Cα Atoms Research Articles

Representations of protein structure for exploring the conformational space: A speed–accuracy trade-off

The recent breakthrough in the field of protein structure prediction shows the relevance of using knowledge-based based scoring functions in combination with a low-resolution 3D representation of protein macromolecules. The choice of not using all atoms is barely supported by any data in the literature, and is mostly motivated by empirical and practical reasons, such as the computational cost of assessing the numerous folds of the protein conformational space. Here, we present a comprehensive study, carried on a large and balanced benchmark of predicted protein structures, to see how different types of structural representations rank in either accuracy or calculation speed, and which ones offer the best compromise between these two criteria. We tested ten representations, including low-resolution, high-resolution, and coarse-grained approaches. We also investigated the generalization of the findings to other formalisms than the widely-used “potential of mean force” (PMF) method. Thus, we observed that representing protein structures by their β carbons—combined or not with Cα—provides the best speed–accuracy trade-off, when using a “total information gain” scoring function. For statistical PMFs, using MARTINI backbone and side-chains beads is the best option. Finally, we also demonstrated the necessity of training the reference state on all atom types, and of including the Cα atoms of glycine residues, in a Cβ-based representation.

How Pure and Hydrated Reline Deep Eutectic Solvents Affect the Conformation and Stability of Lysozyme: Insights from Atomistic Molecular Dynamics Simulations.

Factors governing the stability and activity of proteins and enzymes in nonaqueous solvents have just been started to be explored. Because of their benign and economically viable nature, deep eutectic solvents (DESs) are being seen as an alternative media in many biotransformation processes. The present study exploits the changes in the conformation and stability of hen egg white lysozyme (HEWL) in the presence of reline (a eutectic mixture of choline chloride and urea) and reline/water mixtures using atomistic molecular dynamics simulations. The lysozyme structure was found to be partially folded in both reline and reline/water mixtures. Root-mean-square deviation (RMSD) of the positions of Cα atoms of lysozyme indicate that 50/50 reline/water solvent induces more destabilization in the conformation of HEWL than that by pure reline and 75/25 reline/water mixture. From the root-mean-square fluctuation (RMSF) analysis, it is found that the lysozyme active site (Glu35-Asp52) is quite stable in the presence of pure reline but it is least stable in the presence of 50/50 reline/water mixture. Our results show that the secondary structure of the lysozyme is significantly affected in the presence of reline. Our further analysis reveals that the hydrogen bonding interaction between HEWL-[Ch]+ dominates over HEWL-urea and HEWL-Cl- in pure reline than in reline/water mixtures.

Protein secondary structure motifs: A kinematic construction.

The kinematic geometry of protein backbone structures, constrained by either single or multiple hydrogen bonds (H-bonds), possibly in a periodic array, is discussed. These structures include regular secondary structure elements α-helices and β-sheets but also include other short H-bond stabilized irregular structural elements like β-turns. The work here shows that the variations observed in such structures have simple geometrical correlations consistent with constrained motion kinematics. A new classification of the ideal helices is given, in terms of the parameter α, the angle at a Cα atom to its two neighboring Cα 's along the helix, and shown how it can be generalized to include nonideal helices. Specifically, we derive an analytical expression of the backbone dihedrals, (ϕ, ψ), in terms of the parameter α subject to the constraint that the peptide planes are parallel to the helical axis. Helices constructed in this way exhibit near-vertical alignment of the C = O and N - H units and are the canonical objects of this study. These expressions are easily modifiable to include perturbations of parameters relevant to nonplanar peptide units and noncanonical angles. The addition of a second parameter, ε0 , inclination of successive peptide planes along a helix with respect to the helical axis leads to a generalization of the previous expression and provides an efficient parametrization of such structures in terms of coordinates consistent with H-bond parameters. An analogs parametrization of β-turns, using inverse kinematic methods, is also given. Besides offering a unifying viewpoint, our results may find useful applications to protein and peptide design.

