共查询到20条相似文献,搜索用时 15 毫秒
1.
Margot J. Zaccardi Kathleen F. O'Rourke Eric M. Yezdimer Laura J. Loggia Svenja Woldt David D. Boehr 《Protein science : a publication of the Protein Society》2014,23(3):302-311
Substrate binding, product release, and likely chemical catalysis in the tryptophan biosynthetic enzyme indole‐3‐glycerol phosphate synthase (IGPS) are dependent on the structural dynamics of the β1α1 active‐site loop. Statistical coupling analysis and molecular dynamic simulations had previously indicated that covarying residues in the β1α1 and β2α2 loops, corresponding to Arg54 and Asn90, respectively, in the Sulfolobus sulfataricus enzyme (ssIGPS), are likely important for coordinating functional motions of these loops. To test this hypothesis, we characterized site mutants at these positions for changes in catalytic function, protein stability and structural dynamics for the thermophilic ssIGPS enzyme. Although there were only modest changes in the overall steady‐state kinetic parameters, solvent viscosity and solvent deuterium kinetic isotope effects indicated that these amino acid substitutions change the identity of the rate‐determining step across multiple temperatures. Surprisingly, the N90A substitution had a dramatic effect on the general acid/base catalysis of the dehydration step, as indicated by the loss of the descending limb in the pH rate profile, which we had previously assigned to Lys53 on the β1α1 loop. These changes in enzyme function are accompanied with a quenching of ps‐ns and µs‐ms timescale motions in the β1α1 loop as measured by nuclear magnetic resonance studies. Altogether, our studies provide structural, dynamic and functional rationales for the coevolution of residues on the β1α1 and β2α2 loops, and highlight the multiple roles that the β1α1 loop plays in IGPS catalysis. Thus, substitution of covarying residues in the active‐site β1α1 and β2α2 loops of indole‐3‐glycerol phosphate synthase results in functional, structural, and dynamic changes, highlighting the multiple roles that the β1α1 loop plays in enzyme catalysis and the importance of regulating the structural dynamics of this loop through noncovalent interactions with nearby structural elements. 相似文献
2.
Kathleen F. O'Rourke Rebecca N. D'Amico Debashish Sahu David D. Boehr 《Protein science : a publication of the Protein Society》2021,30(3):543
Experimental observations of enzymes under active turnover conditions have brought new insight into the role of protein motions and allosteric networks in catalysis. Many of these studies characterize enzymes under dynamic chemical equilibrium conditions, in which the enzyme is actively catalyzing both the forward and reverse reactions during data acquisition. We have previously analyzed conformational dynamics and allosteric networks of the alpha subunit of tryptophan synthase under such conditions using NMR. We have proposed that this working state represents a four to one ratio of the enzyme bound with the indole‐3‐glycerol phosphate substrate (E:IGP) to the enzyme bound with the products indole and glyceraldehyde‐3‐phosphate (E:indole:G3P). Here, we analyze the inactive D60N variant to deconvolute the contributions of the substrate‐ and products‐bound states to the working state. While the D60N substitution itself induces small structural and dynamic changes, the D60N E:IGP and E:indole:G3P states cannot entirely account for the conformational dynamics and allosteric networks present in the working state. The act of chemical bond breakage and/or formation, or possibly the generation of an intermediate, may alter the structure and dynamics present in the working state. As the enzyme transitions from the substrate‐bound to the products‐bound state, millisecond conformational exchange processes are quenched and new allosteric connections are made between the alpha active site and the surface which interfaces with the beta subunit. The structural ordering of the enzyme and these new allosteric connections may be important in coordinating the channeling of the indole product into the beta subunit. 相似文献
3.
Martin Sagermann Richard R. Chapleau Elaine DeLorimier Margarida Lei 《Protein science : a publication of the Protein Society》2009,18(1):217-228
Conformational changes play important roles in the regulation of many enzymatic reactions. Specific motions of side chains, secondary structures, or entire protein domains facilitate the precise control of substrate selection, binding, and catalysis. Likewise, the engineering of allostery into proteins is envisioned to enable unprecedented control of chemical reactions and molecular assembly processes. We here study the structural effects of engineered ionizable residues in the core of the glutathione‐S‐transferase to convert this protein into a pH‐dependent allosteric protein. The underlying rational of these substitutions is that in the neutral state, an uncharged residue is compatible with the hydrophobic environment. In the charged state, however, the residue will invoke unfavorable interactions, which are likely to induce conformational changes that will affect the function of the enzyme. To test this hypothesis, we have engineered a single aspartate, cysteine, or histidine residue at a distance from the active site into the protein. All of the mutations exhibit a dramatic effect on the protein's affinity to bind glutathione. Whereas the aspartate or histidine mutations result in permanently nonbinding or binding versions of the protein, respectively, mutant GST50C exhibits distinct pH‐dependent GSH‐binding affinity. The crystal structures of the mutant protein GST50C under ionizing and nonionizing conditions reveal the recruitment of water molecules into the hydrophobic core to produce conformational changes that influence the protein's active site. The methodology described here to create and characterize engineered allosteric proteins through affinity chromatography may lead to a general approach to engineer effector‐specific allostery into a protein structure. 相似文献
4.
