures of the major binding protein for the immunosuppressant FK506 (tacrolimus) in unliganded form and in complex with FK506 and rapamycin[END_REF]) and with a cis-peptide [35] have defined this as the likely site for cis-trans isomerization. In contrast, this site is occupied by a valine side chain (Figure 2B). Furthermore, the overall conformation of the peptide is highly diverged from the expected peptide orientation, suggested by structures of rapamycin or peptide bound FKBPs (Figure 2C [START_REF] Ceymann | Solution structure of the Legionella pneumophila Mip-rapamycin complex[END_REF]35,[START_REF] Wilson | Comparative X-ray structures of the major binding protein for the immunosuppressant FK506 (tacrolimus) in unliganded form and in complex with FK506 and rapamycin[END_REF]). We conclude that, although BpML1 is a bona fide PPIase, this peptide binding is not representative of the binding of substrates for pre-prolyl peptide cis-trans isomerization.

However, the peptide binding is strikingly similar to the structure of human FKBP12 in complex with the transforming growth factor receptor I, bone morphogenic protein receptor 1B, and the activin type I receptor (A Chaikuad, I Alfano, B Shrestha, J Muniz, K Petrie, O Federov, C Phillips, S Bishop, P Mahajan, A Pike, F von Delft, C Arrowsmith, A Edwards, J Weigelt, C Bountra, S Knapp, A Bullock, unpublished work; Figure 2D; see Supplementary Figure 3 online [START_REF] Huse | Crystal structure of the cytoplasmic domain of the type I TGF beta receptor in complex with FKBP12[END_REF]). Eukaryotic FKBPs form physiologically important complexes with other proteins, binding partner proteins to modulate their activity [START_REF] Chelu | Regulation of ryanodine receptors by FK506 binding proteins[END_REF][START_REF] Huse | The TGF beta receptor activation process: an inhibitor-to substrate-binding switch[END_REF], in addition to their roles in protein folding. In particular, the BpML1 peptide forms a short helix, in a similar conformation to the receptor helices that binds to hFKBP12; and each of these receptors places a leucine side chain into the deep pocket of hFKBP12, reminiscent of the valine observed in BpML1. These observations suggest that, like hFKBP12, BpML1 is likely to form complexes with other proteins in the cell in addition to its role as a PPIase. hFKBP12 masks a multiple phosphorylation peptide (the GS region) in TGF RI, and locks the receptor into an inactive conformation to ensure that there is no activation in the absence of an extracellular signal [START_REF] Huse | The TGF beta receptor activation process: an inhibitor-to substrate-binding switch[END_REF]. As bacteria require robust signalling networks to respond appropriately to their surroundings, similar roles in dampening signalling noise are conceivable for BpML1.

X-ray and NMR structures of BPSS1823 reveal intrinsic flexibility in key active site residues

The X-ray structure presented here diffracted to 0.91 Å. This resolution is high enough to reveal details of structural flexibility that cannot be observed at lower resolution. At this resolution, we can observe that 18 % of the amino acid side chains (not including alanine and glycine residues) display multiple conformations. The majority of these residues are distant from the active site, in largely solvent exposed parts of the molecule. In addition to these amino acids, four alternative conformations are observed in the active site. Firstly, the pair of Leu -6 from the binding peptide and Ile 93 displays correlated alternative conformers (see Supplementary Figure 4 online). More importantly, both Asp 44 and Tyr 89 show significant flexibility, with hydrogen bond donor and acceptor atoms moving by up to 1.5 Å between the observed extremes (Figure 3). The refined positions likely represent maxima within the electron density: the two residues therefore probably sample a larger range of positions. Similar flexibility is not observed in the 0.92 Å structure of unliganded hFKBP12 (see Supplementary Figure 5 online [START_REF] Szep | Structural coupling between FKBP12 and buried water[END_REF]), indicating that this is not an artefact of the software used. These two amino acids provide the only sources of hydrogen bond donors and acceptors in the BpML1 active site; and mutations of these residues in LpMip significantly reduce the activity of the enzyme [START_REF] Wintermeyer | Influence of site specifically altered Mip proteins on intracellular survival of Legionella pneumophila in eukaryotic cells[END_REF]. As many proposed mechanisms of action of the enzyme involve these amino acids, this unexpected flexibility is highly suggestive of a role in catalysis. Accordingly, we examined the NMR structures of free and inhibitor-bound BpML1 to examine whether these also are consistent with a pronounced flexibility for Asp 44 and Tyr 89 . Strikingly, the side chain of Asp 44 shows a wide range of conformations (see Supplementary Figure 6 online). This is quite unusual for a residue that is apparently making a hydrogen bond interaction with the bound ligand. In contrast, the side chain of Tyr 89 adopts two preferred rotamers, showing a classical alternative conformation. This contrasts with other well conserved, ligand interacting residues (e.g. Trp 66 ), which show only a single conformation across 20 models consistent with the data. This unusual flexibility of these highly conserved side chains in the active site strongly suggests a role for this flexibility in function. The side chains make hydrogen bonding interactions in at least one of their conformers, indicating that they do not lack a stable conformation. The conformations observed in the structures offer a tantalizing possible role in catalysis: superimposition of the extended prolyl peptide from the recently solved FKBPpeptide structure [35] shows that one of the Tyr 89 conformers would be perfectly placed to stabilize the peptide nitrogen in an intermediate state between the cis and trans peptide. One alternative conformation of Asp 44 could interact with and stabilize the peptide backbone of the substrate. These observations suggest that Tyr 89 and Asp 44 are natively flexible in BpML1, and that this flexibility is likely to be relevant for catalysis.