Virtual screening of phytoconstituents from miracle herb nigella sativa targeting nucleocapsid protein and papain-like protease of SARS-CoV-2 for COVID-19 treatment

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel etiological agent of coronavirus disease 2019 (COVID-19). Nigella sativa, commonly known as black seed or black cumin, has been a historical and traditional plant since thousands of years. Based on their therapeutic efficacy, the chief components of terpenoids and flavonoids were selected from N. sativa seeds and seed oil. This study was designed to check the antiviral efficacy of N. sativa main phytoconstituents against five potential targets of SARS-CoV-2 using in silico structure-based virtual screening approach. Out of twenty five phytocomponents, ten components showed best binding affinity against two viral proteins viz. N-terminal RNA binding domain (NRBD; PDB ID: 6M3M) of nucleocapsid protein and papain-like protease (PL-PRO; PDB ID: 6W9C) of SARS-CoV-2 using AutoDock 4.2.6, AutoDock Vina and iGEMDOCK. PASS analyses of all ten phytocomponents using Lipinski’s Rule of five showed promising results. Further, druglikeness and toxicity assessment using OSIRIS Data Warrior v5.2.1 software exhibited the feasibility of phytocomponents as drug candidates with no predicted toxicity. Molecular dynamics simulation study of NRBD of SARS-CoV-2 nucleocapsid protein-alpha-spinasterol complex and PL-PRO-cycloeucalenol complex displayed strong stability at 300 K. Both these complexes exhibited constant root mean square deviation (RMSDs) of protein side chains and Cα atoms throughout the simulation run time. Interestingly, PL-PRO and NRBD are key proteins in viral replication, host cell immune evasion and viral assembly. Thus, NRBD and PL-PRO have the potential to serve as therapeutic targets for N. sativa phytoconstituents in drug discovery process against COVID-19.

Exploring nature’s bounty: identification of Withania somnifera as a promising source of therapeutic agents against COVID-19 by virtual screening and in silico evaluation

Coronaviruses are etiological agents of extreme human and animal infection resulting in abnormalities primarily in the respiratory tract. Presently, there is no defined COVID-19 intervention and clinical trials of prospective therapeutic agents are still in the nascent stage. Withania somnifera (L.) Dunal (WS), is an important medicinal plant in Ayurveda. The present study aimed to evaluate the antiviral potential of selected WS phytoconstituents against the novel SARS-CoV-2 target proteins and human ACE2 receptor using in silico methods. Most of the phytoconstituents displayed good absorption and transport kinetics and were also found to display no associated mutagenic or adverse effect(s). Molecular docking analyses revealed that most of the WS phytoconstituents exhibited potent binding to human ACE2 receptor, SAR-CoV and SARS-CoV-2 spike glycoproteins as well as the two main SARS-CoV-2 proteases. Most of the phytoconstituents were predicted to undergo Phase-I metabolism prior to excretion. All phytoconstituents had favorable bioactivity scores with respect to various receptor proteins and target enzymes. SAR analysis revealed that the number of oxygen atoms in the withanolide backbone and structural rearrangements were crucial for effective binding. Molecular simulation analyses of SARS-CoV-2 spike protein and papain-like protease with Withanolides A and B, respectively, displayed a stability profile at 300 K and constant RMSDs of protein side chains and Cα atoms throughout the simulation run time. In a nutshell, WS phytoconstituents warrant further investigations in vitro and in vivo to unravel their molecular mechanism(s) and modes of action for their future development as novel antiviral agents against COVID-19.

Exploring the intrinsic dynamics of SARS-CoV-2, SARS-CoV and MERS-CoV spike glycoprotein through normal mode analysis using anisotropic network model

COVID-19 caused by SARS-CoV-2 have become a global pandemic with serious rate of fatalities. SARS-CoV and MERS-CoV have also caused serious outbreak previously but the intensity was much lower than the ongoing SARS-CoV-2. The main infectivity factor of all the three viruses is the spike glycoprotein. In this study we have examined the intrinsic dynamics of the prefusion, lying state of trimeric S protein of these viruses through Normal Mode Analysis using Anisotropic Network Model. The dynamic modes of the S proteins of the aforementioned viruses were compared by root mean square inner product (RMSIP), spectral overlap and cosine correlation matrix. S proteins show homogenous correlated or anticorrelated motions among their domains but direction of Cα atom among the spike proteins show less similarity. SARS-CoV-2 spike shows high vertically upward motion of the receptor binding motif implying its propensity for binding with the receptor even in the lying state. MERS-CoV spike shows unique dynamical motion compared to the other two S protein indicated by low RMSIP, spectral overlap and cosine correlation value. This study will guide in developing common potential inhibitor molecules against closed state of spike protein of these viruses to prevent conformational switching from lying to standing state.

