Insights derived from molecular dynamics simulation into the molecular motions of serine protease proteinase K

Serine protease proteinase K, a member of the subtilisin family of enzymes, is of significant industrial, agricultural and biotechnological importance. Despite the wealth of structural information about proteinase K provided by static X-ray structures, a full understanding of the enzymatic mechanism requires further insight into the dynamic properties of this enzyme. Molecular dynamics simulations and essential dynamics (ED) analysis were performed to investigate the molecular motions in proteinase K. The results indicate that the internal core of proteinase K is relatively rigid, whereas the surface-exposed loops, most notably the substrate-binding regions, exhibit considerable conformational fluctuations. Further ED analysis reveals that the large concerted motions in the substrate-binding regions cause opening/closing of the substrate-binding pockets, thus supporting the proposed induced-fit mechanism of substrate binding. The distinct electrostatic/hydrogen-bonding interactions between Asp39 and His69 and between His69 and Ser224 within the catalytic triad lead to different thermal motions and orientations of these three catalytic residues, which can be related to their different functional roles in the catalytic process. Statistical analyses of the geometrical/functional properties as well as evolutionary conservation of the glycines in proteinase K-like proteins reveal that glycines may play an important role in determining the folding architecture and structural flexibility of this class of enzymes. Our simulation study complements the biochemical and structural studies and provides new insights into the dynamic structural basis of the functional properties of this class of enzymes.     

Protein dynamics and motions in relation to their functions: several case studies and the underlying mechanisms

Proteins are dynamic entities in cellular solution with functions governed essentially by their dynamic personalities. We review several dynamics studies on serine protease proteinase K and HIV-1 gp120 envelope glycoprotein to demonstrate the importance of investigating the dynamic behaviors and molecular motions for a complete understanding of their structure–function relationships. Using computer simulations and essential dynamic (ED) analysis approaches, the dynamics data obtained revealed that: (i) proteinase K has highly flexible substrate-binding site, thus supporting the induced-fit or conformational selection mechanism of substrate binding; (ii) Ca²⁺ removal from proteinase K increases the global conformational flexibility, decreases the local flexibility of substrate-binding region, and does not influence the thermal motion of catalytic triad, thus explaining the experimentally determined decreased thermal stability, reduced substrate affinity, and almost unchanged catalytic activity upon Ca²⁺ removal; (iii) substrate binding affects the large concerted motions of proteinase K, and the resulting dynamic pocket can be connected to substrate binding, orientation, and product release; (iv) amino acid mutations 375 S/W and 423 I/P of HIV-1 gp120 have distinct effects on molecular motions of gp120, facilitating 375 S/W mutant to assume the CD4-bound conformation, while 423 I/P mutant to prefer for CD4-unliganded state. The mechanisms underlying protein dynamics and protein–ligand binding, including the concept of the free energy landscape (FEL) of the protein–solvent system, how the ruggedness and variability of FEL determine protein’s dynamics, and how the three ligand-binding models, the lock-and-key, induced-fit, and conformational selection are rationalized based on the FEL theory are discussed in depth.     

The effect of calciums on molecular motions of proteinase K

The native serine protease proteinase K binds two calcium cations. It has been reported that Ca²⁺ removal decreased the enzyme’s thermal stability and to some extent the substrate affinity, but has discrepant effects on catalytic activity of the enzyme. Molecular dynamics simulations were performed on the Ca²⁺-bound and Ca²⁺-free proteases to investigate the mechanism by which the calciums affect the structural stability, molecular motions, and catalytic activity of proteinase K. Very similar structural properties were observed between these two forms of proteinase K during simulations; and several long-lived hydrogen bonds and salt bridges common to both forms of proteinase K were found to be crucial in maintaining the local conformations around these two Ca²⁺ sites. Although Ca²⁺ removal enhanced the overall flexibility of proteinase K, the flexibility in a limited number of segments surrounding the substrate-binding pockets decreased. The largest differences in the equilibrium structures of the two simulations indicate that, upon the removal of Ca²⁺, the large concerted motion originating from the Ca1 site can transmit to the substrate-binding regions but not to the catalytic triad residues. In conjunction with the large overlap of the essential subspaces between the two simulations, these results not only provide insight into the dynamics of the underlying molecular mechanism responsible for the unchanged enzymatic activity as well as the decreased thermal stability and substrate affinity of proteinase K upon Ca²⁺ removal, but also complement the experimentally determined structural and biochemical data.     

