Role of methionine 71 in substrate recognition and structural integrity of bacterial peptidyl-tRNA hydrolase

Accepted Manuscript Role of methionine 71 in substrate recognition and structural integrity of bacterial peptidyl-tRNA hydrolase

Salman Shahid, Ashish Kabra, Surbhi Mundra, Ravi Kant Pal, Sarita Tripathi, Anupam Jain, Ashish Arora PII: DOI: Reference:

S1570-9639(18)30067-0 doi:10.1016/j.bbapap.2018.05.002 BBAPAP 40092

To appear in: Received date: Revised date: Accepted date:

3 January 2018 19 April 2018 3 May 2018

Please cite this article as: Salman Shahid, Ashish Kabra, Surbhi Mundra, Ravi Kant Pal, Sarita Tripathi, Anupam Jain, Ashish Arora , Role of methionine 71 in substrate recognition and structural integrity of bacterial peptidyl-tRNA hydrolase. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Bbapap(2018), doi:10.1016/j.bbapap.2018.05.002

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ACCEPTED MANUSCRIPT Role of Methionine 71 in substrate recognition and structural integrity of bacterial peptidyl-tRNA hydrolase Salman Shahida , Ashish Kabraa , Surbhi Mundraa,c, Ravi Kant Palb, Sarita Tripathia , Anupam Jaina , Ashish Aroraa* a

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Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Lucknow 226031, India. X-ray crystallography facility, National Institute of Immunology, New Delhi - 110067, India.

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Department of Science and Technology, New Delhi - 110016, India.

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* Corresponding author: Ashish Arora, Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Lucknow – 226031, E-mail: [email protected] , Tel: 91 -522-277- 2450-18 ext.4479; Fax: 91-522-2771941.

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ABSTRACT

Background: Bacterial peptidyl-tRNA hydrolase (Pth) is an essential enzyme that alleviates

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tRNA starvation by recycling prematurely dissociated peptidyl-tRNAs. The specificity of Pth for N-blocked-aminoacyl-tRNA has been proposed to be contingent upon conserved residue N14

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forming a hydrogen bond with the carbonyl of the first peptide bond in the substrate. M71 is involved in forming a conserved hydrogen bond with N14. Other interactions facilitating this

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recognition are not known.

Methods: The structure, dynamics, and stability of the M71A mutant of Pth from Vibrio

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cholerae (VcPth) were characterized by X-ray crystallography, NMR spectroscopy, MD simulations and DSC.

Results: Crystal structure of M71A mutant was determined. In the structure, the dimer interface is formed by the insertion of six C-terminal residues of one molecule into the active site of another molecule. The side-chain amide of N14 was hydrogen bonded to the carbonyl of the last peptide bond formed between residues A196 and E197, and also to A71. The CSP profile of mutation was similar to that observed for the N14D mutant. M71A mutation lowered the thermal stability of the protein. 1

ACCEPTED MANUSCRIPT Conclusion: Our results indicate that the interactions of M71 with N14 and H24 play an important role in optimal positioning of their side-chains relative to the peptidyl-tRNA substrate. Overall, these interactions of M71 are important for the activity, stability, and compactness of the protein. Significance: The work presented provides original and new structural and dynamics information that significantly enhances our understanding of the network of interactions that

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govern this enzyme’s activity and selectivity.

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Keywords

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Peptidyl-tRNA hydrolase, NMR spectroscopy, Differential scanning calorimetry, Molecular

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dynamics simulation.

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Abbreviations

Pth, peptidyl-tRNA hydrolase; RRF, ribosome recycling factor; HSQC, heteronuclear single

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quantum coherence; CSP, chemical shift perturbation; DSC, differential scanning calorimetry; MD, molecular dynamics; RMSF, root mean square fluctuation; RMSD, root mean square

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1. Introduction

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deviation.

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Protein translation and related processes are promising targets for the discovery of new antimicrobial agents. Studies have shown that on an average 10% of protein translation aborts prematurely due to ribosome stalling, which leads to accumulation of peptidyl-tRNA inside the cell [1-5]. This accumulation slows down the process of protein synthesis due to scarcity of free tRNA, which is toxic for the cell [6].The release of peptidyl-tRNA from stalled ribosomes is rescued by many factors like ribosome recycling factor (RRF) and elongation factor-G [3-4]. However, unique hydrolyzing activity of an enzyme, peptidyl-tRNA hydrolase (Pth), which hydrolyzes the ester linkage between the C-terminal carboxyl group of the peptide and the 2’- or 3’-hydroxyl of the ribose at the 3’-end of tRNA, is essential for the release of free peptide and 2

ACCEPTED MANUSCRIPT tRNA from peptidyl-tRNA accumulated in the cytoplasm [7-8]. Pth enzyme was first identified in Escherichia coli and later its essentiality was confirmed for Bacillus subtilis, Escherichia coli, and Mycobacterium tuberculosis [9-12]. Many structural, mutational, and biochemical studies have been carried out for bacterial Pths in order to understand its mechanism of action. X-ray crystal structures of Pth from Escherichia

coli,

Mycobacterium

tuberculosis,

Mycobacterium

smegmatis,

Francisella

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tularensis, Burkholderia thailandensis, Pseudomonas aeruginosa, Acinetobacter baumannii,