A comparative study of structural and conformational properties of WNK kinase isoforms bound to an inhibitor: insights from molecular dynamic simulations

The with-no-lysine (WNK) kinase causes pseudohypoaldosteronism type II, a genetic form of hypertension. Due to ∼80% similarity among four isoforms (WNK1/2/3/4) of the WNK protein family, the discovery of an ATP-competitive inhibitor renders a significant challenge. Here, we combined molecular modeling and molecular dynamics simulations to study the structural and conformational properties of the WNK kinase isoforms bound to an ATP competitive inhibitor (WNK463). We have also investigated the effect of phosphorylation on the conformational properties of each isoform. The largest deviation of Cα atoms is observed for the unphosphorylated uWNK4 complex, while the least deviation is obtained for uWNK3. The G-loop and αC-helix regions are also more flexible in uWNK4 compared to the other three unphosphorylated isoforms. However, in uWNK1, the A-loop region is the most flexible compared to other complexes. In all cases, phosphorylation stabilizes different regions of the protein–inhibitor complexes. In the case of uWNK4, relatively higher anti-correlated motions are observed compared to the other three unphosphorylated complexes. Furthermore, in the case of uWNK4, the distance between N- and C-lobes is found to be slightly higher than other complexes. This distance is reduced in all four complexes after the phosphorylation. Principal component analyses suggest that the phosphorylation leads to structural stabilization in WNK1 and WNK4, while it causes more flexibility in WNK2 and WNK3. Overall, our study provides comprehensive and comparative information on the structural dynamics of the WNK isoform family with the known competitive inhibitor that would aid in the development of a new inhibitor. Communicated by Ramaswamy H. Sarma

Combination of C21 and ARBs with rhACE2 as a therapeutic protocol: A new promising approach for treating ARDS in patients with coronavirus infection.

Background: Coronavirus disease 2019 (COVID-19) is caused by a new severe acute respiratory syndrome Coronavirus. COVID-19 patients are at risk for acute respiratory distress syndrome and death from respiratory failure. Methods: In this study the complete genome of the SARS-CoV-2 reference sequence, geologically isolated types, and Coronavirus related to human diseases were compared by the Molecular Phylogenetic Maximum Likelihood method. The secondary and tertiary structures of the main protease of SARS-CoV were defined as the most similar viruses to SARS-CoV-2, aligned with chimera software. Therefore, considering ineffective antiviral medications used for SARS-CoV and the importance of preventing acute respiratory distress syndrome as the main cause of mortality, 2 strategies were adopted to acquire the most effective drug combination. Results: The results of phylogenic analysis showed that SARS-CoV is the most similar virus to SARS-CoV-2. The secondary structure and superimposing of tertiary structure did not show a significant difference between SARS and SARS-CoV-2 3C-like main protease and the root means square deviation between Cα atoms did not support the difference between the 2 protein structures. Thus, these 2 mechanisms were fostered in accordance with the correlation between acute respiratory distress syndrome-related Coronavirus, angiotensin-converting enzyme 2 on one side and the possible treatments for reducing the respiratory side effects on the other. The analysis of renin-angiotensin system as well as the tested drugs applied to acute respiratory distress syndrome cases, indicated that angiotensin II receptor blockers, angiotensin-converting enzyme inhibitors, and C21 as nonpeptide agonist might possess a promising modality of treatment for acute respiratory distress syndrome. Furthermore, implementing recombinant human ACE2 as a competitive receptor might be an effective way to trap and chelate the SARS-CoV-2 particles. Conclusion: The data suggest that combination therapy of angiotensin II receptor blockers and C21 could be a potential pharmacologic regimen to control and reduce acute respiratory distress syndrome. Moreover, rhACE2 can be recommended as an effective protective antiviral therapy in the treatment of COVID-19 and its complications.

