Catalytic center assembly of HPPK as revealed by the crystal structure of a ternary complex at 1.25 A resolution

BACKGROUND: Folates are essential for life. Unlike mammals, most microorganisms must synthesize folates de novo. 6-Hydroxymethyl-7, 8-dihydropterin pyrophosphokinase (HPPK) catalyzes pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), the first reaction in the folate pathway, and therefore is an ideal target for developing novel antimicrobial agents. HPPK from Escherichia coli is a 158-residue thermostable protein that provides a convenient model system for mechanistic studies. Crystal structures have been reported for HPPK without bound ligand, containing an HP analog, and complexed with an HP analog, two Mg(2+) ions, and ATP. RESULTS: We present the 1.25 A crystal structure of HPPK in complex with HP, two Mg(2+) ions, and AMPCPP (an ATP analog that inhibits the enzymatic reaction). This structure demonstrates that the enzyme seals the active center where the reaction occurs. The comparison with unligated HPPK reveals dramatic conformational changes of three flexible loops and many sidechains. The coordination of Mg(2+) ions has been defined and the roles of 26 residues have been derived. CONCLUSIONS: HPPK-HP-MgAMPCPP mimics most closely the natural ternary complex of HPPK and provides details of protein-substrate interactions. The coordination of the two Mg(2+) ions helps create the correct geometry for the one-step reaction of pyrophosphoryl transfer, for which we suggest an in-line single displacement mechanism with some associative character in the transition state. The rigidity of the adenine-binding pocket and hydrogen bonds are responsible for adenosine specificity. The nonconserved residues that interact with the substrate might be responsible for the species-dependent properties of an isozyme.     

600 ps Molecular dynamics reveals stable substructures and flexible hinge points in cAMP dependent protein kinase

Molecular dynamics simulations of the catalytic subunit of cAMP dependent protein kinase (cAPK) have been performed in an aqueous environment. The relations among the protein hydrogen‐bonding network, secondary structural elements, and the internal motions of rigid domains were examined. The values of fluctuations of protein dihedral angles during dynamics show quite distinct maxima in the regions of loops and minima in the regions of α‐helices and β‐strands. Analyses of conformation snapshots throughout the run show stable subdomains and indicate that these rigid domains are constrained during the dynamics by a stable network of hydrogen bonds. The most stable subdomain during the dynamics was in the small lobe including part of the carboxy‐terminal tail. The most significant flexible region was the highly conserved glycine‐rich loop between β strands 1 and 2 in the small lobe. Many of the main chain dihedral angle changes measured in a comparison of the crystallographic structures of “open” and “closed” conformations of cAPK correspond to the highly flexible residues found during dynamics. © 1999 John Wiley & Sons, Inc. Biopoly 50: 513–524, 1999     

Comparison of full-atomic and coarse-grained models to examine the molecular fluctuations of c-AMP dependent protein kinase

Molecular fluctuations of the native conformation of c-AMP dependent protein kinase (cAPK) have been investigated with three different approaches. The first approach is the full atomic normal mode analysis (NMA) with empirical force fields. The second and third approaches are based on a coarse-grained model with a single single-parameter- harmonic potential between close residues in the crystal structure of the molecule without any residue specificity. The second method calculates only the magnitude of fluctuations whereas the third method is developed to find the directionality of the fluctuations which are essential to understand the functional importance of biological molecules. The aim, in this study, is to determine whether using such coarse-grained models are appropriate for elucidating the global dynamic characteristics of large proteins which reduces the size of the system at least by a factor of ten. The mean-square fluctuations of C(alpha) atoms and the residue cross-correlations are obtained by three approaches. These results are then compared to test the results of coarse grained models on the overall collective motions. All three of the approaches show that highly flexible regions correspond to the activation and solvent exposed loops, whereas the conserved residues (especially in substrate binding regions) exhibit almost no flexibility, adding stability to the structure. The anti-correlated motions of the two lobes of the catalytic core provide flexibility to the molecule. High similarities among the results of these methods indicate that the slowest modes governing the most global motions are preserved in the coarse grained models for proteins. This finding may suggest that the general shapes of the structures are representative of their dynamic characteristics and the dominant motions of protein structures are robust at coarse-grained levels.     