The structure of BpML1 bound to cycloheximide N-ethylethanoate reveals a novel mode of inhibitor binding

We previously showed that BpML1 binds to rapamycin, which inhibits all known FKBPs (Norville, I., Harmer, N., Harding, S., Fischer, G., Keith, K., Brown, K., Sarkar-Tyson, M., Titball, R., unpublished work). Cycloheximide, an inhibitor of eukaryotic protein synthesis, is an unrelated compound that has previously been shown to inhibit the PPIase activity of both hFKBP12 and LpMip (Figure 4A [START_REF] Christner | Synthesis and cytotoxic evaluation of cycloheximide derivatives as potential inhibitors of FKBP12 with neuroregenerative properties[END_REF]). Cycloheximide N-ethylethanoate, which is also active against hFKBP12, inhibited the PPIase activity of BpML1 (Figure 4B): the observed K I of 6.5 ± 1.0 M is similar to the K I reported for this compound towards FKBP12 (4.4 M). As there is no extant structure of cycloheximide N-ethylethanoate with an FKBP, we determined the structure of BpML1 bound to cycloheximide N-ethylethanoate by NMR. This structure reveals that the compound binds to the active site (Figure 4C). However, although the compound binds to most of the highly conserved side chains, the major interactions occur with the 3b-1 and 4-5 loops (Figure 4D). In fact, hydrophobic contacts are formed with residues Ala 94 and Ile 98 , outside the core active site. No significant interactions are formed with the other sides of the catalytic bowl of the FKBP, with the highly conserved active site residues Tyr 33 and Phe 57 in particular having no contact with the inhibitor. In comparison, previously observed inhibitors such as FK506, rapamycin, and other synthetic inhibitors [START_REF] Wilson | Comparative X-ray structures of the major binding protein for the immunosuppressant FK506 (tacrolimus) in unliganded form and in complex with FK506 and rapamycin[END_REF][START_REF] Sich | Solution structure of a neurotrophic ligand bound to FKBP12 and its effects on protein dynamics[END_REF][START_REF] Sun | Design and structure-based study of new potential FKBP12 inhibitors[END_REF], tend to bind more broadly across the catalytic bowl, and contact conserved active site residues across it (Figure 5). Many of these inhibitors also make extensive contacts with the same two loops as cycloheximide N-ethylethanoate.

Remarkably, the conformation of cycloheximide N-ethylethanoate mimics that of the peptide found in the BpML1 crystal structure, and the structure of hFKBP12 bound to TGF R (Figure 4E). Thus, cycloheximide N-ethylethanoate represents a new class of FKBP inhibitors, mimicking the binding of FKBPs to proteins to modulate activity, rather than proline-peptides as rapamycin and FK506 do.