Zeynep Kurkcuoglu Ahmet Bakan Duygu Kocaman Ivet Bahar Pemra Doruker 《PLoS computational biology》2012,8(9)
Catalytic loop motions facilitate substrate recognition and binding in many enzymes. While these motions appear to be highly flexible, their functional significance suggests that structure-encoded preferences may play a role in selecting particular mechanisms of motions. We performed an extensive study on a set of enzymes to assess whether the collective/global dynamics, as predicted by elastic network models (ENMs), facilitates or even defines the local motions undergone by functional loops. Our dataset includes a total of 117 crystal structures for ten enzymes of different sizes and oligomerization states. Each enzyme contains a specific functional/catalytic loop (10–21 residues long) that closes over the active site during catalysis. Principal component analysis (PCA) of the available crystal structures (including apo and ligand-bound forms) for each enzyme revealed the dominant conformational changes taking place in these loops upon substrate binding. These experimentally observed loop reconfigurations are shown to be predominantly driven by energetically favored modes of motion intrinsically accessible to the enzyme in the absence of its substrate. The analysis suggests that robust global modes cooperatively defined by the overall enzyme architecture also entail local components that assist in suitable opening/closure of the catalytic loop over the active site. 相似文献
5.
The emergence of compensatory drug-resistant mutations in HIV-1 protease challenges the common view of the reaction mechanism of this enzyme. Here, we address this issue by performing classical and ab initio molecular dynamics simulations (MD) on a complex between the enzyme and a peptide substrate. The classical MD calculation reveals large-scale protein motions involving the flaps and the cantilever. These motions modulate the conformational properties of the substrate at the cleavage site. The ab initio calculations show in turn that substrate motion modulates the activation free energy barrier of the enzymatic reaction dramatically. Thus, the catalytic power of the enzyme does not arise from the presence of a pre-organized active site but from the protein mechanical fluctuations. The implications of this finding for the emergence of drug-resistance are discussed. 相似文献
6.
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. 相似文献
7.
Karthik K. Shanmuganatham Rachel S. Wallace Ann Ting‐I Lee Bryce V. Plapp 《Protein science : a publication of the Protein Society》2018,27(3):750-768
The dynamics of enzyme catalysis range from the slow time scale (~ms) for substrate binding and conformational changes to the fast time (~ps) scale for reorganization of substrates in the chemical step. The contribution of global dynamics to catalysis by alcohol dehydrogenase was tested by substituting five different, conserved amino acid residues that are distal from the active site and located in the hinge region for the conformational change or in hydrophobic clusters. X‐ray crystallography shows that the structures for the G173A, V197I, I220 (V, L, or F), V222I, and F322L enzymes complexed with NAD+ and an analogue of benzyl alcohol are almost identical, except for small perturbations at the sites of substitution. The enzymes have very similar kinetic constants for the oxidation of benzyl alcohol and reduction of benzaldehyde as compared to the wild‐type enzyme, and the rates of conformational changes are not altered. Less conservative substitutions of these amino acid residues, such as G173(V, E, K, or R), V197(G, S, or T), I220(G, S, T, or N), and V222(G, S, or T) produced unstable or poorly expressed proteins, indicating that the residues are critical for global stability. The enzyme scaffold accommodates conservative substitutions of distal residues, and there is no evidence that fast, global dynamics significantly affect the rate constants for hydride transfers. In contrast, other studies show that proximal residues significantly participate in catalysis. 相似文献
8.
