Dynamics of the thumb-finger regions in a GH11 xylanase Bacillus circulans: comparison between the Michaelis and covalent intermediate

Abstract

The structure and dynamics of B. circulans β-1,4-xylanase (BCX) were comparatively studied utilizing molecular dynamics. Simulations of the free enzyme, non-covalently bound, and covalently bound xylobiose intermediate were conducted and post-dynamically studied to comprehend structural changes adopted during a reaction. Results showed that (a) covalent association of the substrate with the receptor induces a change in the structural conformation of the receptor; (b) the thumb region is highly flexible in the non-covalent complex compared to the covalent complex, drawing a conformational distinction between the two systems, a characteristic brought about by a more compact covalent complex structure in contrast to the non-covalent complex. This is most likely the result of a rigid covalent bond in addition to the hydrogen bond interactions between the substrate and receptor in the latter, (c) the distance between the thumb-finger residues Asp11-Pro116 is shortened upon substrate binding indicating that the flaps are drawn towards each other resulting in partial closing of the flaps. This study provides an invaluable contribution to the understanding of the dynamics of glycosidase enzymes which could largely contribute to the design of potent inhibitors targeting GH enzymes implicated in the orchestration of disease and disorders.

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Introduction

Glycoside hydrolases (GH) are a complex group of enzymes that are responsible for the hydrolysis of the glycosidic bond in sugars.¹ These enzymes possess a diverse range of functions in living organisms, both pathological and physiological, thus making them important therapeutic targets.² The inhibition of some of these enzymes has been utilized for functional drugs against diseases such as influenza³ and diabetes, viral infections, as well as for potential anticancer drugs. For instance, oseltamivir (commercially known as Tamiflu) inhibits the influenza neuraminidase enzyme, a type of glycoside hydrolase responsible for breaking down glycoproteins that are required for influenza virus replication.⁴ Comprehensive knowledge of the structure and function of these enzymes is essential if we are to design efficient enzyme inhibitors for a greater variety of enzyme targets. Here, B. circulans β-1,4-xylanase (BCX) is examined to better understand the mechanism and function of glycoside hydrolase enzymes in general, as it is experimentally well-characterised^5–7 and somewhat computationally.^8,9 Therefore, this enzyme was used as a model for the family of GHs, despite of it being a bacterial enzyme responsible for breaking down plant matter, thus its inhibition serves no medicinal purpose. Previous studies have concluded that intermolecular motions of enzymes, in-terms of substrate binding and succeeding chemical and dissociation steps, are important for their function.^10–12 The conformational transition experienced by the enzyme during catalysis is therefore one aspect that warrants deeper investigation.^13,14

BCX is a small enzyme with two conserved carboxylic residues Glu78 and Glu172 that act as the nucleophile and acid/base residues respectively, leading to the hydrolysis of the glycosidic link with the net retention of the anomeric carbon configuration. This is a two-step mechanism that occurs via a covalent glycosyl-enzyme intermediate^7,15,16 (Fig. 1). The X-ray crystal structure of BCX covalently bonded with 2-deoxyfluoroxylose (2DFX) has been resolved and studied.⁶

Fig. 1 Mechanism of retaining endo-β-1,4-xylanase: catalytic residues are Glu78 and Glu172 (sugar-ring distortion not shown). R = xylopyranose.

We have previously reported the effect of active site mutant Tyr69Phe⁸ as well as the ring distortion on the enzyme structural and mechanistic features. Herein, we expand on our previous reports by providing more insight into the enzyme dynamics associated with the catalytic reaction pathway.

The structure of the GH11 xylanase can be described as a partially closed ‘hand’ consisting of one domain folding into two sheets, which is twisted to form a cleft on one side of the protein where the active site is found.¹⁵ The cleft is covered by a long loop called the thumb region, shown in Fig. 2, which involves 11 residues (residue Tyr113-Thr124) connecting β-strands B7 and B8. The thumb is structured as a classical hairpin, containing a Type 1 β-turn (residue Ser117-Gly120) and six internal hydrogen bonds. The finger region is formed from A and B β-sheets (residue 10–15) and the palm is made up of an α-helix and a twisted β-sheet.

Fig. 2 Structure of the BCX thumb (res 113–124) and finger (res 10–15) regions as well as the catalytic residues Glu78 and Glu172.