Conclusions

Here, we report the structure of BpML1, a Mip homologue of Burkholderia pseudomallei, which was determined by X-ray crystallography and NMR in parallel. We show that this enzyme is competent in binding to peptides or the class inhibitor cycloheximide Nethylethanoate. Intriguingly, we show that the peptide bound in the structure is not a cisproline, but a small helical peptide that mimics TGF receptors bound to hFKBP12; and that cycloheximide N-ethylethanoate binds in the active site in a way that mimics this peptide, rather than the catalytic peptides which are mimicked by other class inhibitors. These data suggest that Mips (and potentially other bacterial FKBPs), like their eukaryotic cousins, are competent to bind peptides in a helical conformation. This suggests that BpML1 and other Mips have roles in protein-protein interactions, perhaps as modulators of signalling, in addition to their chaperone functions. Consistent with this observation, deletion of BpML1 abolished B. pseudomallei showed a pleiotrophic phenotype, including loss of motility which might suggest a role in the control of the flagellar motors (Norville, I., Harmer, N., Harding, S., Fischer, G., Keith, K., Brown, K., Sarkar-Tyson, M., Titball, R., unpublished work).

The ultra-high resolution crystal structure of BpML1 revealed that two highly conserved active site side chains, Asp 44 and Tyr 89 , show unusual flexibility. These observations were confirmed by NMR, and suggest that these side chains require flexibility, even in a ligand bound form, for performing their roles in catalysis. However, it is not clear whether this represents a general mechanism for FKBPs, or a specific case for BpML1: this enzyme is up to 6-fold more active than previously described FKBPs, and so the flexibility that we observed may be an adaptation of this enzyme to provide it with a greater rate enhancement.

The structure of the cycloheximide N-ethylethanoate complex with BpML1 suggests that this compound might provide an excellent starting point for novel drug development. The binding site for cycloheximide N-ethylethanoate, uniquely for FKBP inhibitors of known structure, is focused on the 3b-1 and 4-5 loops, and contacts residues outside the active site in the 4-5 loop. This new interaction offers a greater possibility of obtaining a selective compound that would not inhibit human proteins: the 4-5 loop, in particular, shows much greater diversity between species than the active site. Furthermore, we show here that both of these loops show considerable flexibility, increasing the likelihood of a species-specific induced fit.

We conclude that the bacterial FKBPs are likely to be a more diverse and functionally rich protein family than has previously been appreciated. In consequence of this, their validated role in the pathogenesis of several micro-organisms, and the availability of multiple inhibitors for this protein class, they show great promise for antimicrobial drug discovery. (a) Asp 44 , which accepts a key hydrogen bond from the bound peptide backbone, shows two conformers for the side chain. The distance between the two conformations for O 2 is 0.9 Å. The higher occupancy conformation makes an additional hydrogen bond to the bound peptide backbone, while the other makes two additional hydrogen bonds to other residues of BpML1. (b) Tyr 89 , the only hydrogen bond donor in the active site pocket, also shows a range of conformations. The distance between the O atoms is 1.5 Å. One conformation is consistent with a direct (3.0 Å) hydrogen bond to the peptide, while the other makes a water-mediated hydrogen bond to the peptide. Lower occupancy forms are indicated by a black arrow. (c) Representative electron density for a multiple conformer residue (Tyr 89 , left) and the peptide binding to the active site (right). Colours: BpML1, yellow; binding peptide, orange; multiple conformation at lower occupancy, cyan; mF o -DF c simulated annealing omit map (side chains truncated to C ) contoured at 3 , green. 2mF o -DF c map for the final structure contoured at 1 , blue. Inhibitors are shown as all heavy atom spherical models, with carbon coloured yellow, oxygen red, and nitrogen blue; proteins are shown as white, semi-transparent surfaces, with cartoon beneath. Cartoon colours: BpMip, yellow; FKBP12, cyan. The nine most conserved active site residues of FKBPs are shown as sticks, with carbon coloured as for the main chain, and other atoms as above. Side chains Tyr 33 and Phe 57 of BpMip do not contact the inhibitor, and are highlighted with carbon coloured orange and with black arrows; equivalent FKBP12 residues are highlighted with carbon coloured orange and with black arrowheads. (a) BpMip binding to cycloheximide N-ethylethanoate (this work; PDB ID: 2KO7). (b) FKBP12 binding to rapamycin (PDB ID: 2DG3). (c) FKBP12 binding to FK-506 (PDB ID: 1FKJ). (d) FKBP12 binding to the neurotrophic ligand GPI-1046 (PDB ID: 1F40). (e, f, g) FKBP12 binding to synthetic, high affinity ligands (PDB IDs: 1FKG, 1FKI, 1J4R). (h) FKBP12 binding to a synthetic, dimeric ligand (PDB ID: 1A7X). Note that cycloheximide N-ethylethanoate occupies a distinct area of the protein, and in particular makes no interaction with the 3b strand (black dashed arrow). A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 16 May 2011 as manuscript BJ20110345 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110345