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Salmonella typhimurium, Streptococcus pyogenes, Staphylococcus aureus and Vibrio cholera

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have been determined [9, 13-23], while NMR solution structures have been solved for Mycobacterium tuberculosis, Mycobacterium smegmatis, and Vibrio cholerae Pth [23-25]. To

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elucidate the interactions in the enzyme-substrate complex, crystal structures of Pth with bound bases, substrate mimics, and an acceptor-TψC fragment of tRNA have been characterized, and

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NMR studies with minimal substrate analog and uncharged fragments of tRNA have been performed [19, 21, 26-29]. The catalytic site of Pth is bifurcated into the peptide binding and

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tRNA binding regions. The peptide binding site is defined by residues N14, N72, and N118 on one side of the catalytic diad of H24 and D97 [9, 26, 30]. On the other side of this, there is a

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clamping region for the 5’-phosphate of the tRNA, which is composed of basic residues K107, K109, and R137 (The numbering is with reference to VcPth sequence). Other residues Y19, F70,

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M71, and H117 form the active site periphery, which may help in proper docking of the peptide region of the substrate in the active site cleft. The active site crevice is surrounded by three loops

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named as base loop (segment G111-H117) forming the base, gate loop (segment L99-V105) that is opposite to the base loop, and lid loop (segment H142-L154), which forms the roof of the

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molecule [23]. The lid loop is the most flexible segment of the protein and it is connected to the rest of the molecule by two hinge residues, H142 and L154, at its two ends. Mutational studies have been performed to elucidate the role of strictly conserved residues corresponding to N14, H24, N72, D97 and N118, and also the residues indirectly involved in the catalysis either by stacking or stabilizing the substrate in the active site groove, i.e. Y19, N25, F70, M71, H117 [9, 30-32]. Schmitt et al. [9] have shown that in EcPth, the N10A, H20A, and D93A mutations reduce the catalytic efficiency by >100 folds, with respect to the wild type (wt) protein. The M67A mutation, on the other hand, reduces the catalytic

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ACCEPTED MANUSCRIPT efficiency by ~15 folds [9]. Goodall et al. [32] have shown that M67E mutation in EcPth reduces the catalytic efficiency by ~130 folds [32]. In order to characterize the role of M71 residue in Pth structure and function, we have solved the crystal structure of the M71A mutant from peptidyl-tRNA hydrolase from Vibrio cholerae (VcPth) and have compared it to the recently determined structure of the wild-type protein [23]. We have also assigned the backbone chemical shifts for this mutant. This has

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allowed us to map the chemical shift perturbations (CSPs), with reference to the wild type

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protein, on to the structure, and to explore the underlying networks that give rise to them. 100 ns

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MD simulation were performed for the M71A mutant structure to analyze the changes in local flexibility of this protein. We have further analyzed the thermal stability of the M71A mutant The results of this study improve our

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using differential scanning calorimetry (DSC).

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understanding about the network of interactions that govern the activity of the Pth enzyme.

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2. Material and methods

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2.1. Mutagenesis and Purification of M71A mutant

The mutant M71A was constructed by site directed mutagenesis by using the pET-NH6

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vector having the wt-vcpth gene. The amplified PCR product having desired mutation was ligated into the pET-NH6 vector. The sequence verified plasmid was transformed into BL21

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(λDE3) cells. The cells were grown in LB media and were induced at a particular cell density with 0.3 mM IPTG, after which they were left to grow overnight. The recombinant protein was

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purified by the two-step Ni-NTA chromatography followed by the gel filtration chromatography as described in Kabra et al. [23]. The purity of the protein was checked on the 15 % SDS-PAGE and by UV absorbance for nucleotide contamination.

2.2. Crystallization, Data collection, and Structure refinement

The recombinant M71A mutant protein was concentrated to varied concentration, and crystallization was set by the hanging drop vapor diffusion method. Crystallization was achieved at a protein concentration of 8 mg/mL, with crystallization conditions exactly matching to that 4

ACCEPTED MANUSCRIPT used for the crystallization of wt-VcPth [23]. Suitable crystals were screened and data were collected with Rigaku FR-E+ SuperBright with a wavelength (λ) of 1.54 Å using R-AXIS IV++ detector. A complete data set was collected for 2.55 Å resolution, and HKL-2000 software was used for indexing, integrating, and scaling the images. The molecular replacement method was done for phase determination, using the wt-VcPth structure (PDB ID: 4ZXP) as a template. Iterative cycles of refinement were carried out using REFMAC5 and COOT, and progress was

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monitored by evaluating the Rfree factor [33-34].