Crystallographic structures give a rigid view of the location of the atoms that are often partly disturbed by crystal packing and represent only one particular snapshot of many possible conformations. A study of several X-ray structures of the enzyme provides some information about the dynamic movement that should occur going from one conformation to another.

The crystallographic B-factors reveal some clues to the general mobility of residues. However, the precise dynamic features of enzyme structures are still difficult to infer. In this regard, molecular dynamics simulations provide a robust tool to explore the conformational landscape of a biological system. Previous experimental and computational studies on the function of the thumb-like structure have resulted in many inconsistent conclusions.^17–19 Most of these studies have not considered the different enzyme states in the simulation or they may not have used a long enough MD time scale (maximum 1 ns was used).¹⁸

In this study we investigate different stages of the reaction pathway to accurately probe the dynamics of the thumb-finger region. Here, we perform 200 ns (50 ns × 4) multiple-trajectory MD simulations for the free-enzyme state, non-covalently bound as well as the covalently bound intermediate (see Fig. 1). It is worth mentioning that, in the previous reports, estimating the thumb-finger dynamics was mainly based on measuring one parameter which is the distance between the two central residues of the thumb and the finger regions.¹⁸ In this study, we use various metrics to describe the motion of the thumb-finger region. Results obtained from this study should therefore provide better understanding of the role of the thumb-finger regions in the GH catalytic process. This study will aid in the design of potent inhibitors against glycoside hydrolases involved in biochemical pathways of disorders and diseases.

Materials and computational methods

Enzyme models

Modeling of the three enzyme systems, namely, a free enzyme, non-covalent and covalent intermediate (see Fig. 3) has been described in our previous publication.⁸ In our previous work, however, a phenolate moiety was used as a leaving group to approximate the experimental kinetic data,⁸ in this work we have used a xylopyranose moiety to retain the structure of the natural substrate.

Fig. 3 BCX comparative structures for the free enzyme (A), non-covalently bound (B) and covalently bound (C) intermediates.²³ (Free enzyme and non-covalent complex were constructed with reference to the covalent X-ray structure using Chimera software.²⁴)

Systems set-up and molecular dynamics simulations

The X-ray crystallographic structure of the covalent enzyme–inhibitor complex of wild type BCX, PDB code 1BVV, was obtained from the Protein Data Bank. Due to the importance of protonation, an acid/base residue Glu172 in the non-covalent system was protonated and visually inspected in Chimera software.²⁰

Molecular dynamics simulation settings and parameters

In this study we conducted a multiple MD trajectory approach,²¹ which has been previously applied in enzyme system simulations.²² This approach was preferred over continuous MD trajectory approach since the latter is notorious of high statistical errors acquired during a simulation. This approach was conducted as explained in our previous study.²²

Simulations of the free enzyme, noncovalent complex and covalent complex were conducted on the GPU version of PMEMD of the Amber12 package.^23,24 The AMBER force field²⁵ FF99SB derivative was applied for protein description. The Amber12-implemented LEaP module was used for hydrogen atoms addition to the protein and counter ions addition to neutralize the systems. A TIP3P water box²⁶ was used to contain the respective systems with protein atoms located 10 Å away from the water box edge. Periodic boundary conditions were applied with long-range electrostatics treated with particle-mesh Ewald method²⁷ of Amber12 with direct space and 12 Å van der Waals cut-off. System minimizations were performed with a restraint potential of 500 kcal mol⁻¹ Å⁻² to treat the solute for 1000 steepest descent steps, followed by 1000 steps of conjugate gradient minimization. The systems were subsequently minimized with unrestrained conjugate gradient over 1000 steps. MD simulations with canonical ensemble (NVT) were conducted for 50 ps, gradually heated from 0 to 300 K, harmonic restrains of 5 kcal mol⁻¹ Å⁻² for solute atoms and a Langevin thermostat with 1 ps collision random frequency. System equilibration was conducted at 300 K in the unrestrained NPT ensemble for 500 ps. The pressure was maintained at 1 bar using a Berendsen barostat. The SHAKE algorithm²⁸ was used was applied to constrain all hydrogen bonds and 2 fs MD runs with the SPFP precision model²⁹ were conducted. A 2 ns production run was conducted prior to a 50 ns × 4 multiple-trajectory MD from configurations with random velocities read at 500 ps, 1 ns, 1.5 ns and 2 ns intervals from the initial 2 ns production run with NPT ensemble and a Berendsen³⁰ barostat-measured pressure of 1 bar with a 2 ps pressure coupling constant for each case. Coordinates were captured every 1 ps interval and trajectories analysed every 1 ps. Advanced analysis including RMSD, RMSF, PCA, radius of gyration were performed using the Amber12-implemented PTRAJ and CPPTRAJ modules.³¹ Molecular visualizations and plotting were respectively conducted with Chimera²⁰ and Origin software.³²