Molecular dynamics simulations of two GH74 endo-processive xyloglucanases and the mutated variants to understand better the mechanism of the enzyme action

BackgroundGH74 xyloglucanases are composed of two separate domains connected by two unstructured peptides. Previously, a hypothesis was made that the movement of domains may affect the enzyme mechanism of catalysis. MethodsThe molecular dynamics (MD) simulations of endo-processive xyloglucanases from Paenibacillus odorifer (PoGH74cat) and Myceliophthora thermophila (MtXeg74A) were carried out. ResultsMD simulations for both enzymes in complex with XXLG and XGXXLG oligosaccharides confirmed the possibility of domain movement. In the case of MtXeg74A, changes in the distances between Cα atoms of aromatic residues involved in xyloglucan binding in −3 and +3 subsites of the active site cleft and those of selected residues on the opposite side of the cleft reached values up to 10–12 Å. For PoGH74cat the conformational changes were less pronounced. In MtXeg74A variants, the deletion of loop 1, which partially closes the entrance to the cleft, and the additional double mutation of two Trp residues in +3 and +5 subsites caused the enhanced mobility of the XGXXLG and also induced changes in topography of the cleft. ConclusionsThese findings demonstrate the possibility of existence of GH74 xyloglucanases in a more open and more closed enzyme conformation. The enzyme in an open conformation may more easily accommodate the branched polysaccharide, while its transition to the closed conformation, together with loop 1 function, should aid processivity. General significanceOur results provide an insight into a mechanism of action of GH74 xyloglucanases and may be useful for discussing the catalytic mechanisms of glycoside hydrolases from other families.

Discovery of Evodiamine Derivatives as Highly Selective PDE5 Inhibitors Targeting a Unique Allosteric Pocket.

Clinical use of phosphodiesterase-5 (PDE5) inhibitors is limited by several side effects due to weak isoform selectivity. Herein, a unique allosteric pocket of PDE5 is identified by molecular modeling and structural biology, which enables the discovery of highly selective PDE5 inhibitors from natural product evodiamine (EVO). The crystal structure of PDE5 with bound EVO derivative (S)-7e revealed that binding of (S)-7e to the novel allosteric pocket induced dramatic conformation changes in the H-loop with a maximum 24 Å movement of their Cα atoms. This movement directly blocks the binding of substrate/inhibitors to the PDE5 active site, which is different from all traditional PDE5 inhibitors such as sildenafil, tadalafil, and vardenafil. These derivatives showed >570-fold selectivity over PDE6C and PDE11A and achieved potent efficacy for the effective treatment of pulmonary hypertension in vivo.

Tightening the Crosslinking Distance Restraints for Better Resolution of Protein Structure and Dynamics

Chemical crosslinking coupled with mass spectrometry (CXMS) has been increasingly used in structural biology. CXMS distance restraints are usually applied to Cα or Cβ atoms of the crosslinked residues, with upper bounds typically over 20Å. The incorporation of loose CXMS restraints only marginally improves the resolution of the calculated structures. Here, we present a revised format of CXMS distance restraints, which works by first modifying the crosslinked residue with a rigid extension derived from the crosslinker. With the flexible side chain explicitly represented, the reformatted restraint can be applied to the modification group instead, with an upper bound of 6Å or less. The short distance restraint can be represented and back-calculated simply with a straight line. The use of tighter restraints not only afford better-resolved structures but also uncover protein dynamics. Together, our approach enables more information extracted from the CXMS data.

Role of Thermolysin in Catalytic-Controlled Self-Assembly of Fmoc-Dipeptides

In recent years, short peptide self-assembled materials, prepared under the control of the thermolysin catalyst, have been investigated extensively and shown to acquire various morphologies and fun...

Tightening the Cross-Linking Distance Restraints for Better Resolution of Protein Structure and Dynamics

Chemical cross-linking coupled with mass spectrometry (CXMS) has been increasingly used in structural biology. CXMS distance restraints are usually applied to Cα or Cβ atoms of the cross-linked residues, with upper bounds typically over 20 A. The incorporation of loose CXMS restraints only marginally improves the resolution of the calculated structures. Here we present a new format of CXMS distance restraints, which works by first modifying the cross-linked residue with a rigid extension derived from the cross-linker. With the flexible side-chain explicitly represented, the reformatted restraint can be applied to the modification group instead, with an upper bound of 6 A or less. The short distance restraint can be represented and back-calculated simply with a straight-line. The use of tighter restraints not only afford better-resolved structures but also uncover protein dynamics. Together, the new approach enables more information extracted from the CXMS data.