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2.3. Chemical Shift assignment and assessment of chemical shift perturbation for the M71A

Single

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N-labeled and

15

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mutant

N/13 C labeled samples of M71A mutant at concentrations of 0.5 15

N-HSQC was

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mM and 0.8 mM, respectively, were prepared as described earlier [23]. The

recorded at 25 ˚C on a 600 MHz Varian NMR Spectrometer. CSPs were analyzed to check the

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effect of point mutation on each residue with the cutoff value of 0.07 ppm. All shifted peaks were confirmed in HNCACB, HNCACO, HNCA spectra. All peaks in the HSQC were centered

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and peak list was generated. Combined CSP ΔT otal of 1 HN and

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N nuclei were weighted

according to ΔT otal = [[Δ1 H]2 +[0.2×Δ15 N]2 ]1/2 to normalize the larger chemical shift range of

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N

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[35]. All of the peaks that were assigned for the wt-VcPth could also be assigned for the M71A

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mutant.

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2.4. MD simulations

All the water molecules were removed from crystal structure prior to MD simulation, which was performed by using the structure of the B chain of M71A mutant with GROMACS 4.5.6 package and Gromos43a1 force field for 100 nanoseconds (ns) [36]. The protein system was placed in the cubic box of dimension 1.0 nm and filled with SPC water molecules followed by charge neutralization of system with appropriate number of chloride ions. Subsequently, the entire system was subjected to energy minimization. The system was heated from 0K to 300K followed by equilibration for 100 picoseconds (ps) using NVT ensemble and then by using NPT ensemble. Finally, the MD production was carried out for an equilibrated system for 100 ns. 5

ACCEPTED MANUSCRIPT After the simulation, various parameters were computed and examined like total energy, RMSD, the radius of gyration (Rg) and residue-wise root mean square fluctuations (RMSF) profiles. 2.5. Thermal denaturation study

Thermal stability of the M71A mutant was measured with the high precision Microcal

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VP-DSC calorimeter with 0.5 mL cell volume. Before the experiment, the protein was

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exhaustively dialyzed against phosphate buffer (20 mM phosphate buffer, pH 6.5, containing 50

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mM NaCl, 1 mM DTT and 0.1% sodium azide) and its concentration was brought to 0.02 mM. DSC scans were performed from 20 ˚C to 65 ˚C at heating rate of 60 ˚C per hour, using the same

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procedure as described in Kabra et al. [23].

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3. Results

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3.1. Overall structure of the M71A mutant of VcPth

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The crystal structure of M71A mutant was determined by molecular replacement method with the template (4ZXP) in the space group C2221 with two molecules in the asymmetric unit.

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The calculated Matthews coefficient was 2.52 Å3 Da-1 . The secondary structure elements and tertiary fold of M71A mutant are very similar to that of the wt-VcPth structure. The root mean

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square deviation (RMSD) upon superimposition between wt-VcPth and M71A mutant is ~ 0.9 Å, while it is ~ 0.6 Å between chain A and chain B for M71A mutant, for Cα of 192 residues [23].

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The final model comprises of 390 amino acids and 118 water molecules. Electron density for G144-D147 could not be traced in chain A due to high flexibility of helix α4, so these residues were not built in the structure during refinement. The data collection and refinement parameters are given in Table 1. The P17-K19 residues form 310 helix in both the chains, while in the wild type structure it was formed only in the chain A. Structural analysis for the M71A mutant was done with the chain B, since it is complete and better refined. The dimer formed in the M71A C2221 crystal is substantially different from the dimer formed in the wt-VcPth crystal [23]. The two molecules in the unit cell are oriented in a manner such that the C-terminal of one molecule is entering into the active site cleft of the other molecule as shown in Fig. 1-A. A similar 6

ACCEPTED MANUSCRIPT orientation was observed in the EcPth crystal structure, in which the C-terminal of a symmetry related molecule entered into the active site cleft of the other molecule [9]. Therefore, comparison with both wt-VcPth and EcPth structures was performed to distinguish the changes that result from point mutation as opposed to those that result from differences in intermolecular packing. The Ramachandran plot analysis shows 94.0 % residues in the allowed region, but F70 in both the chains falls in the disallowed region, as analyzed with PROCHECK [37]. Significant

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residue wise RMSD for backbone is also observed near the site of mutation for T69 and F70

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(0.68 Å and 0.57 Å), and also for the C and N termini. F70 is present at the sharp turn between

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the sheet β4 and helix α2, and clear density is observed for F70 in both the chains as shown in Fig. 1-B. The dihedral angles (ɸ, ψ) for F70 in wt-VcPth and M71A mutant are -53.52°, 147.67°

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and 72.14°, 147.87°, respectively [23]. Their unusual conformation is stabilized by the hydrogen bonds between main chain O atom of F70 with hydroxyl group (OG1) and N atoms of T69 and

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L73, respectively. The phenyl ring of F70 forms Pi-alkyl hydrophobic interaction with pyrrolidine ring of P15, which is not observed in the wt-VcPth. This directly relates to the

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minimum interatomic distance between P15 and F70, which are 3.3 Å and 6.3 Å in the M71A mutant and wt-VcPth, respectively [23]. However, a similar distance of 3.6 Å between the