Distance metrics

The magnitude of opening and closing of the active site was determined by the movement and position of the thumb-finger regions. Five different distances were carefully chosen and monitored to provide information pertaining the dynamics of the thumb-finger regions. These distances are between alpha-carbon atoms of four residues, two representing the thumb-finger region, Asp11 and Pro116; and two representing the active site, Glu172 and Glu78. These distances are (a) Asp11-Pro116 (the thumb-finger distance), (b) Asp11-Glu172, (c) Asp11-Glu78, (d) Pro116-Glu172 and (e) Pro116-Glu78 (see Fig. 3).

The thumb-finger distance, Asp11-Pro116, provides the extent to which the enzyme opens and closes. Asp11-Glu172 cross-distance gives a measure of whether the finger region becomes compressed in response to the substrate. The Pro116-Glu78 cross-distance is a similar metric but for the thumb region. The distances, Pro116-Glu172 and Asp11-Glu78, were also monitored to roughly estimate the vector of the motion. Collective monitoring of all these distances along the MD trajectory will provide a comprehensive picture on the entire dynamics of the thumb-finger regions, hence the conformational changes upon substrate binding.

Angle analysis

Consideration of distance and angle measurements between the thumb-finger residues and catalytic residues may assist to effectively describe the flap dynamics of BCX. The angle together with the distance parameter were previously used to provide insight on the flap dynamics in proteins.³³ Therefore we measured the angles constructed by Pro116-Glu172-Asp11 and Asp11-Glu78-Pro116 alpha-carbon atoms along the simulation trajectory for each system. Plotted results were deposited in the ESI† Section.

Principal component analysis (PCA)

PCA, also known as essential dynamics analysis, is a widely applied technique to understand the dynamics of biological systems at molecular level.³⁴ PCA describes harmonic atomic displacement and identifies major conformational changes between structures.^22,26 The 50 ns MD trajectories from the respective systems were stripped of water molecules and ions using the PTRAJ³¹ module implemented in Amber12. Stripped trajectories were aligned against a fully minimized structure. PCA was subsequently performed on alpha-carbon atoms for 1000 snapshots at 5 ps time interval, respectively. The foremost two principal components were calculated and in-house scripts were used to construct covariance matrices. PC1 and PC2 generated from the respective trajectories correspond to the covariance matrix of the foremost two Eigenvectors. PCA scatter plots were constructed with the Origin software and the structural diagrams were subsequently constructed in VMD.³⁵ A ProDy interface of the VMD-intergrated Normal Mode Wizard (NMW)³⁶ was used to construct porcupine plots.

Dynamic cross correlation

Dynamic cross correlation method has been previously applied in the analyses of backbone fluctuations and domain motions by focusing on alpha carbon atoms.³⁷ In order to obtain understanding of the thumb-finger region dynamics, we constructed dynamic cross correlation matrices to represent cross-correlated displacements of alpha atoms in trajectories across the respective systems. Cross correlation elements for two i and j C_α atoms are given by the following equation:
Where r_i = C_αⁱ, 〈 〉 = average time over MD trajectory. The generated matrix was constructed in Origin software (cite). Highly correlated motions are denoted by C_ij = 1 whereas C_ij = −1 denotes highly anti-correlated motions. High correlation is congruent to motion of the same phase and period. Motion deviation from 1 or −1 insinuate that i and j motions are simply less correlated (or anti-correlated).

Results and discussion

System stability and MD simulations

To verify the equilibration of the systems prior to molecular dynamics analyses, we monitored the root of mean square deviation (RMSD) for the free enzyme, non-covalent complex and covalent complex. RMSD plots of simulated systems are provided in the ESI† Section (S1).