Insights into Actin Polymerization and Nucleation Using a Coarse-Grained Model

We studied actin filament polymerization and nucleation with molecular dynamics simulations and a previously established coarse-grained model having each residue represented by a single interaction site located at the Cα atom. We approximate each actin protein as a fully or partially rigid unit to identify the equilibrium structural ensemble of interprotein complexes. Monomers in the F-actin configuration bound to both barbed and pointed ends of a short F-actin filament at the anticipated locations for polymerization. Binding at both ends occurred with similar affinity. Contacts between residues of the incoming subunit and the short filament were consistent with expectation from models based on crystallography, x-ray diffraction, and cryo-electron microscopy. Binding at the barbed and pointed end also occurred at an angle with respect to the polymerizable bound structure, and the angle range depended on the flexibility of the D-loop. Additional barbed end bound states were seen when the incoming subunit was in the G-actin form. Consistent with an activation barrier for pointed end polymerization, G-actin did not bind at an F-actin pointed end. In all cases, binding at the barbed end also occurred in a configuration similar to the antiparallel (lower) dimer. Individual monomers bound each other in a short-pitch helix complex in addition to other configurations, with several of them apparently nonproductive for polymerization. Simulations with multiple monomers in the F-actin form show assembly into filaments as well as transient aggregates at the barbed end. We discuss the implications of these observations on the kinetic pathway of actin filament nucleation and polymerization and possibilities for future improvements of the coarse-grained model.

Conserved buried water molecules enable the β-trefoil architecture.

Available high-resolution crystal structures for the family of β-trefoil proteins in the structural databank were queried for buried waters. Such waters were classified as either: (a) unique to a particular domain, family, or superfamily or (b) conserved among all β-trefoil folds. Three buried waters conserved among all β-trefoil folds were identified. These waters are related by the threefold rotational pseudosymmetry characteristic of this protein architecture (representing three instances of an identical structural environment within each repeating trefoil-fold motif). The structural properties of this buried water are remarkable and include: residing in a cavity space no larger than a single water molecule, exhibiting a positional uncertainty (i.e., normalized B-factor) substantially lower than the average Cα atom, providing essentially ideal H-bonding geometry with three solvent-inaccessible main chain groups, simultaneously serving as a bridging H-bond for three different β-strands at a point of secondary structure divergence, and orienting conserved hydrophobic side chains to form a nascent core-packing group. Other published work supports an interpretation that these interactions are key to the formation of an efficient folding nucleus and folded thermostability. The fundamental threefold symmetric structural element of the β-trefoil fold is therefore, surprisingly, a buried water molecule.

Molecular dynamics simulation of the heart type fatty acid binding protein in a crystal environment

Crystallographic data comes from a space-time average over all the unit cells within the crystal, so dynamic phenomena do not contribute significantly to the diffraction data. Many efforts have been made to reconstitute the movement of the macromolecules and explore the microstates that the confined proteins can adopt in the crystalline network. We explored different strategies to simulate a heart fatty acid binding protein (H-FABP) crystal by means of Molecular Dynamics (MD) simulations. We evaluate the effect of introducing restraints according to experimental isotropic B-factors and we analyzed the H-FABP motions in the crystal using Principal Component Analysis (PCA), isotropic and anisotropic B-factors. We compared the behavior of the protein simulated in the crystal confinement versus in solution, and we observed the effect of that confinement in the mobility of the protein residues. Restraining one-third of Cα atoms based on experimental B-factors produce lower B-factors than simulations without restraints, showing that the position restraint of the atoms with the lowest experimental B-factor is a good strategy to maintain the geometry of the crystal with an obvious decrease in the degrees of motion of the protein. PCA shows that, as position restraint reduces the conformational space explored by the system, the motion of the crystal is better recovered, for an essential subspace of the same size, in the simulations without restraints. Restraining only one Cα seems to be a good balance between giving flexibility to the system and preserving its structure. Communicated by Ramaswamy H. Sarma

Inactive dimeric structure of the protease domain of stomatin operon partner protein.

The N-terminal region of the stomatin operon partner protein (STOPP) PH1510 (1510-N) from the hyperthermophilic archaeon Pyrococcus horikoshii is a serine protease with a catalytic Ser-Lys dyad (Ser97 and Lys138) and specifically cleaves the C-terminal hydrophobic region of the p-stomatin PH1511. In a form of human hemolytic anemia known as hereditary stomatocytosis, stomatin is deficient in the erythrocyte membrane owing to mis-trafficking. Stomatin is thought to act as an oligomeric scaffolding protein to support cell membranes. The cleavage of stomatin by STOPP might be involved in a regulatory system. Several crystal structures of 1510-N have previously been determined: the wild type, the K138A mutant and its complex with a substrate peptide. Here, the crystal structure of the S97A mutant of 1510-N (1510-N S97A) was determined at 2.25 Å resolution. The structure contained two 1510-N S97A molecules in the asymmetric unit. On the superposition of one monomer of the 1510-N S97A and wild-type dimers, the S97A Cα atom of the other monomer of 1510-N S97A deviated by 23 Å from that of the wild type. This result indicates that 1510-N can greatly change the form of its dimer. Because of crystallographic symmetry in space group P65, a sixfold helical structure is constructed using the 1510-N dimer as a basic unit. This helical structure may be common to STOPP structures.