Comparison of Full-atomic and Coarse-grained Models to Examine the Molecular Fluctuations of c-AMP Dependent Protein Kinase

Abstract

Molecular fluctuations of the native conformation of c-AMP dependent protein kinase (cAPK) have been investigated with three different approaches. The first approach is the full atomic normal mode analysis (NMA) with empirical force fields. The second and third approaches are based on a coarse-grained model with a single single-parameter- harmonic potential between close residues in the crystal structure of the molecule without any residue specificity. The second method calculates only the magnitude of fluctuations whereas the third method is developed to find the directionality of the fluctuations which are essential to understand the functional importance of biological molecules. The aim, in this study, is to determine whether using such coarse-grained models are appropriate for elucidating the global dynamic characteristics of large proteins which reduces the size of the system at least by a factor of ten. The mean-square fluctuations of C^α atoms and the residue cross-correlations are obtained by three approaches. These results are then compared to test the results of coarse grained models on the overall collective motions. AH three of the approaches show that highly flexible regions correspond to the activation and solvent exposed loops, whereas the conserved residues (especially in substrate binding regions) exhibit almost no flexibility, adding stability to the structure. The anti-correlated motions of the two lobes of the catalytic core provide flexibility to the molecule. High similarities among the results of these methods indicate that the slowest modes governing the most global motions are preserved in the coarse grained models for proteins. This finding may suggest that the general shapes of the structures are representative of their dynamic characteristics and the dominant motions of protein structures are robust at coarse-grained levels.     

Unusual conformational changes in 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase as revealed by X-ray crystallography and NMR

The crystal structure of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) in complex with MgADP has been determined at 1.5-A resolution with a crystallographic R factor of 0.191. The solution structure of HPPK in complex with Mg(2+) and beta,gamma-methyleneadenosine 5'-triphosphate (MgAMPPCP) has been determined using a simulated annealing protocol with 3,523 experimental NMR restraints. The root mean square deviation of the ensemble of 20 refined conformers that represent the solution structure from the mean coordinate set derived from them is 0.74 +/- 0.26 A for all backbone atoms and 0.49 +/- 0.22 A when residues Pro(14), Pro(44)-Gln(50), and Arg(84)-Pro(91) are excluded. Binding of MgADP causes significant changes in the conformation and dynamical property of three loops of HPPK that are involved in catalysis. A dramatic, unusual conformational change is that loop 3 moves away from the active center significantly with some residues moving by >17 A. The binding of MgADP also stabilizes loop 1 and loop 3 but makes loop 2 more mobile. Very similar conformational and dynamical changes are observed in the NMR solution structure of HPPK.MgAMPPCP. The conformational and dynamical changes may play important roles in both substrate binding and product release in the catalytic cycle.     

Mechanism of the conformational transitions in 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase as revealed by NMR spectroscopy