Thumb-finger dynamics

Root of mean square fluctuation (RMSF). The RMSF of amino acid residues from simulations of the free enzyme, non-covalent and covalent systems are presented in Fig. 4. The free enzyme displayed a highest degree of fluctuation compared to the substrate–bound complexes. This is due to the absence of a substrate to interact with the active site to reduce residue fluctuations.

Fig. 4 RMSF plot of the BCX free enzyme (blue), enzyme non-covalently bonded to the substrate (red) and covalently bonded (green): T₁, T₂, T₃, T₄ and T_avg are representative of the four distinctive 50 ns MD trajectories and overall average, respectively. The respective thumb-finger regions are highlighted in T_avg.

Interestingly, for the thumb region (res 113–124), the amino acid Pro116 shows a relatively high degree of residue fluctuation compared to the finger region (res 10–15) amino acid Asp11. This is accordance with the idea that the thumb region of the GH11 xylanases largely contribute to overall protein flexibility.³⁸ Moreover, a higher degree of overall residue fluctuation was observed for the non-covalent complex than in the covalent complex. This is possibly due to stronger hydrogen bond and other electrostatic interactions between the more immobilized glycocyl ligand after covalent anchoring to the catalytic site (see S9†).

Asp11-Pro116 distance. The distances measured between the thumb-finger residues of the free enzyme, non-covalent complex and covalent complex systems are presented in S2 and average values in Table S1.† Evidently, the free enzyme exhibits a comparatively larger distance, averaging 11.41 Å, as compared to the complexed systems thus indicating a more open conformation. This observation is comprehensible since the enzyme is not complexed with a substrate to induce closing of the active site. Conversely, the non-covalent complex had a slightly longer average distance (11.07 Å) compared to the covalent system (10.48 Å), thus implying a slightly more open enzyme conformation for the non-covalent complex.

Asp11-Glu172 distance. As evident in S3 and Table S1,† the free enzyme system exhibited a relatively large cross-distance, averaging 11.76 Å, as compared to the non-covalent substrate (11.71 Å) and covalent substrate (11.55 Å) system. Moreover, the non-covalent and covalent systems displayed a minute difference in average distance (0.16 Å) signifying a magnitude of conformational dissimilarity between the two systems upon substrate binding. This also indicates that the substrate induces minor flexibility to the finger region.

Asp11-Glu78 distance. A distance between a finger region residue Asp11 and a catalytic residue Glu78 is graphically presented in S4 and average values in Table S1.† The free enzyme displayed a relatively large distance, averaging 18.80 Å, in contrast to the non-covalently bound substrate (17.75 Å) and a covalently bound substrate (10.79 Å). A substantial difference in average distance between the non-covalent and covalent complexes (6.96 Å) may imply that the finger region is pulled towards the active site consequently lending conformational support to substrate processing in the active site.

Pro116-Glu78 distance. A plot of the comparative cross-distance between a thumb region residue Pro116 and a catalytic residue Glu78 is presented in S5 and average values in Table S1.† The free enzyme displayed a comparatively large distance, measuring 12.05 Å in average, in comparison to the non-covalently bound complex (11.24 Å) and covalently bound complex (6.38 Å).

A significant difference of 4.86 Å in average distance is evident between a covalent and a non-covalent complex, indicating a large conformational difference between the two systems. The distance of the covalent system is in accordance with the distance observed in the Asp11-Glu78 distance for the covalent complex to support the compact conformational and substrate binding.

Pro116-Glu172 distance. A distance between a thumb region residue Pro116 and a catalytic residue Glu172 is graphically presented in S6 and average values in Table S1.† A larger distance was observed for the free enzyme (16.64 Å) compared to the non-covalent complex (15.97 Å) and the covalent complex (15.59 Å). A distance difference of 0.38 Å was observed between the non-covalent and covalent systems drawing a magnitude of functional and conformational distinction between the thumb and finger regions of these systems.

Glu78-Glu172 distance. A distance between the active site catalytic residue Glu78 and Glu172 is graphically presented in S7 and average values in Table S1.† A comparatively large distance was observed for the free enzyme (14.44 Å) compared to a non-covalently bound complex (14.14 Å) and a covalently bound complex (11.72 Å). Interestingly, an average distance of 2.42 Å was observed between a non-covalently bound complex and covalently bound complex. This minor difference in inter-catalytic residue distance between such systems was expected since the transition state conformation of the sugar is expected to be the same for both non-covalent and covalent systems.