Biochemical

  Journal Immediate Publication. Published on 16 May 2011 as manuscript BJ20110345 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110345

Biochemical

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Biochemical

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Biochemical

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  Final 20/100 structures selected based on the lowest CYANA target function. b Dihedral angle restraints derived from TALOS [47]. c Maximum violation shown in parentheses. d Ramachandran analysis was performed with PROCHECKNMR. e The average RMSD to the mean of the protein was calculated over regions 13-117. Biochemical Journal Immediate Publication. Published on 16 May 2011 as manuscript BJ20110345 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110345A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure Legends Figure 1 :

 1 Figure LegendsFigure 1: Structures of B. pseudomallei Mip (BpML1). (a) Cartoon of the 0.91 Å structure of BpML1 (left, green; PDB ID: 2Y78) compared with hFKBP12 (right, cyan; PDB ID: 2DG3 [48]). The overall fold is identical, with BpML1 containing all of the secondary structural elements, and an additional N-terminal -strand (arrowed). (b) BpML1 shows a nearly identical active site conformation to LpMip (purple; PDB ID: 1FD9). All of the most conserved side chains are identical in conformation, except for Phe 46 (arrow), which is replaced functionally in LpMip with a neighbouring Phe. (c): The NMR structure of BpML1 confirms the crystallographic data and identifies flexibility within the active site. Left: Cartoon of the NMR structure of BpML1 bound to ligand (yellow; PDB ID: 2KO7) shows a very similar architecture to the crystal structure (a). Right: the comparison of bound (yellow) and apo (pink: PDB ID: 2KE0) shows significant conformational flexibility in the ligand-binding 3a strand (arrow) and the 4-5 loop (arrowhead). Both structures shown are representative for an ensemble of 20 structures consistent with the data.

Figure 2 :

 2 Figure 2: BpML1 binding to thrombin recognition peptide suggests that the protein is involved in protein-protein interactions.(a) Analysis of crystal packing in the X-ray structure of BpML1 (PDB ID: 2Y78) shows a helical peptide binding to the active site. Cartoon is shown of BpML1 (green) and the peptide derived from cloning (orange). (b) The peptide binds with a valine side chain (black arrow) in the heart of the enzyme active site. The peptide is shown in orange sticks, with BpML1 as green cartoon. The grey arrow indicates the direction of the peptide, N to C terminus. (c) The peptide binding is unlikely to represent a substrate for the PPIase activity. Comparison with the structure of a cis-prolyl peptide bound to SlyD from T. thermophilus (left, yellow; PDB ID: 3LUO) and FK-506 bound to hFKBP12 (right, cyan; FK-506 pseudopeptide shown in yellow; PDB ID: 1FKJ) shows that a bona fide cis-proline peptide lying in the opposite orientation (grey arrows); and that a prolyl or pseudoprolyl side chain occupies the active site (black arrowheads). (d) BpML1 peptide binding mimics hFKBP12 binding to TGF RI. Left: BpML1 (green) binding to peptide (white sticks). Right: hFKBP12 (cyan) binding to a helix from TGF RI (pink sticks). The side chains binding to the heart of the active sites are coloured yellow. Note the similarity of the positions of the carbonyls in the red boxes. Six peptide main chain atoms making direct or through single water-mediated hydrogen bonds with BpML1 are shown as spheres on top of the sticks, and are further indicated by black arrowheads.