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), leading to the biosynthesis of folate cofactors. HPPK undergoes dramatic conformational changes during its catalytic cycle, and the conformational changes are essential for enzymatic catalysis. Thus, the enzyme is not only an attractive target for developing antimicrobial agents but also an excellent model system for studying the catalytic mechanism of enzymatic pyrophosphoryl transfer as well as the role of protein dynamics in enzymatic catalysis. In the present study, we report the NMR solution structures of the binary complex HPPK*MgAMPCPP and the ternary complex HPPK*MgAMPCPP*DMHP, where alpha,beta-methyleneadenosine triphosphate (AMPCPP) and 7, 7-dimethyl-6-hydroxypterin (DMHP) are the analogues of the substrates ATP and HP, respectively. The results suggest that the three catalytic loops of the binary complex of HPPK can assume multiple conformations in slow exchanges as evidenced by multiple sets of NMR signals for several residues in loops 2 and 3 and the very weak or missing NH cross-peaks for several residues in loops 1 and 3. However, the ternary complex shows only one set of NMR signals, and the cross-peak intensities are rather uniform, suggesting that the binding of the second substrate shifts the multiple conformations of the binary complex to an apparently single conformation of the ternary complex. The NMR behaviors and conformations of the binary complex HPPK*MgAMPCPP are significantly different from those of HPPK in complex with Mgbeta,gamma-methyleneadenosine triphosphate (MgAMPPCP). It is suggested that the conformational properties of the binary substrate complex HPPK*MgATP be represented by those of HPPK*MgAMPCPP, because MgAMPCPP is a better MgATP analogue for HPPK with respect to both binding affinity and bound conformation.     

Backbone dynamics of the ribonuclease binase active site area using multinuclear ((15)N and (13)CO) NMR relaxation and computational molecular dynamics

The nano-pico second backbone dynamics of the ribonuclease binase, homologous to barnase, is investigated with (15)N, (13)C NMR relaxation at 11.74 and 18.78 T and with a 1.1 ns molecular dynamics simulation. The data are compared with the temperature factors reported for the X-ray structure of this enzyme. The molecular dynamics and X-ray data correspond well and predict motions in the loops 56-61 and 99-104 that contain residues that specifically recognize substrate and are catalytic (His101), respectively. In contrast, the (15)N relaxation data indicate that these loops are mostly ordered at the nano-pico second time scale. Nano-pico second motions in the recognition loop 56-61 are evident from (13)CO-(13)C cross relaxation data, but the mobility of the catalytic loop 99-104 is not detected by (13)CO cross relaxation either. From the results of this and previous work [Wang, L., Pang, Y., Holder, T., Brender, J. R., Kurochkin, A., and Zuiderweg, E. R. P. (2001) Proc. Natl. Acad. Sci. U.S.A., 98, 7684-7689], the following dynamical characterization of the active site area of binase emerges: a beta sheet, rigid at all probed time scales, supports the catalytic residue Glu 72. Both substrate-encapsulating loops are mobile on both fast and slow time scales, but the fast motions of the loop which contains the other catalytic residue, His 101, as predicted by B-factors and computational molecular dynamics is not detected by NMR relaxation. This work strongly argues for the use of several measures in the study of protein dynamics.     

Structure of S. aureus HPPK and the discovery of a new substrate site inhibitor

The first structural and biophysical data on the folate biosynthesis pathway enzyme and drug target, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (SaHPPK), from the pathogen Staphylococcus aureus is presented. HPPK is the second essential enzyme in the pathway catalysing the pyrophosphoryl transfer from cofactor (ATP) to the substrate (6-hydroxymethyl-7,8-dihydropterin, HMDP). In-silico screening identified 8-mercaptoguanine which was shown to bind with an equilibrium dissociation constant, K_d, of ∼13 µM as measured by isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). An IC₅₀ of ∼41 µM was determined by means of a luminescent kinase assay. In contrast to the biological substrate, the inhibitor has no requirement for magnesium or the ATP cofactor for competitive binding to the substrate site. The 1.65 Å resolution crystal structure of the inhibited complex showed that it binds in the pterin site and shares many of the key intermolecular interactions of the substrate. Chemical shift and ¹⁵N heteronuclear NMR measurements reveal that the fast motion of the pterin-binding loop (L2) is partially dampened in the SaHPPK/HMDP/α,β-methylene adenosine 5′-triphosphate (AMPCPP) ternary complex, but the ATP loop (L3) remains mobile on the µs-ms timescale. In contrast, for the SaHPPK/8-mercaptoguanine/AMPCPP ternary complex, the loop L2 becomes rigid on the fast timescale and the L3 loop also becomes more ordered – an observation that correlates with the large entropic penalty associated with inhibitor binding as revealed by ITC. NMR data, including ¹⁵N-¹H residual dipolar coupling measurements, indicate that the sulfur atom in the inhibitor is important for stabilizing and restricting important motions of the L2 and L3 catalytic loops in the inhibited ternary complex. This work describes a comprehensive analysis of a new HPPK inhibitor, and may provide a foundation for the development of novel antimicrobials targeting the folate biosynthetic pathway.     