It should be noted that in all of the distances reported here, the covalent system is relatively the shortest and the non-covalent the longest. This may be explained by the gradual pulling of the finger region towards the ligand through hydrogen bond interactions between the ligand and finger region residues which form the active site, as well as the formation of a rigid covalent bond between the ligand and Glu78. The steric effects of the non-covalently bound ligand may have contributed to the lengthening of the distances in the non-covalent complex. The sugar moiety in this complex is not as much immobilized which results in less and weaker hydrogen bonds formed during the MD simulation. This may have resulted in a less contracted complex. Since the movement of the sugar in the covalently bound case is limited (a more rigid system), more hydrogen bonds and other electrostatic forces form between the enzyme and the tightly bound ligand (see S10†). This may have contributed to the contraction of the active site.

The work by Paes et al. suggests that the thumb holds the substrate in place and then opens up to facilitate the expulsion of the product. The RMSF findings presented in this study show that the finger region exhibits minor movement in comparison to the thumb region in all three systems, thus supporting a Paes et al. suggestion. The finger-active site Asp11-Glu172 cross-distance was slightly larger (0.16 Å) in the non-covalently bound complex than in the covalently bound complex. However, for the thumb-active site cross-distance (Pro116-Glu78), a significantly longer distance (4.86 Å) was seen for the non-covalent complex in contrast to the covalent complex. The thumb region may also be a participant in the propelling of the substrate during the reaction.

Pro116-Glu172-Asp11 angle. An angle constructed by the thumb-finger region residues Pro116 and Asp11 as well as the catalytic residue Glu172 is graphically presented in S8 and average values in Table S2.† A free enzyme, expectedly, shows a comparatively larger angle averaging 45.34° compared to the non-covalent (42.11°) and the covalent (41.33°) complex system. A reduced angle of the covalent complex is suggestive of the constriction of the active site imposed by the covalent bond, thus supporting induced pulling of the finger region towards the active site.

Asp11-Glu78-Pro116 angle. An angle formed by the thumb-finger region residues Asp11 and Pro116 as well as the catalytic residue Glu78 is graphically presented in S9 and average values in Table S2.† Large angle fluctuations are seen for the free enzyme averaging 81.87° in contrast to the non-covalent complex (35.22°) and steady covalent complex (33.60°) systems. A considerable difference in average angle (7.73°) between the Pro116-Glu172-Asp11 (S8†) and Asp11-Glu78-Pro116 (S9†) in the covalent complex is in accordance with the active site constriction and shortening of the Asp11-Glu78 distance.

The covalently bound complex shows a relatively reduced angle with minimal constant fluctuation compared to the more fluctuating non-covalent complex with an average difference of 1.62° between the two systems, thus drawing a conformational difference adopted during the reaction.

Radius of gyration

The radius of gyration is the moment of inertia of alpha-carbon atoms from their centre of mass. Fig. 5 presents the comparative radius of gyration for the free enzyme, non-covalent and covalent complex systems, respectively. The free enzyme shows the largest radius of gyration (15.27 Å) compared to the bound systems (see average values in Table S5†). Interestingly, the non-covalent complex exhibited a slightly larger radius of gyration (15.26 Å) in comparison to the covalent complex (15.19 Å), highlighting a slightly less compact nature of the non-covalent complex over the covalent one. This also complies with the insinuation of a disproportionate opening and closing of the thumb-finger region and active site in the covalent and non-covalent systems, whereby the thumb-finger region and active site are more open in the non-covalent system and less open in the covalent system. In the covalent system, a tighter protein structure arrangement exists, owing to the much reduced moment of inertia for the alpha-carbon atoms.

Fig. 5 Comparative radius of gyration for the respective free enzyme, non-covalent and covalent intermediate systems: T₁, T₂, T₃, T₄ and T_avg are representative of the four distinctive 50 ns MD trajectories and overall average, respectively.