Figure 3 :

 3 Figure3: The ultra-high resolution X-ray structure of BpML1 (PDB ID: 2Y78) shows multiple conformers of key ligand-binding residues. (a) Asp44 , which accepts a key hydrogen bond from the bound peptide backbone, shows two conformers for the side chain. The distance between the two conformations for O 2 is 0.9 Å. The higher occupancy conformation makes an additional hydrogen bond to the bound peptide backbone, while the other makes two additional hydrogen bonds to other residues of BpML1. (b) Tyr 89 , the only hydrogen bond donor in the active site pocket, also shows a range of conformations. The distance between the O atoms is 1.5 Å. One conformation is consistent with a direct (3.0 Å) hydrogen bond to the peptide, while the other makes a water-mediated hydrogen bond to the peptide. Lower occupancy forms are indicated by a black arrow. (c) Representative electron density for a multiple conformer residue (Tyr 89 , left) and the peptide binding to the active site (right). Colours: BpML1, yellow; binding peptide, orange; multiple conformation at lower occupancy, cyan; mF o -DF c simulated annealing omit map (side chains

Figure 4 :

 4 Figure 4: Cycloheximide N-ethylethanoate identifies a novel binding mode for FKBP inhibitors. (a) Structures of cycloheximide (left) and its derivative cycloheximide N-ethylethanoate. (b) Inhibition of BpML1 by cycloheximide N-ethylethanoate: cycloheximide N-ethylethanoate inhibited the PPIase activity of BpML1 in a dose dependent manner, showing a K I of 6.5 ± 1.0 M. All experiments were performed in triplicate, and standard errors of the mean are shown. The solid line shows the inhibition curve for fit values of the inhibition parameters. (c) Overview of the NMR structure of cycloheximide N-ethylethanoate (orange spheres) bound to BpML1 (PDB ID: 2KO7). Left: BpML1 shown as yellow cartoon. Right: Surface of BpML1 shown, coloured by charge density (red: negative; blue: positive). Charge density was calculated using APBS [49] and PDB2PQR [50]. (d) Close-up view of active site residues binding to cycloheximide N-ethylethanoate. Residues within 3.5 Å of cycloheximide Nethylethanoate are shown as sticks. Left: BpML1 shown as cartoon, cycloheximide Nethylethanoate shown as sticks. The hydrogen bond between the cycloheximide Nethylethanoate hydroxyl and Asp 44 is especially noteworthy; and that some functional groups of cycloheximide N-ethylethanoate (arrowed) do not make any interactions with the protein, and could be altered. Right: BpML1 is shown as semi-transparent surface, cycloheximide Nethylethanoate as spheres. cycloheximide N-ethylethanoate shows good shape complementarity to BpML1, consistent with its micromolar inhibitory activity. (e) Cycloheximide N-ethylethanoate mimics the binding of peptides to FKBPs. Left: cycloheximide N-ethylethanoate (light orange sticks; atoms mimicking the peptide coloured bolder and shown thicker) bound to BpMip (yellow cartoon; PDB ID: 2KO7). Centre: peptide (white sticks; atoms equivalent to those highlighted left shown light grey and thicker) bound to BpMip (green cartoon; PDB ID: 2Y78). Right: helix from TGF RI (pink sticks) binding to hFKBP12 (cyan cartoon; PDB ID: 1B6C). The relevant portions of each peptide are highlighted with black arrowheads, whilst hydrophobic side chains/cycloheximide Nethylethanoate atoms binding to the protein are highlighted with open arrowheads. The black arrow indicates a section of cycloheximide N-ethylethanoate that binds to a BpML1 pocket that is not accessed by the peptides. Note how the cycloheximide N-ethylethanoate molecule binds closer to the 4-5 loop than the peptides.

Figure 5 :