Method for identification of rigid domains and hinge residues in proteins based on exhaustive enumeration

Many proteins undergo large‐scale motions where relatively rigid domains move against each other. The identification of rigid domains, as well as the hinge residues important for their relative movements, is important for various applications including flexible docking simulations. In this work, we develop a method for protein rigid domain identification based on an exhaustive enumeration of maximal rigid domains, the rigid domains not fully contained within other domains. The computation is performed by mapping the problem to that of finding maximal cliques in a graph. A minimal set of rigid domains are then selected, which cover most of the protein with minimal overlap. In contrast to the results of existing methods that partition a protein into non‐overlapping domains using approximate algorithms, the rigid domains obtained from exact enumeration naturally contain overlapping regions, which correspond to the hinges of the inter‐domain bending motion. The performance of the algorithm is demonstrated on several proteins. Proteins 2015; 83:1054–1067. © 2015 Wiley Periodicals, Inc.     

Stability domains, substrate-induced conformational changes, and hinge-bending motions in a psychrophilic phosphoglycerate kinase. A microcalorimetric study

The cold-active phosphoglycerate kinase from the Antarctic bacterium Pseudomonas sp. TACII18 exhibits two distinct stability domains in the free, open conformation. It is shown that these stability domains do not match the structural N- and C-domains as the heat-stable domain corresponds to about 80 residues of the C-domain, including the nucleotide binding site, whereas the remaining of the protein contributes to the main heat-labile domain. This was demonstrated by spectroscopic and microcalorimetric analyses of the native enzyme, of its mutants, and of the isolated recombinant structural domains. It is proposed that the heat-stable domain provides a compact structure improving the binding affinity of the nucleotide, therefore increasing the catalytic efficiency at low temperatures. Upon substrate binding, the enzyme adopts a uniformly more stable closed conformation. Substrate-induced stability changes suggest that the free energy of ligand binding is converted into an increased conformational stability used to drive the hinge-bending motions and domain closure.     

Multiscale modeling of macromolecular conformational changes combining concepts from rigidity and elastic network theory

The development of a two-step approach for multiscale modeling of macromolecular conformational changes is based on recent developments in rigidity and elastic network theory. In the first step, static properties of the macromolecule are determined by decomposing the molecule into rigid clusters by using the graph-theoretical approach FIRST and an all-atom representation of the protein. In this way, rigid clusters are not limited to consist of residues adjacent in sequence or secondary structure elements as in previous studies. Furthermore, flexible links between rigid clusters are identified and can be modeled as such subsequently. In the second step, dynamical properties of the molecule are revealed by the rotations-translations of blocks approach (RTB) using an elastic network model representation of the coarse-grained protein. In this step, only rigid body motions are allowed for rigid clusters, whereas links between them are treated as fully flexible. The approach was tested on a data set of 10 proteins that showed conformational changes on ligand binding. For efficiency, coarse-graining the protein results in a remarkable reduction of memory requirements and computational times by factors of 9 and 27 on average and up to 25 and 125, respectively. For accuracy, directions and magnitudes of motions predicted by our approach agree well with experimentally determined ones, despite embracing in extreme cases >50% of the protein into one rigid cluster. In fact, the results of our method are in general comparable with when no or a uniform coarse-graining is applied; and the results are superior if the movement is dominated by loop or fragment motions. This finding indicates that explicitly distinguishing between flexible and rigid regions is advantageous when using a simplified protein representation in the second step. Finally, motions of atoms in rigid clusters are also well predicted by our approach, which points to the need to consider mobile protein regions in addition to flexible ones when modeling correlated motions.     