Principal component analysis

Conformational transitions of the free enzyme non-covalently bound and covalently bound substrate complexes were characterised using PCA, a technique that has been widely employed to present experimentally detected conformational variations. Principal components of the free enzyme, non-covalent complex and covalent complex along a path of two components are presented in Fig. 6.

Fig. 6 Principal component analysis scatter plots of 1000 snapshots along the first two components, PC1 and PC2 for the respective free enzyme, non-covalent and covalent complexes displaying differences in eigenvectors: T₁, T₂, T₃, T₄ & T_avg are represent four distinctive 50 ns MD trajectories and overall average, respectively.

PCA revealed that the free enzyme inhabits a comparatively wider phase space and displays higher fluctuation in contrast to the non-covalently bound and covalently bound complex, respectively. This observation is congruent to the presented radius of gyration results and limited residue fluctuation in the covalent complex system in contrast to the non-covalent complex system. Such observation may also be explained by the number of hydrogen bonds formed between the ligand and active site residues in the non-covalent (see Table S3†) and covalent system (see Table S4†) respectively.

Changes in the direction of residue motion presented by porcupine plots could provide information about residue dynamics of targeted regions in an enzyme during a simulation. Conformational differences between the three systems upon substrate binding will also be highlighted. Generally, the first two normal modes represent the overall motion exhibited by a macromolecule during a simulation. Here were constructed porcupine plots corresponding to three modes of low frequency to study the comparative distinction in motion between the free enzyme, non-covalent complex and covalent complex.

Fig. 7 presents porcupine plots showing the direction of residue mobility (A) and their frequencies (B) across divergent modes in a free enzyme system. In mode 1, high residue mobility frequency is seen in the thumb region, where residues are pointed in the direction of the palm region. High mobility frequency is seen in the residues sitting at the tips of the thumb and finger region (mode 2). Residue behavior at these locations implies that there are intermittent sessions of partial opening and closing of the thumb-finger flaps. This observation also corresponds to the RMSF results which deemed the thumb region highly vibrant when compared to the finger region. The magnitude of residue fluctuation observed in the free enzyme and the recorded distances corresponds to the less compact structure of the free enzyme system. Prominent residues exhibiting high frequency fluctuations across divergent normal modes in the three distinctive systems are presented in Table S6.†

Fig. 7 Porcupine plots depicting prominent motions averaged across the first three normal modes (A) and their corresponding mobility frequencies (B) of the free enzyme.

Porcupine plots showing prominent residue motion direction and their corresponding frequency across three different modes in the non-covalent complex system are presented in Fig. 8. Mode 1 shows a twisting motion of the thumb-finger region based on the direction of residue movement in these regions. A change in direction of motion is seen in mode 2 for the residues in the thumb-finger region, both pointing towards each other thus pronouncing the shrinking of the active site. In mode 3, the direction and frequency of residues sitting at the tips of the thumb-finger region implies that there is closing of the flaps. In addition, the motion of the thumb-finger region is pointed towards the center of active site implying the drawing of both flaps towards each other to engulf the ligand. This observation is in agreement with the measured distances, angle and radius of gyration for the non-covalent complex, thus discriminating it from the other two systems. Residues displaying high mobility frequency are presented in Table S6.†

Fig. 8 Porcupine plots depicting prominent motions averaged across the first three normal modes (A) and their corresponding mobility frequencies (B) of the non-covalently bound system.

Fig. 9 presents porcupine plots showing the direction of residue mobility and their frequencies across three divergent modes in a covalent complex system. In mode 1, the direction of residue motion in the finger region and thumb region is pointed away from the active site cavity, with the palm region residues pointed towards the active site. This implies that there is an opening of the active site cavity. The highest mobility is displayed by the thumb region residues across all three modes which are dispatched in different directions per mode. The finger region showed relatively low mobility across the three modes.

Fig. 9 Porcupine plots depicting prominent motions averaged across the first three normal modes (A) and their corresponding mobility frequencies (B) of the covalently bound system.