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corresponding residues was found in EcPth structure [9]. Insertion of a peptide chain or minisubstrates or small molecules in the putative peptide binding cleft region of the protein leads to a

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significant change in the torsional angles of residue F70. When the peptide binding cleft is filled, the F70 torsional angles fall in the disallowed region of the Ramachandran plot, as is the case for

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AbPth (PDB ID:4JY7; ɸ, ψ are 76.6° and 148.3°), EcPth (PDB ID:2PTH; ɸ, ψ are 92.5°, 147.5°), and M71A mutant (PDB ID:5ZK0; ɸ, ψ are 72.14°, 147.87°). On the other hand, when the site is

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empty, the F70 torsional angles fall in the allowed region of the Ramachandran plot, as is the case for structures of wt-VcPth (PDB ID:4ZXP; ɸ, ψ are -53.52º, 147.67º) and EcPth in complex with CCA-acceptor-TpsiC domain of tRNA (PDB ID:3VJR; ɸ, ψ are -60.1°, 157.3°). Overall, it appears that the conformation of F70 is a fair indicator of whether the peptide-binding site is empty or filled. (Kaushik et al. 2013, Schmitt et al. 1997, Ito et al. 2012) [19,9,26]. The dihedral angles of F70 for N14D, H24N, D97N and N118D mutants were similar to that observed for the wt-VcPth [23]. Upon mutation, 3 and 4 new intrachain salt bridges are observed in chain A and chain B, respectively. The salt bridge between ND1 and OD1 atoms of H142 and D100, respectively, is 7

ACCEPTED MANUSCRIPT formed only in the M71A mutant. A stronger salt bridge is observed between these pairs of residues in the case of MtPth crystal structure in which the histidine residue is substituted with arginine (R139 and D96). It was mentioned in the latter case that this interaction maintained the aspartyl group in the closed position of the gate [14]. The distance between OD1 atom of N14 and N atom of M71 is shortened in the M71A mutant to 2.84 Å from 3.29 Å in the wt-VcPth structure. However, corresponding distance in

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EcPth is 2.80 Å. The position occupied by alanine after mutation is nearly the same as that of the

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methionine in the wt-VcPth structure, as shown in Fig. 1-B, however, most of the interactions

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that involve the side chain of methionine are lost upon mutation. Interestingly, slight change in the orientation of helix α6 (L184-T193) from helix α5 (A159-D182) is observed for M71A

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crystal structure, which leads to formation of new hydrogen bonds with the core structure. The NZ atom of K107 is hydrogen bonded to the main chain O atoms of L191, H192, and F194,

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while these interactions were not observed in the wt-VcPth and other mutant structures. In EcPth interaction between NZ atom of K103 and O atom of H192 was found, but the other two interactions were not present. This may be due to deviation in the χ2 angle of side chain of K107.

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χ2 observed for K107 in wt-VcPth, EcPth (K103), and M71A mutant structures are -173.9˚, -

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171.9˚, and -77.3˚, respectively. The change in K107 χ2 in M71A mutant structure aligns the side chain of K107 towards the helix α6 as shown in Fig. S1 [9, 23]. The change in orientation is

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indicated by the angle formed by Cα for triad M180-181-D182 between the two helices. This angle is 106˚, 102˚, 103˚, 103˚, 105˚, and 106˚ for the wt-VcPth, EcPth, N14D, H24N, D97N and

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N118D mutants, respectively, while it is 99˚ for the M71A mutant [23]. The entry of the C-terminal of chain A into the active site cleft of chain B leads to change

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in the orientation of Y19 and F70. These residues come closer in a way so as to allow the stacking of the substrate in between their aromatic rings. The side chain orientation of Y19 and F70 are shown in Fig. 1-B. F70 interacts with P15, while the main chain of N14 interacts with the side chain of N25. This results in the formation of a new hydrogen bond between ND2 atom of N25 with the main chain O atom of Y19. These interactions possibly bring the aromatic rings of Y19 and F70 closer as compared to other mutant structures. Similar interactions were also found in EcPth structure. This is indicated by the minimum interatomic distance between F70 and Y19, which for the wt-VcPth, N14D, H24N, D97N, and N118D mutants is 8.3 Å, 8.7 Å, 8.4 Å, 8.4 Å, and 8.4 Å, respectively, while it is 3.9 Å for M71A mutant and EcPth [9, 23]. 8

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Table 1 Summary of Diffraction data and Structure Refinement Statistics for M71A mutant of VcPth.