Crystal contact-free conformation of an intrinsically flexible loop in protein crystal: Tim21 as the case study

BackgroundIn protein crystals, flexible loops are frequently deformed by crystal contacts, whereas in solution, the large motions result in the poor convergence of such flexible loops in NMR structure determinations. We need an experimental technique to characterize the structural and dynamic properties of intrinsically flexible loops of protein molecules.MethodsWe designed an intended crystal contact-free space (CCFS) in protein crystals, and arranged the flexible loop of interest in the CCFS. The yeast Tim 21 protein was chosen as the model protein, because one of the loops (loop 2) is distorted by crystal contacts in the conventional crystal.ResultsYeast Tim21 was fused to the MBP protein by a rigid α-helical linker. The space created between the two proteins was used as the CCFS. The linker length provides adjustable freedom to arrange loop 2 in the CCFS. We re-determined the NMR structure of yeast Tim21, and conducted MD simulations for comparison. Multidimensional scaling was used to visualize the conformational similarity of loop 2. We found that the crystal contact-free conformation of loop 2 is located close to the center of the ensembles of the loop 2 conformations in the NMR and MD structures.ConclusionsLoop 2 of yeast Tim21 in the CCFS adopts a representative, dominant conformation in solution.General significanceNo single powerful technique is available for the characterization of flexible structures in protein molecules. NMR analyses and MD simulations provide useful, but incomplete information. CCFS crystallography offers a third route to this goal.     

Relating molecular flexibility to function: a case study of tubulin

Microtubules (MT), along with a variety of associated motor proteins, are involved in a range of cellular functions including vesicle movement, chromosome segregation, and cell motility. MTs are assemblies of heterodimeric proteins, alpha beta-tubulins, the structure of which has been determined by electron crystallography of zinc-induced, pacilitaxel-stabilized tubulin sheets. These data provide a basis for examining relationships between structural features and protein function. Here, we study the fluctuation dynamics of the tubulin dimer with the aim of elucidating its functional motions relevant to substrate binding, polymerization/depolymerization and MT assembly. A coarse-grained model, harmonically constrained according to the crystal structure, is used to explore the global dynamics of the dimer. Our results identify six regions of collective motion, comprised of structurally close but discontinuous sequence fragments, observed only in the dimeric form, dimerization being a prerequisite for domain identification. Boundaries between regions of collective motions appear to act as linkages, found primarily within secondary-structure elements that lack sequence conservation, but are located at minima in the fluctuation curve, at positions of hydrophobic residues. Residue fluctuations within these domains identify the most mobile regions as loops involved in recognition of the adjacent regions. The least mobile regions are associated with nucleotide binding sites where lethal mutations occur. The functional coupling of motions between and within regions identifies three global motions: torsional and wobbling movements, en bloc, between the alpha- and beta-tubulin monomers, and stretching longitudinally. Further analysis finds the antitumor drug pacilitaxel (TaxotereR) to reduce flexibility in the M loop of the beta-tubulin monomer; an effect that may contribute to tightening lateral interactions between protofilaments assembled into MTs. Our analysis provides insights into relationships between intramolecular tubulin movements of MT organization and function.     