Insight derived from molecular dynamics simulation into dynamics and molecular motions of cuticle-degrading serine protease Ver112

Cuticle-degrading serine protease Ver112, which derived from a nematophagous fungus Lecanicillium psalliotae, has been exhibited to have high cuticle-degrading and nematicidal activities. We have performed molecular dynamics (MD) simulation based on the crystal structure of Ver112 to investigate its dynamic properties and large-scale concerted motions. The results indicate that the structural core of Ver112 shows a small fluctuation amplitude, whereas the substrate binding sites, and the regions close to and opposite the substrate binding sites experience significant conformational fluctuations. The large concerted motions obtained from essential dynamics (ED) analysis of MD trajectory can lead to open or close of the substrate binding sites, which are proposed to be linked to the functional properties of Ver112, such as substrate binding, orientation, catalytic, and release. The significant motion in the loop regions that is located opposite the binding sites are considered to play an important role in modulating the dynamics of the substrate binding sites. Furthermore, the bottom of free energy landscape (FEL) of Ver112 are rugged, which is mainly caused by the fluctuations of substrate binding regions and loops located opposite the binding site. In addition, the mechanism underlying the high flexibility and catalytic activity of Ver112 was also discussed. Our simulation study complements the biochemical and structural studies, and provides insight into the dynamics-function relationship of cuticle-degrading serine protease Ver112.     

Subtle functional collective motions in pancreatic-like ribonucleases: from ribonuclease A to angiogenin

The analysis of the dynamic behavior of enzymes is fundamental to structural biology. A direct relationship between protein flexibility and biological function has been shown for bovine pancreatic ribonuclease (RNase A) (Rasmussen et al., Nature 1992;357:423-424). More recently, crystallographic studies have shown that functional motions in RNase A involve the enzyme beta-sheet regions that move concertedly on substrate binding and release (Vitagliano et al., Proteins 2002;46:97-104). These motions have been shown to correspond to intrinsic dynamic properties of the native enzyme by molecular dynamics (MD) simulations. To unveil the occurrence of these collective motions in other members of pancreatic-like superfamily, we carried out MD simulations on human angiogenin (Ang). Essential dynamics (ED) analyses performed on the trajectories reveal that Ang exhibits collective motions similar to RNase A, despite the limited sequence identity (33%) of the two proteins. Furthermore, we show that these collective motions are also present in ensembles of experimentally determined structures of both Ang and RNase A. Finally, these subtle concerted beta-sheet motions were also observed for other two members of the pancreatic-like superfamily by comparing the ligand-bound and ligand-free structures of these enzymes. Taken together, these findings suggest that pancreatic-like ribonucleases share an evolutionary conserved dynamic behavior consisting of subtle beta-sheet motions, which are essential for substrate binding and release.     

Specificity of trypsin and chymotrypsin: loop-motion-controlled dynamic correlation as a determinant

Trypsin and chymotrypsin are both serine proteases with high sequence and structural similarities, but with different substrate specificity. Previous experiments have demonstrated the critical role of the two loops outside the binding pocket in controlling the specificity of the two enzymes. To understand the mechanism of such a control of specificity by distant loops, we have used the Gaussian network model to study the dynamic properties of trypsin and chymotrypsin and the roles played by the two loops. A clustering method was introduced to analyze the correlated motions of residues. We have found that trypsin and chymotrypsin have distinct dynamic signatures in the two loop regions, which are in turn highly correlated with motions of certain residues in the binding pockets. Interestingly, replacing the two loops of trypsin with those of chymotrypsin changes the motion style of trypsin to chymotrypsin-like, whereas the same experimental replacement was shown necessary to make trypsin have chymotrypsin's enzyme specificity and activity. These results suggest that the cooperative motions of the two loops and the substrate-binding sites contribute to the activity and substrate specificity of trypsin and chymotrypsin.     