Diffraction Data Space group

C2221 1.5418Å

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Wave length [Å] Cell parameters

36.71,117.50,206.62

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a, b, c [Å] No. of protein molecule per

2

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asymmetric unit Vm [Å3 Da-1 ] a

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Solvent content [%] Resolution range [Å]

No. of unique reflections

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No. of observed reflections

2.5 51.1

50.00-2.55 [2.64-2.55] 68284 14447 [1185] 4.7 [2.1]

Average I/ σ [I]

26.9 [4.6]

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Rmerge

97.3 [81.7]

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Completeness [%]

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Average redundancy

0.061[0.206]

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Refinement and Structural Model

19.2

Free R factor [%]

25.7

No. of reflection [Fo≥0σ [Fo]] R factor [%]

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c

No. of atoms Overall

3064

Protein

2944

Water

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Average B factor [Å2 ] Overall

38.6 9

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37.3

Side chain

41.0

Water

33.0

94.0

Allowed

6.0

Disallowed

0.0

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Favored

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Ramachandran plot [%]

PDB ID

5ZK0

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a Numbers in parentheses represent the highest-resolution shell b Rmerge = ∑hkl∑i|Ii[hkl]i−|/∑hkl∑iIi[hkl]

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c R = ∑hkl ||Fo|−|Fc||/∑hkl|Fo|

Fig. 1. Crystal structure of peptidyl-tRNA hydrolase M71A mutant from Vibrio cholerae (PDB ID:5ZK0): [A] Cartoon representation of M71A mutant crystal structure showing the C-terminal 10

ACCEPTED MANUSCRIPT of chain A (blue) entering into the active site cleft of chain B (orange) in the crystallographic unit cell. Superimposition of wt-VcPth and M71A mutant structure: [B] The stick model showing orientation of active site residues including residues Y19 and F70. M71A mutant structure is depicted by yellow sticks. The wt-VcPth structure is depicted by blue sticks, and 2Fo-Fc electron density map of the M71A mutant contoured at 2σ is shown in yellow colour.

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3.2. Crystal packing and Intermolecular interactions

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The packing of the molecules in the asymmetric unit in the structure of M71A mutant is different with respect to the wt-VcPth crystal structure. The solvation energy of the polypeptide

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chains for the mutant structure is reduced by 2.2 kCal/mol, approximately, upon complex formation, as analyzed by the PDBePISA server. The residues involved in the formation of the

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interface in the wt-VcPth crystal are nearly same for the two chains as described in Kabra et al. [23], while in the case of M71A mutant structure the interface involves different sets of residues

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for the chains A and B. In the M71A mutant structure, chain A residues N14, P15, E18, Y19, H24, P46, T68-L73, K76, N118, V149, A150, V153, L154 are involved in interfacial interaction.

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While for chain B, the interfacial residues are L101-V105, K107-K109, R137, H142-G144, C164, and H192-E197. This indicates that the interface is formed between the active site of chain

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A and the C-terminal of chain B. The intermolecular bonding network for the M71A mutant is completely different from wt-VcPth. In the M71A mutant structure, the interface has 8 hydrogen

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bonds, 2 salt bridges and 6 hydrophobic interactions, as given in Table S1, and shown in Fig. S2. The three hydrogen bonds that stabilize the interface are formed by strictly conserved residues

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involved in the enzyme catalysis, i.e., N and ND2 atoms of N72 with the O atoms of K195 and E197, respectively. Likewise, ND2 atom of N14 forms hydrogen bond with the O atom of A196. Electrostatic interactions are observed between NH1 and OE2 of R137 and E18. π-anion interaction is observed between H117 and OE2 of E197.

3.3. Chemical shift perturbation (CSPs) of M71A mutant

For the M71A mutant, NMR backbone assignments show very high CSP for the A71 amide NH resonance, which could be due to change in the residue, change in conformation, 11

ACCEPTED MANUSCRIPT change in hydrogen bonding, or a combination of all three. Overall, CSPs were mainly clustered in four regions: loop before 310 -1 helix (G11-N14), loop before helix α1 (R23-A26), loop before helix α2 to helix α2 (T68-A77) and base loop to helix α3 (G116-D122), as shown in Fig. 2. The first cluster G11-N14 results from perturbation of N14, which directly interacts with M71 in the wild-type protein. M71 also has hydrophobic interactions with H24 in the wild-type protein which accounts for perturbations in the second cluster R23-A26. The third cluster (T68-A77)

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results from primary and secondary mutation effects, change in the orientation of N72 side chain

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and change in conformation or hydrogen bonding. The fourth cluster (G116-D122) results from

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alterations in the interaction of H24 and H117. Beside these clustered regions, residues I6, K47, R54, D97, L99, V105, T123, S125-K126, E132, L138, D147 and V153-L154 also showed low to

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moderate CSPs. The dynamics of the N118 amide vector remained in the intermediate range on the NMR time scale after M71A mutation as no amide cross peak for N118 was observed in 1 HN HSQC spectrum. The range of CSP amplitudes for the M71A mutant was similar to that

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observed for the N14D mutant [23].

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ACCEPTED MANUSCRIPT Fig. 2. CSP analysis for M71A mutant of VcPth. [A] Overlapped 1 H- 15 N HSQC spectra of wtVcPth with M71A mutant. Red peaks represent the wt-VcPth and blue peaks represent the M71A mutant. Some residues, which show significant CSPs are labeled. [B] Residues showing CSP > 0.1 ppm are mapped on to the VcPth solution struct ure in blue color and are labeled. [C] CSP analysis as a function of residue number for M71A mutant. Residues showing CSPs above the

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cutoff of 0.07 ppm are labeled.