The cyclooxygenase (COX) enzymes are responsible for the committed step in prostaglandin biosynthesis, the generation of prostaglandin H(2). As a result, these enzymes are pharmacologically important targets for nonsteroidal antiinflammatory drugs, such as aspirin and newer COX-2 selective inhibitors. The cyclooxygenases are functional homodimers, and each subunit contains both a cyclooxygenase and a peroxidase active site. These enzymes are quite interesting mechanistically, as the conversion of arachidonic acid to prostaglandin H(2) requires two oxygenation and two cyclization reactions, resulting in the formation of five new chiral centers with nearly absolute regio- and stereochemical fidelity. We have used molecular dynamics (MD) simulations to investigate the equilibrium behavior of both COX-1 and COX-2 enzyme isoforms with bound arachidonate. These simulations were compared with reference simulations of arachidonate in solution to explore the effect of enzyme on substrate conformation and positioning in the active site. The simulations suggest that the substrate has greater conformational freedom in the COX-2 active site, consistent with the larger COX-2 active site volume observed in X-ray crystal structures. The simulations reveal different conformational behavior for arachidonate in each subunit over the course of extended equilibrium MD simulations. The simulations also provide detailed information for several protein channels that might be important for oxygen and water transport to or from active sites or for intermediate trafficking between the cyclooxygenase and peroxidase active sites. The detailed comparisons for COX-1 versus COX-2 active site structural fluctuations may also provide useful information for design of new isozyme-selective inhibitors. 相似文献
9.
Protein function is a dynamic property closely related to the conformational mechanisms of protein structure in its physiological environment. To understand and control the function of target proteins, it becomes increasingly important to develop methods and tools for predicting collective motions at the molecular level. In this article, we review computational methods for predicting conformational dynamics and discuss software tools for data analysis. In particular, we discuss a high-throughput, web-based system called iGNM for protein structural dynamics. iGNM contains a database of protein motions for more than 20 000 PDB structures and supports online calculations for newly deposited PDB structures or user-modified structures. iGNM allows dynamics analysis of protein structures ranging from enzymes to large complexes and assemblies, and enables the exploration of protein sequence-structure-dynamics-function relations. 相似文献
10.
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. 相似文献
11.
Src tyrosine kinases are essential in numerous cell signaling pathways, and improper functioning of these enzymes has been implicated in many diseases. The activity of Src kinases is regulated by conformational activation, which involves several structural changes within the catalytic domain (CD): the orientation of two lobes of CD; rearrangement of the activation loop (A-loop); and movement of an alpha-helix (alphaC), which is located at the interface between the two lobes, into or away from the catalytic cleft. Conformational activation was investigated using biased molecular dynamics to explore the transition pathway between the active and the down-regulated conformation of CD for the Src-kinase family member Lyn kinase, and to gain insight into the interdependence of these changes. Lobe opening is observed to be a facile motion, whereas movement of the A-loop motion is more complex requiring secondary structure changes as well as communication with alphaC. A key result is that the conformational transition involves a switch in an electrostatic network of six polar residues between the active and the down-regulated conformations. The exchange between interactions links the three main motions of the CD. Kinetic experiments that would demonstrate the contribution of the switched electrostatic network to the enzyme mechanism are proposed. Possible implications for regulation conferred by interdomain interactions are also discussed. 相似文献
12.
The decryption of sequence of structural events during protein conformational transitions is essential to a detailed understanding of molecular functions ofvarious biological nanomachines. Coarse‐grained models have proven useful by allowing highly efficient simulations of protein conformational dynamics. By combining two coarse‐grained elastic network models constructed based on the beginning and end conformations of a transition, we have developed an interpolated elastic network model to generate a transition pathway between the two protein conformations. For validation, we have predicted the order of local and global conformational changes during key ATP‐driven transitions in three important biological nanomachines (myosin, F1 ATPase and chaperonin GroEL). We have found that the local conformational change associated with the closing of active site precedes the global conformational change leading to mechanical motions. Our finding is in good agreement with the distribution of intermediate experimental structures, and it supports the importance of local motions at active site to drive or gate various conformational transitions underlying the workings of a diverse range of biological nanomachines. Proteins 2010. © 2010 Wiley‐Liss, Inc. 相似文献
13.