 5 Figure 5: Comparison of the binding of FKBP inhibitors to FKBPs indicates that cycloheximide N-ethylethanoate represents a novel class of inhibitor.Inhibitors are shown as all heavy atom spherical models, with carbon coloured yellow, oxygen red, and nitrogen blue; proteins are shown as white, semi-transparent surfaces, with cartoon beneath. Cartoon colours: BpMip, yellow; FKBP12, cyan. The nine most conserved active site residues of FKBPs are shown as sticks, with carbon coloured as for the main chain, and other atoms as above. Side chains Tyr33 and Phe 57 of BpMip do not contact the inhibitor, and are highlighted with carbon coloured orange and with black arrows; equivalent FKBP12 residues are highlighted with carbon coloured orange and with black arrowheads. (a) BpMip binding to cycloheximide N-ethylethanoate (this work; PDB ID: 2KO7). (b) FKBP12 binding to rapamycin (PDB ID: 2DG3). (c) FKBP12 binding to FK-506 (PDB ID: 1FKJ). (d) FKBP12 binding to the neurotrophic ligand GPI-1046 (PDB ID: 1F40). (e, f, g) FKBP12 binding to synthetic, high affinity ligands (PDB IDs: 1FKG, 1FKI, 1J4R). (h) FKBP12 binding to a synthetic, dimeric ligand (PDB ID: 1A7X). Note that cycloheximide N-ethylethanoate occupies a distinct area of the protein, and in particular makes no interaction with the 3b strand (black dashed arrow).

Biochemical

  Journal Immediate Publication. Published on 16 May 2011 as manuscript BJ20110345 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110345

Table 1 : Data collection and refinement statistics

 1

		BpML1 (PDB ID: 2Y78)
	Data collection
	Space group	P4 3 2 1 2
	Cell dimensions †
	a, b, c (Å)	54.68, 54.68, 119.2
	( )	90, 90, 90
	Resolution (Å)	40.3-0.91 (0.96-0.91)*
	R merge	0.080 (0.398)
	I / I	13.8 (2.2)
	Completeness (%)	98.3 (88.2)
	Redundancy	5.9 (2.7)
	Refinement
	Resolution (Å)	40.3-0.91
	No. reflections	125,244
	R work / R free	0.109/0.116
	No. atoms
	Protein	2032
	Ligand/ion	58
	Water	275
	B-factors
	Protein	12.7
	Ligand/ion	27.2
	Water	24.2
	R.m.s. deviations
	Bond lengths (Å)	0.014
	Bond angles ( )	1.586

† All data were collected from one crystal. High and low resolution passes were merged. *Values in parentheses are for highest-resolution shell. Biochemical Journal Immediate Publication. Published on 16 May 2011 as manuscript BJ20110345 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110345

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Table 2 : NMR and refinement statistics

 2

		Apo-	BpML1-CNE
		BpML1	complex (PDB ID:
		(PDB ID:	2KO7)
		2KE0)
	NMR distance and dihedral
	Constraints
	Structure constraints
	Distance constraints a
	Total meaningful NOE	1792	1757
	Restraints
	Short range (|i -j| <= 1)	777	808
	Medium range (1<|i -j| <5)	216	205
	Long range (|i -j| >= 5 )	799	744
	Hydrogen bond restraints	78	92
	Dihedral angle restraints ( , ) b	134	136
	Total number of constraints	2004	1985
	Constraints per residue	17.1	16.8
	Inter molecular NOE restraints		39
	Residual constraint violations c
	Cyana target function	0.84	1.54
	Distance violations (>0.2 Å)	3	4
	Torsion angles (>5.0 º)	0	0
	Ramachandran statistics (%) d
	Favoured	80.8	80.7
	Additionally allowed	19.0	19.0
	Generously allowed	0.1	0.3
	Disallowed	0.0	0.0
	Average RMSD to the mean (Å) e
	Backbone	0.39±0.05	0.52±0.15
	Heavy atom	0.89±0.08	0.98±0.15
	Ligand heavy atom		1.11±0.14
	a

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2011 The Authors Journal compilation © 2011 Portland Press Limited

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

Acknowledgements

N.H. acknowledges the Diamond Synchrotron (Oxford, U.K.) for X-ray data collection. GV acknowledges the protein expression team at the Seattle Structural Genomics Center for Infectious Disease (SSGCID), and access to facilities at Pacific Northwest National Laboratory (Richland, WA) for NMR data collection.

Funding K.O'S. and N.H. were supported by the University of Exeter. I.N. and M.S-T. were supported by funding from the UK Ministry of Defence. S.Z. and G.V. were supported by the National Institute for Health-National Institute of Allergy and Infectious Disease [Federal Contract HHSN272200700057C]. The protein structures reported in this manuscript have been submitted to the PDB under IDs 2KE0, 2KO7, and 2Y78. Biochemical Journal Immediate Publication. Published on 16 May 2011 as manuscript BJ20110345 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110345

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.