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3.4. Molecular dynamics (MD) simulation of M71A mutant in comparison to wild type

Molecular dynamics (MD) is a tool to evaluate the atomic and molecular movement over

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a given period. The difference between initial structural conformations of the protein backbone to the final state is calculated by the root mean square deviation (RMSD). The stability of the

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protein during its simulation can be determined by its deviation from initial conformation. Smaller deviation implies more stable nature of the protein. RMSD value for Cα backbone was

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calculated over 100 ns simulation for M71A mutant and wt-VcPth to check the stability of both the protein systems [23]. The wild type system equilibrated within 20 ns of simulation. Beyond

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20 ns, the average deviation was approximately 2.5 Å, till the end of simulation. While in the case of mutant, the equilibration was attained at around 10 ns, beyond which the average

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deviation was approximately 3.0 Å, as shown in Fig. 3-A [23]. The residue-specific root mean square fluctuations (RMSF) for backbone were calculated for determining the level of flexibility.

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Fig. 3-B shows residue wise RMSF plot for the duration of 70-90 ns. The plot indicates higher flexibility for the residues in the 310 -1 helix (P17-K19) and the loop region between α1 and β2. A

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significant fluctuation is also observed for the residues present in the region from the loop before α2 to β5 (including the site of mutation, A71) and residues surrounding the helix α3.

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Fig. 3. Overlay of RMSD and RMSF graphs for the backbones of wt-VcPth and M71A mutant systems: [A] Plot of RMSD for molecular dynamics simulation (100 ns) for wild type (red) and

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M71A mutant (black) showing increase in RMSD value with deviations for the mutant stabilizing earlier in comparison to the wild type protein. [B] Plot of residue specific RMSF for

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mutation, A71.

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wild type and M71A mutant, over the 70-90 ns duration of simulation showing the site of

3.5. Thermal stability of M71A mutant

The thermal stability of M71A was investigated in phosphate buffer, pH 6.5, by differential scanning calorimetry (DSC). Thermograms of M71A mutant show irreversible two state thermal unfolding pathway. The crystal structure of M71A shows overall fold similar to the wild type structure, but changes in the internal interaction network can influence the thermal stability, which is reflected from the melting temperature (Tm) of the mutant. The calorimetric 14

ACCEPTED MANUSCRIPT profiles for M71A and wild type protein show a single peak with the Tm values of 46.81˚C and 52.08˚C, respectively, as shown in Fig. 4 [23]. The thermal stability of M71A mutant is comparable to that of the N14D mutant of the VcPth (Tm = 46.18 ˚C) [23]. The ΔG change (ΔΔG) predicted by the I-Mutant 2.0 server [38] for M71A mutant protein, is qualitatively comparable with the thermal stability change monitored by the DSC, with respect to the wild

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type protein.

Fig. 4. Differential scanning calorimetry (DSC) thermograms overlay for wt-VcPth (red) and

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M71A mutant (black) showing decrease in melting temperature (Tm) for M71A mutant (46.81˚C)

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in comparison to wt-VcPth (52.08˚C).

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4. Discussion

Pth performs the essential function of hydrolyzing the peptidyl-tRNAs released in the

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cytoplasm because of premature termination of translation. Hydrolysis of the substrate is achieved by the coordinated action of highly conserved residues N14, H24, D97, and N118 [9, 23, 26-27, 29, 32]. Based on the structural characterization of Pth in complex with tRNA fragment, substrate mimic and small bases, the mechanism of its action has been postulated as follows. When Pth binds to its peptidyl-tRNA substrate, the free 5' phosphate and acceptor stem of t-RNA are held by a basic patch formed by residues K107, K109, and R137, and residues G119 and D100, and the CCA is held in place by K145. The carbonyl of the scissile ester bond is hydrogen bonded to the side chains of N72 and N118. A conserved hydrogen bond between D97 15

ACCEPTED MANUSCRIPT and H24 stabilizes the anionic form of the latter, which then extracts a proton from a proximal hydrogen bonded water molecule. The resulting hydroxyl attacks the carbon of the scissile ester. The intermediate tetrahedral state is stabilized by N72 and N118 [26, 29], and its decomposition by general acid hydrolysis completes the hydrolysis reaction. The residues N14, N72, N118, H24, and D97, together form a pocket and in the crystal structure of EcPth, which was the first Pth structure to be determined, this pocket was occupied by the C-end of a neighboring molecule

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[9]. One of the packing contacts involved the interaction of the N14 (N10 in EcPth) residue with

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the main chain carbonyl of the last peptidic bond of the other molecule. Since this bond of the

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substrate is absolutely required for Pth enzyme action, it was proposed that N14 is the residue responsible for discrimination between the N-protected amino-acyl-tRNA and free aminoacyl-

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tRNA. This was later confirmed by biochemical studies wherein it was shown that along with loss in activity, the discrimination between diacetyl-Lys-tRNAlys and Lys-tRNAlys was lowered