Acquired Functional Capsid Structures in Metazoan Totivirus-like dsRNA Virus

Non-enveloped icosahedral double-stranded RNA (dsRNA) viruses possess multifunctional capsids required for their proliferation. Whereas protozoan/fungal dsRNA viruses have a relatively simple capsid structure, which suffices for the intracellular phase in their life cycle, metazoan dsRNA viruses have acquired additional structural features as an adaptation for extracellular cell-to-cell transmission in multicellular hosts. Here, we present the first atomic model of a metazoan dsRNA totivirus-like virus and the structure reveals three unique structural traits: a C-terminal interlocking arm, surface projecting loops, and an obstruction at the pore on the 5-fold symmetry axis. These traits are keys to understanding the capsid functions of metazoan dsRNA viruses, such as particle stability and formation, cell entry, and endogenous intraparticle transcription of mRNA. On the basis of molecular dynamics simulations of the obstructed pore, we propose a possible mechanism of intraparticle transcription in totivirus-like viruses, which dynamically switches between open and closed states of the pore(s).

Revealing binding selectivity of inhibitors toward bromodomain-containing proteins 2 and 4 using multiple short molecular dynamics simulations and free energy analyses

ABSTRACT Emerging evidences indicate bromodomain-containing proteins 2 and 4 (BRD2 and BRD4) play critical roles in cancers, inflammations, cardiovascular diseases and other pathologies. Multiple short molecular dynamics (MSMD) simulations combined with molecular mechanics generalized Born surface area (MM-GBSA) method were applied to investigate the binding selectivity of three inhibitors 87D, 88M and 89G towards BRD2 over BRD4. The root-mean-square fluctuation (RMSF) analysis indicates that the structural flexibility of BRD4 is stronger than that of BRD2. Moreover the calculated distances between the Cα atoms in the centres of the ZA_loop and BC_loop of BRD4 are also bigger than that of BRD2. The rank of binding free energies calculated using MM-GBSA method agrees well with that determined by experimental data. The results show that 87D can bind more favourably to BRD2 than BRD4, while 88M has better selectivity on BRD4 over BRD2. Residue-based free-energy decomposition method was utilized to estimate the inhibitor-residue interaction spectrum and the results not only identify the hot interaction spots of inhibitors with BRD2 and BRD4, but also demonstrate that several common residues, including (W370, W374), (P371, P375), (V376, V380) and (L381, L385) belonging to (BRD2, BRD4), generate significant binding difference of inhibitors to BRD2 and BRD4.

Extracellular alpha/beta-hydrolase from Paenibacillus species shares structural and functional homology to tobacco salicylic acid binding protein 2

An alpha/ beta hydrolase annotated as a putative salicylate esterase within the genome of a species of Paenibacillus previously identified from differential and selective growth on Kraft lignin was structurally and functionally characterised. Feruloyl esterases are key to the degradation of lignin in several bacterial species and although this activity was investigated, no such activity was observed. The crystal structure of the Paenibacillus esterase, here denoted as PnbE, was determined at 1.32 Å resolution, showing high similarity to Nicotiana tabacum salicylic acid binding protein 2 from the protein database. Structural similarities between these two structures across the core domains and key catalytic residues were observed, with superposition of catalytic residues giving an RMSD of 0.5 Å across equivalent Cα atoms. Conversely, the cap domains of PnbE and Nicotiana tabacum SABP2 showed greater divergence with decreased flexibility in the PnbE cap structure. Activity of PnbE as a putative methyl salicylate esterase was supported with binding studies showing affinity for salicylic acid and functional studies showing methyl salicylate esterase activity. We hypothesise that this activity could enrich Paenibacillus sp. within the rhizosphere by increasing salicylic acid concentrations within the soil.