The eigenvectors presented clear differences in the direction of residue motion across all three modes in all three systems. A clear distinction in the frequency of amino acid mobility is evident hence marking conformational differences between the three systems induced by substrate binding and hence the mode of binding. Evidently, the eigenvectors present a clear dissimilarity in the direction of motion which is in congruency with the PCA plots across the three systems. The observed residue fluctuations in the covalent system, as well as the shortened distances measured during a simulation are corresponding to the slightly increased compactness of this system. Conformational differences between individual systems upon substrate binding are presented in S11.†

To further explore the opening and closing of the thumb-finger flaps, we measured interatomic distance between residues at the tip of the thumb and finger regions (Asp11-Pro116) from MD trajectory across the three systems in relation to the first principal component (PC1) since it ideally describes system dynamics. This could help elaborate on the relative motion between the thumb-finger region and conformational evolution of the three systems. Fig. 10 presents the interatomic distances between Asp11-Pro116 versus PC1 during simulations of the free enzyme, non-covalent and covalent complex. As seen in Fig. 10, the free enzyme structure sampled a broad conformational phase distribution and distance distributions indicated that the structure is predominant in its open form with very small populations in its partially closed form. Interestingly, substrate binding to the enzyme resulted in conformational changes of the free enzyme.

Fig. 10 Interatomic distances between Asp11-Pro116 vs. PC1 during simulations of the free enzyme, non-covalent and covalent complex.

The distribution of the Asp11-Pro116 distance instantiates the transformation of the free enzyme into a closed conformation upon substrate binding. A covalent complex adopts a relatively more closed conformation for most of the duration of the simulation in comparison to the free enzyme and non-covalent complex systems.

Structural differences between the three systems were further investigated where we examined dynamically correlated motions in the individual systems. As evident in Fig. 11, the global dynamics of the free enzyme system were similar to that of the non-covalent complex, implying that substrate binding did not impose any significant secondary structure alterations to the protein. However, disparities were spotted between the covalent complex and the other two systems. The free enzyme displayed less correlated motions compared to the non-covalent and covalent complex. A covalent complex presented highly correlated motions in contrast to the non-covalent complex.

Fig. 11 Dynamic cross-correlation matrix analyses for the free enzyme, non-covalent complex and covalent complex systems.

Moreover, the thumb region (res 113–124) showed a combination of highly correlated and less correlated motions accompanied by very low anti-correlated motions in both free enzyme and non-covalent complex systems, while only highly correlated motions were observed in the covalent complex system thumb region. The finger region (res 10–15) of both the free enzyme and non-covalent complex displayed highly correlated motions, whereas the covalent complex showed less correlated motions. The observed motions are consistent with the PCA results, which are indicative of the opening and closing of the thumb-finger region in the respective systems. They are also suggestive of partially reduced conformational flexibility upon substrate binding which is also influenced by the mode in which the substrate binds on the enzyme.

Conclusion

Molecular dynamics of B. circulans β-1,4-xylanase were studied with a multiple-trajectory MD approach to explore differences in the thumb-finger regions in the free enzyme, non-covalently bound and covalently bound complex. Findings from this study show that, (a) covalent association of the substrate with the receptor induces a change in structural conformation of the receptor, (b) the thumb region is highly flexible in the non-covalent complex than in the covalent complex, drawing a conformational distinction between the two systems, a character brought about by a more compact covalent complex structure in contrast to the non-covalent complex. This is most likely the result of a rigid covalent bond in addition to the hydrogen bond interactions between the substrate and receptor in the latter, (c) the distance between the thumb-finger residues Asp11-Pro116 is shortened upon substrate binding indicating that the flaps are drawn towards each other resulting in partial closing of the flaps, (d) based on the radius of gyration results, a covalent complex is more compact when compared to the non-covalent complex due to the presence of a rigid covalent bond in addition to the hydrogen bond interactions formed between the substrate and receptor. This study provides an invaluable contribution to the understanding of the dynamics of glycosidase enzymes which could largely contribute to the design of potent inhibitors targeting GH enzymes implicated in the orchestration of disease and disorders.

Conflict of interest

Authors declare no potential financial and other conflict of interest.

Acknowledgements

The authors acknowledge the UKZN School of Health Sciences, NRF for funding and the Center for High Performance Computing (http://www.chpc.ac.za) for computational resources.

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Footnote

† Electronic supplementary information (ESI) available: RMSD vs. time, distance metrics, angle metrics, active site interactions and porcupine plots for B. circulans β-1,4-xylanase free enzyme, non-covalent and covalent intermediate complexes are provided with the supplementary information. See DOI: 10.1039/c5ra16836h

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