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by a factor of 450 for the N10D mutant of EcPth [28]. In order to understand the network of interactions that ensure this discrimination, we recently characterized the structure, dynamics,

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and stability of wild type VcPth and mutants of its N14, H24, D97, and N118 residues, by X-ray crystallography, NMR spectroscopy, MD simulations and DSC [23]. We observed that N118 and

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N14 were involved in orthogonal competing interactions with H24, both of which reduced the nucleophilicity of the latter residue. The effects of both of the former residues were likely to be

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offset by positioning of a peptidyl-tRNA substrate, which would result in hydrolysis. While, binding of an aminoacyl-tRNA would only offset the effect of N118, which would drive H24

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towards N14, as a result of which no hydrolysis would take place. In the course of structural characterization of the N14D mutant of VcPth, we observed

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that in its crystal structure, the side chain of D14 had moved away from M71 after the mutation, with reference to the wild type structure, which lead to loss of the conserved hydrogen bond between OD1 of N14 and N of M71. The loss of this hydrogen bond was also apparent in the solution state as indicated by very high CSP that were observed for the M71 NH cross peak in the HSQC spectrum of the N14D mutant. Further, in our previous studies, we noted that the H24 ring was held in place by formation of a salt bridge with D97, by orthogonal interactions with N14 and N118, and also by hydrophobic interactions with V153 and M71. Given the interaction of M71 with H24 and N14, and loss of activity upon its mutation to alanine, we found it interesting to characterize the affect of M71 residue on the structure, dynamics, and stability of 16

ACCEPTED MANUSCRIPT Pth, by following a strategy of using X-ray crystallography, NMR spectroscopy, MD simulations and DSC, as was reported in our earlier study [23]. To our surprise, the M71A mutant did not crystallize in the dimeric form observed for the wild-type and all other mutants. Rather, the dimer interface involved the active site of one molecule into which the C-terminal region of the other molecule was inserted, much like the EcPth crystal structure [Fig. 1-A]. However, unlike EcPth where there is only one molecule in

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the asymmetric unit and the inserted chain comes from a symmetry related molecule, in the case

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of M71A mutant, both the chains are within the same asymmetric unit. In spite of the mutation of

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the highly conserved residue M71, most of the intermolecular structural interactions observed for EcPth are retained in the structure of M71A mutant. Importantly, the hydrogen bond between

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N14 of chain B and carbonyl of the penultimate residue (A176) of chain A is retained. This interaction has been hypothesized as the basis for discrimination between N-protected

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aminoacyl- and free aminoacyl-tRNA. Correspondingly, stronger hydrogen bonds are observed for M71A mutant and EcPth, in comparison to wt-VcPth, between the OD1 of N14 and NH of

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residue M/A71. However, loss of the side chain of methionine does lead to changes in the chemical environment for the N14 residue. It is observed that the peak of N14 becomes more

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distinct in the HSQC spectrum of the M71A mutant [Fig. 2-A], while in the wt-VcPth, it shows overlap with the peak for residue K160 [23]. The CSP amplitudes and its pattern obtained for the

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M71A mutant are very similar to that of N14D mutant [23]. Unlike the N14D mutant, in which case the breaking of the D14-M71 hydrogen bond was clearly indicated both by X-ray crystal

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structure and NMR CSP analyses, the picture is more complicated for the N14-A71 pair. In this case, the NH is coming from the residue that has been mutated and the high CSP (> 1 ppm)

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observed could be due to change in the residue, change in conformation, change in hydrogen bonding, or a combination of all of the three. Because of the differences in crystal packing between the crystal structures of wt-VcPth and its M71A mutant, these distances cannot be directly compared to unambiguously interpret the strengthening of this hydrogen bond upon mutation of M71 to alanine. The χ1 angle of N72 has changed from 64.16˚ in wt-VcPth to -51.24˚ in the M71A mutant. This change in χ1 angle allows N72 to move in the direction of the catalytic histidine as shown in Fig. 5-A. This change in χ1 angle leads to the formation of hydrogen bond between antepenultimate O atom of K195 and N atom of N72 in M71A mutant structure. This change is a 17

ACCEPTED MANUSCRIPT consequence of peptide chain insertion and similar orientation of N72 side chain is observed for EcPth. The side chain of residue N118 moves away from H24 with a change in its χ2 angle from -24.81º in wild-type to -54.24º in M71A mutant. It is relevant to mention that the cross-peak for N118 amide NH group is not observed in the HSQC spectrum of M71A mutant. Generally, in the absence of cross-peak, it is not possible to comment upon the dynamic nature of the concerned residue. However, in the case of VcPth, we have systematically analysed the dynamics of N118

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amide NH group [23]. Specific NMR assignment have shown that the amide cross-peak for

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residue 118 was observed for H24N, N72D and N118D mutants, while this cross-peak is not

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observed in the HSQC spectrum of wt-VcPth, indicating that in the case of the latter the amide vector is undergoing motions in the intermediate range on the NMR time scale. For the N14D

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and M71A mutants also this cross-peak is not observed, indicating once again that intermediate range NMR time scale motions are maintained for the N118 amide vector upon these mutations.