Molecular Dynamics of Phenylalanine Transfer RNA

Abstract

The atomic motions of yeast phenylalanine transfer RNA have been simulated using the molecular dynamics algorithm. Two simulations were carried out for a period of 12 picoseconds, one with a normal Van der Waals potential and the other with a modified Van der Waals potential intended to mimic the effect of solvent. An analysis of large scale motions, surface exposure, root mean square displacements, helical oscillations and relaxation mechanisms reveals the maintenance of stability in the simulated structures and the general similarity of the various dynamic features of the two simulations. The regions of conformational flexibility and rigidity for tRNA^Phe have been shown in a quantitative measure through this approach.     

Binding mode of Pyridoclax to myeloid cell leukemia-1 (Mcl-1) revealed by nuclear magnetic resonance spectroscopy,docking and molecular dynamics approaches

Abstract

Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 family proteins. Its amplification is one of the most frequent genetic aberrations found in human cancers. Pyridoclax, a promising BH3 mimetic inhibitor, interacts directly with Mcl-1 and induces massive apoptosis at a concentration of 15?µM in combination with anti-Bcl-x_L strategies in chemo-resistant ovarian cancer cell lines. In this study, a combined experimental and theoretical approach was used to investigate the binding mode of Pyridoclax to Mcl-1. The representative poses generated from dynamics simulations compared with NMR data revealed: (i) Pyridoclax bound to P1 and P2 pockets of Mcl-1 BH3 binding groove through its styryl and methyl groups establishing mainly hydrophobic contacts, (ii) one of the ending pyridines interacts through electrostatic interaction with K234 side chain, a negatively charged residue present only in this position in Mcl-1.

Communicated by Ramaswamy H. Sarma     

New structural motifs on the chymotrypsin fold and their potential roles in complement factor B

Factor B and C2 are two central enzymes for complement activation. They are multidomain serine proteases and require cofactor binding for full expression of proteolytic activities. We present a 2.1 A crystal structure of the serine protease domain of factor B. It shows a number of structural motifs novel to the chymotrypsin fold, which by sequence homology are probably present in C2 as well. These motifs distribute characteristically on the protein surface. Six loops surround the active site, four of which shape substrate-binding pockets. Three loops next to the oxyanion hole, which typically mediate zymogen activation, are much shorter or absent. Three insertions including the linker to the preceding domain bulge from the side opposite to the active site. The catalytic triad and non-specific substrate-binding site display active conformations, but the oxyanion hole displays a zymogen-like conformation. The bottom of the S1 pocket has a negative charge at residue 226 instead of the typical 189 position. These unique structural features may play different roles in domain-domain interaction, cofactor binding and substrate binding.     

Alpha7 Helix Plays an Important Role in the Conformational Stability of PTP1B

Abstract

The C-terminus of Protein Tyrosine Phosphatase 1B (PTP1B) includes an α-helix (α7), which forms an allosteric binding site 20 Å away from the active site. This helix is specific to PTP1B and its truncation decreases the catalytic activity significantly. Here, molecular dynamics (MD) simulations in the presence and absence of α7 were performed to investigate the role played by α7. The highly mobile α7 was found to maintain its contacts with loop 11 (L11)- α3 helix throughout the simulations. The interactions of Tyr152 on L11, Tyr176, Thr177 on the catalytically important WPD loop and Ser190 on α3 are important for the conformational stability and the concerted motions of the regions surrounding the WPD loop. In the absence of α7, L11 and WPD loop move away from their crystal structure conformations, resulting in the loss of the interactions in this region, and a decrease in the residue displacement correlations in the vicinity of WPD loop. Therefore, we suggest that one of the functionally important roles of α7 may be to limit the L11 and α3 motions, and, facilitate the WPD loop motions. Truncation of α7 in PTP1B is found to affect distant regions as well, such as the substrate recognition site and the phosphate binding-loop (P-loop), changing the conformations of these regions significantly. Our results show that the PTP1B specific α7 is important for the conformation and dynamics of the WPD loop, and also may play a role in ligand binding.     