Large-scale conformational change is a common feature in the catalytic cycles of enzymes. Many enzymes function as homodimers with active sites that contain elements from both chains. Symmetric and anti-symmetric cooperative motions in homodimers can potentially lead to correlated active site opening and/or closure, likely to be important for ligand binding and release. Here, we examine such motions in two different domain-swapped homodimeric enzymes: the DcpS scavenger decapping enzyme and citrate synthase. We use and compare two types of all-atom simulations: conventional molecular dynamics simulations to identify physically meaningful conformational ensembles, and rapid geometric simulations of flexible motion, biased along normal mode directions, to identify relevant motions encoded in the protein structure. The results indicate that the opening/closure motions are intrinsic features of both unliganded enzymes. In DcpS, conformational change is dominated by an anti-symmetric cooperative motion, causing one active site to close as the other opens; however a symmetric motion is also significant. In CS, we identify that both symmetric (suggested by crystallography) and asymmetric motions are features of the protein structure, and as a result the behaviour in solution is largely non-cooperative. The agreement between two modelling approaches using very different levels of theory indicates that the behaviours are indeed intrinsic to the protein structures. Geometric simulations correctly identify and explore large amplitudes of motion, while molecular dynamics simulations indicate the ranges of motion that are energetically feasible. Together, the simulation approaches are able to reveal unexpected functionally relevant motions, and highlight differences between enzymes. 相似文献
14.
The hydrolysis of GTP in p21(ras) triggers conformational changes that regulate the ras/ERK signaling pathway. An important active site residue is Gln61, which has been found to be mutated in 30% of human tumors. The dynamics of the active site conformation is studied by using molecular dynamics simulation of two independent structures of the GTP-bound uncomplexed enzyme. Two distinct conformations of the enzyme are observed, in which the side-chain residue Gln61 is in different orientations. Essential dynamics analysis is used to describe the essential motions in the transition between the two conformations. Results are compared with earlier simulations of p21(ras) and its complex with GTPase activating protein p21-GAP. 相似文献
15.
Spiwok V Lipovová P Skálová T Dusková J Dohnálek J Hasek J Russell NJ Králová B 《Journal of molecular modeling》2007,13(4):485-497
Enzymes from cold-adapted species are significantly more active at low temperatures, even those close to zero Celsius, but
the rationale of this adaptation is complex and relatively poorly understood. It is commonly stated that there is a relationship
between the flexibility of an enzyme and its catalytic activity at low temperature. This paper gives the results of a study
using molecular dynamics simulations performed for five pairs of enzymes, each pair comprising a cold-active enzyme plus its
mesophilic or thermophilic counterpart. The enzyme pairs included α-amylase, citrate synthase, malate dehydrogenase, alkaline
protease and xylanase. Numerous sites with elevated flexibility were observed in all enzymes; however, differences in flexibilities
were not striking. Nevertheless, amino acid residues common in both enzymes of a pair (not present in insertions of a structure
alignment) are generally more flexible in the cold-active enzymes. The further application of principle component analysis
to the protein dynamics revealed that there are differences in the rate and/or extent of opening and closing of the active
sites. The results indicate that protein dynamics play an important role in catalytic processes where structural rearrangements,
such as those required for active site access by substrate, are involved. They also support the notion that cold adaptation
may have evolved by selective changes in regions of enzyme structure rather than in global change to the whole protein.
Figure Collective motions in Cα atoms of the active site of cold-active xylanase
Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. 相似文献
16.
Relationship between stability and activity of enzymes is maintained by underlying conformational flexibility. In thermophilic enzymes, a decrease in flexibility causes low enzyme activity while in less stable proteins such as mesophiles and psychrophiles, an increase in flexibility is associated with enhanced enzyme activity. Recently, we identified a mutant of a lipase whose stability and activity were enhanced simultaneously. In this work, we probed the conformational dynamics of the mutant and the wild type lipase, particularly flexibility of their active site using molecular dynamic simulations and time-resolved fluorescence techniques. In contrast to the earlier observations, our data show that active site of the mutant is more rigid than wild type enzyme. Further investigation suggests that this lipase needs minimal reorganization/flexibility of active site residues during its catalytic cycle. Molecular dynamic simulations suggest that catalytically competent active site geometry of the mutant is relatively more preserved than wild type lipase, which might have led to its higher enzyme activity. Our study implies that widely accepted positive correlation between conformation flexibility and enzyme activity need not be stringent and draws attention to the possibility that high enzyme activity can still be accomplished in a rigid active site and stable protein structures. This finding has a significant implication towards better understanding of involvement of dynamic motions in enzyme catalysis and enzyme engineering through mutations in active site. 相似文献
17.