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An interesting feature is observed for residue K107, which facilitates the clamping of 5’ phosphate of the substrate. In the structure of M71A mutant, the NZ atom of this residue forms a hydrogen bond with the main chain O atoms of L191, H192, and F194. This is brought about by

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a change in the χ2 angle of lysine chain by approximately 100˚ and a concomitant rotation of

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helix α6 along the axis defined by the residues K186-A187-Q188, as shown in Fig. S1. This interaction is also facilitated by the change in inclination of helix α6 with reference to the helix

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α5 along the axis defined by the residues M180-K181-D182, as shown in Fig. 6. Another potentially important feature that is observed is for the aromatic ring of H24.

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This ring is tilted in the M71A mutant in comparison to the wt-VcPth structure, with reference to the plane of the H117 ring, as shown in Fig. 5-B. This is clearly indicated in the χ2 angles of

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H24, which are 20.78º, 20.98º, and 46.84º in the crystal structures of wt-VcPth, EcPth, and M71A mutant, respectively. The hydrophobic interactions, observed in the wild type structure, between H24 and M71 side-chains may help in optimum relative positioning of the scissile bond of the substrate and the histidine side chain. This may account for the loss in activity upon mutation of M71 to alanine.

18

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[B]

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[A]

Fig. 5. [A]Side chain orientation of active site residues for wt-VcPth (yellow) and M71A mutant

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(blue). The change in χ1 angle allows N72 to move in the direction of the catalytic histidine. [B]

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Change in orientation of the aromatic ring of H24 with reference to the plane of H117 ring.

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Fig. 6. Effect of point mutation M71A on the relative orientation of the C-terminal helices; [A]

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Cartoon representation of wt-VcPth (green) and M71A mutant (pink), showing the inclination of α6 from core structure. The triad M180-K181-A182 is shown by sticks, as labeled. [B] Helices α5 and α6, depicting the change in orientation, are shown as blue and red cylinders, respectively. [C] Stick representation of residues M180, K181 and D182 in the wt-VcPth (top) and M71A mutant (bottom) structures showing the inclination angle between the Cα atoms.

20

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The molecular dynamics (MD) simulations and DSC were used to characterize the effect of point mutation on the stability of the protein. The simulations for M71A mutant show that it reaches the equilibration state earlier (10 ns) than the wild type structure (20 ns), but with higher deviation. The higher flexibility of five regions in the mutant with respect to the wt-VcPth is related to the loss of hydrophobic interaction between the side chain of M71 and H24 ring upon

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mutation. The loss of these interactions reduces stability of M71A mutant in comparison to wt-

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VcPth, as indicated by the DSC studies.

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5. Conclusion

Pth serves an essential role by recycling in the cytoplasm the peptidyl-tRNAs that are

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prematurely dissociated from the ribosomes. The active site of Pth is comprised of conserved residues N14, H24, N72, D97, and N118, which together form a crevice. Residue M71 closely

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interacts with N14 and H24 and its role in enzyme activity is indicated by a loss in catalytic efficiency of 15-130 folds upon its mutation. We have characterized the structure, dynamics, and

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stability of the M71A mutant of VcPth. Our results strengthen the postulate that the interaction between M71 and N14 plays an important role in positioning of the side-chain amide of N14 in

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an orientation that could facilitate hydrogen bonding with the carbonyl of the first amide bond from the scissile ester group of peptidyl-tRNA substrate, while the M71 and H24 interactions

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help in optimal relative positioning of the scissile group and the histidine ring. Overall, these

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interactions of M71 are important for the activity, stability, and compactness of the protein.

5.1. Protein Data Bank accession code The coordinate file of the final structure of VcPth mutant M71A has been deposited in the Protein Data Bank with accession code 5ZK0.

Acknowledgement

We are thankful to the X-ray diffraction facility of National Institute of Immunology (NII), New Delhi, India, for X-ray data collection. This work was supported by grants from CSIR Network 21

ACCEPTED MANUSCRIPT Projects

BSC0113

and

BSC0104,

and

Department

of

Biotechnology

(DBT),

BT/PR12991/BRB/10/748/2009, New Delhi, India. S. S., A. K. and S. M. are recipients of research fellowship from University Grants Commission (UGC), Indian Council of Medical Research (ICMR), Department of Science and Technology (DST), New Delhi, India, respectively.

S.M. is also thankful to DST for her INSPIRE fellowship. This has the

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communication number 9xxx from CSIR-CDRI.

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Highlights Peptidyl-tRNA hydrolase selectively hydrolyzes only N-blocked aminoacyl-tRNAs.



The active site residues of Pth are N14, H24, N72, D97 and N118.



Residue M71 is highly conserved and has important interactions with N14 and H24.



The structure, dynamics and stability of M71A was characterized.



M71 is important for the activity, stability, and compactness of the protein.

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

27