Coupled Global and Local Changes Direct Substrate Translocation by Neurotransmitter-Sodium Symporter Ortholog LeuT

Significant advances have been made in recent years in characterizing neurotransmitter:sodium symporter (NSS) family structure and function. Yet, many time-resolved events and intermediates that control the various stages of transport cycle remain to be elucidated. Whether NSSs harbor one or two sites for binding their substrates (neurotransmitters or amino acids), and what the role of the secondary site S2 is, if any, are still unresolved. Using molecular modeling and simulations for LeuT, a bacterial NSS, we present a comprehensive account of substrate-binding and -stabilization events, and subsequently triggered interactions leading to substrate (alanine) release. LeuT instantaneous conformation as it reconfigures from substrate-receiving (outward-facing) to -releasing (inward-facing) state appears to be a determinant of its affinity to bind substrate at site S2. In the outward-facing state, S1 robustly binds alanine and regulates subsequent redistribution of interactions to trigger extracellular gate closure; whereas S2 is only a transient binding site. The substrate-binding affinity at S2 increases in an intermediate close to inward-facing state. LeuT harbors the two substrate-binding sites, and small displacements of second substrate near S2 are observed to induce concerted small translocations in the substrate bound to primary site S1, although complete release requires collective structural rearrangements that fully expose the intracellular vestibule to the cytoplasm.     

Conformational substates in enzyme mechanism: the 120 K structure of alpha-lytic protease at 1.5 A resolution.

Insight into the dynamic properties of alpha-lytic protease (alpha LP) has been obtained through the use of low-temperature X-ray crystallography and multiple-conformation refinement. Previous studies of alpha LP have shown that the residues around the active site are able to move significantly to accommodate substrates of different sizes. Here we show a link between the ability to accommodate ligands and the dynamics of the binding pocket. Although the structure of alpha LP at 120 K has B-factors with a uniformly low value of 4.8 A2 for the main chain, four regions stand out as having significantly higher B-factors. Because thermal motion should be suppressed at cryogenic temperatures, the high B-factors are interpreted as the result of trapped conformational substates. The active site residues that are perturbed during accommodation of different substrates are precisely those showing conformational substates, implying that substrate binding selects a subset of conformations from the ensemble of accessible states. To better characterize the precise nature of these substates, a protein model consisting of 16 structures has been refined and evaluated. The model reveals a number of features that could not be well-described by conventional B-factors: for example, 40% of the main-chain residue conformations are distributed asymmetrically or in discrete clusters. Furthermore, these data demonstrate an unexpected correlation between motions on either side of the binding pocket that we suggest is a consequence of "dynamic close packing." These results provide strong evidence for the role of protein dynamics in substrate binding and are consistent with the results of dynamic studies of ligand binding in myoglobin and ribonuclease A.     

Coupled Global and Local Changes Direct Substrate Translocation by?Neurotransmitter-Sodium Symporter Ortholog LeuT

Significant advances have been made in recent years in characterizing neurotransmitter:sodium symporter (NSS) family structure and function. Yet, many time-resolved events and intermediates that control the various stages of transport cycle remain to be elucidated. Whether NSSs harbor one or two sites for binding their substrates (neurotransmitters or amino acids), and what the role of the secondary site S2 is, if any, are still unresolved. Using molecular modeling and simulations for LeuT, a bacterial NSS, we present a comprehensive account of substrate-binding and -stabilization events, and subsequently triggered interactions leading to substrate (alanine) release. LeuT instantaneous conformation as it reconfigures from substrate-receiving (outward-facing) to -releasing (inward-facing) state appears to be a determinant of its affinity to bind substrate at site S2. In the outward-facing state, S1 robustly binds alanine and regulates subsequent redistribution of interactions to trigger extracellular gate closure; whereas S2 is only a transient binding site. The substrate-binding affinity at S2 increases in an intermediate close to inward-facing state. LeuT harbors the two substrate-binding sites, and small displacements of second substrate near S2 are observed to induce concerted small translocations in the substrate bound to primary site S1, although complete release requires collective structural rearrangements that fully expose the intracellular vestibule to the cytoplasm.     