Safieh Tork Ladani 《Molecular simulation》2014,40(10-11):765-776
The essential role of enzymes in biological processes has continually ignited sparks of interest in their mechanism of action. Fully understanding the mechanism of enzymes has broad implications in protein engineering and drug design. The more than five order of magnitude speed-up in the rate of peptidyl–prolyl cis–trans isomerisation by cyclophilin A (CypA) has been the target of intense research. CypA serves as a tractable model system, because it reversibly catalyses the rotation around peptidyl–prolyl bonds without any bond breakage or formation. Here, we discuss the results of recent computational approaches used to study the mechanism of CypA. We highlight the critical role of enzyme and substrate conformational dynamics in the developing interactions as the substrate approaches the transition state that results in an astonishing enhancement of isomerisation rate. The rate of isomerisation is affected by the intricate coupling between the dynamics of the substrate, enzyme and solvent. CypA binds its substrates via conformational selection, where rearrangements of key active site residues are necessary for substrate recognition. The conformational plasticity of the active site allows the enzyme to accommodate the most favourable interactions with the transition state that can be exploited for structure-based drug design. 相似文献
18.
Thana Sutthibutpong Triwit Rattanarojpong Pongsak Khunrae 《Journal of biomolecular structure & dynamics》2013,31(15):3978-3992
Local conformational changes and global unfolding pathways of wildtype xyn11A recombinant and its mutated structures were studied through a series of atomistic molecular dynamics (MD) simulations, along with enzyme activity assays at three incubation temperatures to investigate the effects of mutations at three different sites to the thermostability. The first mutation was to replace an unstable negatively charged residue at a surface beta turn near the active site (D32G) by a hydrophobic residue. The second mutation was to create a disulphide bond (S100C/N147C) establishing a strong connection between an alpha helix and a distal beta hairpin associated with the thermally sensitive Thumb loop, and the third mutation add an extra hydrogen bond (A155S) to the same alpha helix. From the MD simulations performed, MM/PBSA energy calculations of the unfolding energy were in a good agreement with the enzyme activities measured from the experiment, as all mutated structures demonstrated the improved thermostability, especially the S100C/N147C proved to be the most stable mutant both by the simulations and the experiment. Local conformational analysis at the catalytic sites and the xylan access region also suggested that mutated xyn11A structures could accommodate xylan binding. However, the analysis of global unfolding pathways showed that structural disruptions at the beta sheet regions near the N-terminal were still imminent. These findings could provide the insight on the molecular mechanisms underlying the enhanced thermostability due to mutagenesis and changes in the protein unfolding pathways for further protein engineering of the GH11 family xylanase enzymes. 相似文献
19.
Despite the increasing number of successful determinations of complex protein structures the understanding of their dynamics properties is still rather limited. Using X-ray crystallography, we demonstrate that ribonuclease A (RNase A) undergoes significant domain motions upon ligand binding. In particular, when cytidine 2'-monophosphate binds to RNase A, the structure of the enzyme becomes more compact. Interestingly, our data also show that these structural alterations are fully reversible in the crystal state. These findings provide structural bases for the dynamic behavior of RNase A in the binding of the substrate shown by Petsko and coworkers (Rasmussen et al. Nature 1992;357:423-424). These subtle domain motions may assume functional relevance for more complex system and may play a significant role in the cooperativity of oligomeric enzymes. 相似文献
20.
A special class of proteins adopts an inactive conformation in aqueous solution and activates at an interface (such as the surface of lipid droplet) by switching their conformations. Lipase, an essential enzyme for breaking down lipids, serves as a model system for studying such interfacial proteins. The underlying conformational switch of lipase induced by solvent condition is achieved through changing the status of the gated substrate‐access channel. Interestingly, a lipase was also reported to exhibit pressure activation, which indicates it is drastically active at high hydrostatic pressure. To unravel the molecular mechanism of this unusual phenomenon, we examined the structural changes induced by high hydrostatic pressures (up to 1500 MPa) using molecular dynamics simulations. By monitoring the width of the access channel, we found that the protein undergoes a conformational transition and opens the access channel at high pressures (>100 MPa). Particularly, a disordered amphiphilic α5 region of the protein becomes ordered at high pressure. This positive correlation between the channel opening and α5 ordering is consistent with the early findings of the gating motion in the presence of a water–oil interface. Statistical analysis of the ensemble of conformations also reveals the essential collective motions of the protein and how these motions contribute to gating. Arguments are presented as to why heightened sensitivity to high‐pressure perturbation can be a general feature of switchable interfacial proteins. Further mutations are also suggested to validate our observations. Proteins 2016; 84:820–827. © 2016 Wiley Periodicals, Inc. 相似文献