FLIPDock: docking flexible ligands into flexible receptors

Conformational changes of biological macromolecules when binding with ligands have long been observed and remain a challenge for automated docking methods. Here we present a novel protein-ligand docking software called FLIPDock (Flexible LIgand-Protein Docking) allowing the automated docking of flexible ligand molecules into active sites of flexible receptor molecules. In FLIPDock, conformational spaces of molecules are encoded using a data structure that we have developed recently called the Flexibility Tree (FT). While the FT can represent fully flexible ligands, it was initially designed as a hierarchical and multiresolution data structure for the selective encoding of conformational subspaces of large biological macromolecules. These conformational subspaces can be built to span a range of conformations important for the biological activity of a protein. A variety of motions can be combined, ranging from domains moving as rigid bodies or backbone atoms undergoing normal mode-based deformations, to side chains assuming rotameric conformations. In addition, these conformational subspaces are parameterized by a small number of variables which can be searched during the docking process, thus effectively modeling the conformational changes in a flexible receptor. FLIPDock searches the variables using genetic algorithm-based search techniques and evaluates putative docking complexes with a scoring function based on the AutoDock3.05 force-field. In this paper, we describe the concepts behind FLIPDock and the overall architecture of the program. We demonstrate FLIPDock's ability to solve docking problems in which the assumption of a rigid receptor previously prevented the successful docking of known ligands. In particular, we repeat an earlier cross docking experiment and demonstrate an increased success rate of 93.5%, compared to original 72% success rate achieved by AutoDock over the 400 cross-docking calculations. We also demonstrate FLIPDock's ability to handle conformational changes involving backbone motion by docking balanol to an adenosine-binding pocket of protein kinase A.     

Conformational changes and allosteric communications in human serum albumin due to ligand binding

It is well recognized that knowledge of structure alone is not sufficient to understand the fundamental mechanism of biomolecular recognition. Information of dynamics is necessary to describe motions involving relevant conformational states of functional importance. We carried out principal component analysis (PCA) of structural ensemble, derived from 84 crystal structures of human serum albumin (HSA) with different ligands and/or different conditions, to identify the functionally important collective motions, and compared with the motions along the low-frequency modes obtained from normal mode analysis of the elastic network model (ENM) of unliganded HSA. Significant overlap is observed in the collective motions derived from PCA and ENM. PCA and ENM analysis revealed that ligand selects the most favored conformation from accessible equilibrium structures of unliganded HSA. Further, we analyzed dynamic network obtained from molecular dynamics simulations of unliganded HSA and fatty acids- bound HSA. Our results show that fatty acids-bound HSA has more robust community network with several routes to communicate among different parts of the protein. Critical nodes (residues) identified from dynamic network analysis are in good agreement with allosteric residues obtained from sequence-based statistical coupling analysis method. This work underscores the importance of intrinsic structural dynamics of proteins in ligand recognition and can be utilized for the development of novel drugs with optimum activity.     

Dynamic roles of arginine residues 82 and 92 of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: crystallographic studies

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), the first reaction in the folate biosynthetic pathway. Arginine residues 82 and 92, strictly conserved in 35 HPPK sequences, play dynamic roles in the catalytic cycle of the enzyme. At 0.89-A resolution, two distinct conformations are observed for each of the two residues in the crystal structure of the wild-type HPPK in complex with two HP variants, two Mg(2+) ions, and an ATP analogue. Structural information suggests that R92 first binds to the alpha-phosphate group of ATP and then shifts to interact with the beta-phosphate as R82, which initially does not bind to ATP, moves in and binds to alpha-phosphate when the pyrophosphoryl transfer is about to occur. The dynamic roles of R82 and R92 are further elucidated by five more crystal structures of two mutant proteins, R82A and R92A, with and without bound ligands. Two oxidized forms of HP are observed with an occupancy ratio of 0.50:0.50 in the 0.89-A structure. The oxidation of HP has significant impact on its binding to the protein as well as the conformation of nearby residue W89.     

Rigid‐body motions of interacting proteins dominate multispecific binding of ubiquitin in a shape‐dependent manner

To understand the dynamic aspects of multispecificity of ubiquitin, we studied nine ubiquitin–ligand (partner protein) complexes by normal mode analysis based on an elastic network model. The coupling between ubiquitin and ligand motions was analyzed by decomposing it into rigid‐body (external) and vibrational (internal) motions of each subunit. We observed that in total the external motions in one of the subunits largely dominated the coupling. The combination of external motions of ubiquitin and the ligands showed general trends of rotations and translations. Moreover, we observed that the rotational motions of ubiquitin were correlated to the ligand orientations. We also identified ubiquitin atomic vibrations that differentiated the orientation of the ligand molecule. We observed that the extents of coupling were correlated to the shapes of the ligands, and this trend was more pronounced when the coupling involved vibrational motions of the ligand. In conclusion, an intricate interplay between internal and external motions of ubiquitin and the ligands help understand the dynamics of multispecificity, which is mostly guided by the shapes of the ligands and the complex. Proteins 2014; 82:77–89. © 2013 Wiley Periodicals, Inc.     