Dramatic differences in the motions of the mouth of open and closed cytochrome P450BM-3 by molecular dynamics simulations

Molecular dynamics trajectories were calculated separately for each of the two molecules in the asymmetric unit of the crystal structure of the hemoprotein domain of cytochrome P450BM-3. Each simulation was 200 ps in length and included a 10 Å layer of explicit solvent. The simulated time-average structure of each P450BM-3 molecule is closer to its crystal structure than the two molecular dynamics time-averaged structures are to each other. In the crystal structure, molecule 2 has a more accessible substrate binding pocket than molecule 1, and this difference is maintained throughout the simulations presented here. In particular, the substrate docking regions of molecule 1 and molecule 2 diverge in the solution state simulations. The mouth of the substrate binding pocket is significantly more mobile in the simulation of molecule 2 than in the simulation of molecule 1. For molecule 1, the width of the mouth is only slightly larger than its X-ray value of 8.7 Å and undergoes fluctuations of about 1 Å. However, in molecule 2, the mouth of the substrate binding pocket is dramatically more open in the time-average molecular dynamics structure (14.7 Å) than in the X-ray structure (10.9 Å). Furthermore, this region of the protein undergoes large amplitude motions during the trajectory that are not seen in the trajectory of molecule 1, repeatedly opening and closing up to 7 Å. Presumably, the binding of different substrates will induce the mouth region to adopt different conformations from within the wide range of structures that are accessible. © 1995 Wiley-Liss, Inc.     

Global and local motions in ribonuclease A: a molecular dynamics study

The understanding of protein dynamics is one of the major goals of structural biology. A direct link between protein dynamics and function has been provided by x-ray studies performed on ribonuclease A (RNase A) (B. F. Rasmussen et al., Nature, 1992, Vol. 357, pp. 423-424; L. Vitagliano et al., Proteins: Structure, Function, and Genetics, 2002, Vol. 46, pp. 97-104). Here we report a 3 ns molecular dynamics simulation of RNase A in water aimed at characterizing the dynamical behavior of the enzyme. The analysis of local and global motions provides interesting insight on the dynamics/function relationship of RNase A. In agreement with previous crystallographic reports, the present study confirms that the RNase A active site is constituted by rigid (His12, Asn44, Thr45) and flexible (Lys41, Asp83, His119, Asp121) residues. The analysis of the global motions, performed using essential dynamics, shows that the two beta-sheet regions of RNase A move coherently in opposite directions, thus modifying solvent accessibility of the active site, and that the mixed alpha/3(10)-helix (residues 50-60) behaves as a mechanical hinge during the breathing motion of the protein. These data demonstrate that this motion, essential for RNase A substrate binding and release, is an intrinsic dynamical property of the ligand-free enzyme.     

Dynamically-Driven Enhancement of the Catalytic Machinery of the SARS 3C-Like Protease by the S284-T285-I286/A Mutations on the Extra Domain

Previously we revealed that the extra domain of SARS 3CLpro mediated the catalysis via different mechanisms. While the R298A mutation completely abolished the dimerization, thus resulting in the inactive catalytic machinery, N214A inactivated the enzyme by altering its dynamics without significantly perturbing its structure. Here we studied another mutant with S284-T285-I286 replaced by Ala (STI/A) with a 3.6-fold activity increase and slightly enhanced dimerization. We determined its crystal structure, which still adopts the dimeric structure almost identical to that of the wild-type (WT), except for slightly tighter packing between two extra-domains. We then conducted 100-ns molecular dynamics (MD) simulations for both STI/A and WT, the longest reported so far for 3CLpro. In the simulations, two STI/A extra domains become further tightly packed, leading to a significant volume reduction of the nano-channel formed by residues from both catalytic and extra domains. The enhanced packing appears to slightly increase the dynamic stability of the N-finger and the first helix residues, which subsequently triggers the redistribution of dynamics over residues directly contacting them. This ultimately enhances the dynamical stability of the residues constituting the catalytic dyad and substrate-binding pockets. Further correlation analysis reveals that a global network of the correlated motions exists in the protease, whose components include all residues identified so far to be critical for the dimerization and catalysis. Most strikingly, the N214A mutation globally decouples this network while the STI/A mutation alters the correlation pattern. Together with previous results, the present study establishes that besides the classic structural allostery, the dynamic allostery also operates in the SARS 3CLpro, which is surprisingly able to relay the perturbations on the extra domain onto the catalytic machinery to manifest opposite catalytic effects. Our results thus imply a promising avenue to design specific inhibitors for 3CL proteases by disrupting their dynamic correlation network.     

Symmetric K+ and Mg2+ ion-binding sites in the 5S rRNA loop E inferred from molecular dynamics simulations

Potassium binding to the 5 S rRNA loop E motif has been studied by molecular dynamics at high (1.0 M) and low (0.2 M) concentration of added KCl in the presence and absence of Mg²⁺. A clear pattern of seven deep groove K⁺ binding sites or regions, in all cases connected with guanine N7/O6 atoms belonging to GpG, GpA, and GpU steps, was identified, indicating that the LE deep groove is significantly more ionophilic than the equivalent groove of regular RNA duplexes. Among all, two symmetry-related sites (with respect to the central G·A pair) were found to accommodate K⁺ ions with particularly long residence times. In a preceding molecular dynamics study by Auffinger et al. in the year 2003, these two sites were described as constituting important Mg²⁺ binding locations. Altogether, the data suggest that these symmetric sites correspond to the loop E main ion binding regions. Indeed, they are located in the deep groove of an important ribosomal protein binding motif associated with a fragile pattern of non-Watson-Crick pairs that has certainly to be stabilized by specific Mg²⁺ ions in order to be efficiently recognized by the protein. Besides, the other sites accommodate monovalent ions in a more diffuse way pointing out their lesser significance for the structure and function of this motif. Ion binding to the shallow groove and backbone atoms was generally found to be of minor importance since, at the low concentration, no well defined binding site could be characterized while high K⁺ concentration promoted mostly unspecific potassium binding to the RNA backbone. In addition, several K⁺ binding sites were located in positions equivalent to water molecules from the first hydration shell of divalent ions in simulations performed with magnesium, indicating that ion binding regions are able to accommodate both mono- and divalent ionic species. Overall, the simulations provide a more precise but, at the same time, a more intricate view of the relations of this motif with its ionic surrounding.     

Water-protein interplay reveals the specificity of α-lytic protease

Wild type and mutant α-lytic protease, differing by only one amino acid, have distinct specificities. Previous studies have shown that motion patterns of the binding pocket play an important role. However, it is still unclear how these differences are generated from a single amino acid mutation. Based on comparative molecular dynamics simulations using explicit and implicit solvent models, we studied the dynamic properties of both protein and water. The explicit solvent simulations showed specificity related differences in the energy landscapes and the power spectra between the two enzymes, whereas implicit solvent simulations did not. Moreover, the explicit solvent simulations demonstrated obvious distinctions in dynamic behaviors of water, such as their residence behaviors and hydrogen bonding. These results suggest that the interplay between water and enzyme is essential in determining the substrate specificity, and the detail knowledge of such interplay can greatly improve our understanding of bio-molecules.     

On the Information Content of Protein Sequences

Abstract

TATA-box binding protein (TBP) in a monomelic form and the complexes it forms with DNA have been elucidated with molecular dynamics simulations. Large TBP domain motions (bend and twist) are detected in the monomer as well as in the DNA complexes; these motions can be important for TBP binding of DNA. TBP interacts with guanine bases flanking the TATA element in the simulations of the complex; these interactions may explain the preference for guanine observed at these DNA positions. Side chains of some TBP residues at the binding interface display significant dynamic flexibility that results in ‘flipflop’ contacts involving multiple base pairs of the DNA. We discuss the possible functional significance of these observations.     

Collective motions in proteins: a covariance analysis of atomic fluctuations in molecular dynamics and normal mode simulations.

A method is described for identifying collective motions in proteins from molecular dynamics trajectories or normal mode simulations. The method makes use of the covariances of atomic positional fluctuations. It is illustrated by an analysis of the bovine pancreatic trypsin inhibitor. Comparison of the covariance and cross-correlation matrices shows that the relative motions have many similar features in the different simulations. Many regions of the protein, especially regions of secondary structure, move in a correlated manner. Anharmonic effects, which are included in the molecular dynamics simulations but not in the normal analysis, are of some importance in determining the larger scale collective motions, but not the more local fluctuations. Comparisons of molecular dynamics simulations in the present and absence of solvent indicate that the environment is of significance for the long-range motions.