Coupling between Catalytic Loop Motions and Enzyme Global Dynamics

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.     

Hierarchical domain‐motion analysis of conformational changes in sarcoplasmic reticulum Ca2+‐ATPase

Sarco(endo)plasmic reticulum Ca²⁺‐ATPase transports two Ca²⁺ per ATP‐hydrolyzed across biological membranes against a large concentration gradient by undergoing large conformational changes. Structural studies with X‐ray crystallography revealed functional roles of coupled motions between the cytoplasmic domains and the transmembrane helices in individual reaction steps. Here, we employed “Motion Tree (MT),” a tree diagram that describes a conformational change between two structures, and applied it to representative Ca²⁺‐ATPase structures. MT provides information of coupled rigid‐body motions of the ATPase in individual reaction steps. Fourteen rigid structural units, “common rigid domains (CRDs)” are identified from seven MTs throughout the whole enzymatic reaction cycle. CRDs likely act as not only the structural units, but also the functional units. Some of the functional importance has been newly revealed by the analysis. Stability of each CRD is examined on the morphing trajectories that cover seven conformational transitions. We confirmed that the large conformational changes are realized by the motions only in the flexible regions that connect CRDs. The Ca²⁺‐ATPase efficiently utilizes its intrinsic flexibility and rigidity to response different switches like ligand binding/dissociation or ATP hydrolysis. The analysis detects functional motions without extensive biological knowledge of experts, suggesting its general applicability to domain movements in other membrane proteins to deepen the understanding of protein structure and function. Proteins 2015; 83:746–756. © 2015 Wiley Periodicals, Inc.     

Structural and functional relationships in two families of beta-1,4-glycanases.

CenA and Cex are beta-1,4-glycanases produced by the cellulolytic bacterium Cellulomonas fimi. Both enzymes are composed of two domains and contain six Cys residues. Two disulfide bonds were assigned in both enzymes by peptide analysis of the isolated catalytic domains. A further disulfide bond was deduced in both cellulose-binding domains from the absence of free thiols under denaturing conditions. Corresponding Cys residues are conserved in eight of nine other known C. fimi-type cellulose-binding domains. CenA and Cex belong to families B and F, respectively, in the classification of beta-1,4-glucanases and beta-1,4-xylanases based on similarities in catalytic domain primary structure. Disulfide bonds in the CenA catalytic domain correspond to the two disulfide bonds in the catalytic domain of Trichoderma reesei cellobiohydrolase II (family B) which stabilize loops forming the active-site tunnel. Sequence alignment indicates the probable occurrence of disulfides at equivalent positions in the two other family B enzymes. Partial resequencing of the gene encoding Streptomyces KSM-9 beta-1,4-glucanase CasA (family B) revealed five errors in the original nucleotide sequence analysis. The corrected amino acid sequence contains an Asp residue corresponding to the proposed proton donor in hydrolysis catalysed by cellobiohydrolase II. Cys residues which form disulfide bonds in the Cex catalytic domain are conserved in XynZ of Clostridium thermocellum and Xyn of Cryptococcus albidus but not in the other eight known family F enzymes. Like other members of its family, Cex catalyses xylan hydrolysis. The catalytic efficiency (kcat/Km) for hydrolysis of the heterosidic bond of p-nitrophenyl-beta-D-xylobioside is 14,385 min-1.mM-1 at 25 degrees C; the corresponding kcat/Km for p-nitrophenyl-beta-D-cellobioside hydrolysis is 296 min-1.mM-1.