Large-scale Conformational Changes Research Articles

Computing ensembles of transitions from stable states: Dynamic importance sampling.

There is an increasing dataset of solved biomolecular structures in more than one conformation and increasing evidence that large-scale conformational change is critical for biomolecular function. In this article, we present our implementation of a dynamic importance sampling (DIMS) algorithm that is directed toward improving our understanding of important intermediate states between experimentally defined starting and ending points. This complements traditional molecular dynamics methods where most of the sampling time is spent in the stable free energy wells defined by these initial and final points. As such, the algorithm creates a candidate set of transitions that provide insights for the much slower and probably most important, functionally relevant degrees of freedom. The method is implemented in the program CHARMM and is tested on six systems of growing size and complexity. These systems, the folding of Protein A and of Protein G, the conformational changes in the calcium sensor S100A6, the glucose-galactose-binding protein, maltodextrin, and lactoferrin, are also compared against other approaches that have been suggested in the literature. The results suggest good sampling on a diverse set of intermediates for all six systems with an ability to control the bias and thus to sample distributions of trajectories for the analysis of intermediate states.

Capturing Functional Motions of Membrane Channels and Transporters with Molecular Dynamics Simulation.

Conformational changes of proteins are involved in all aspects of protein function in biology. Almost all classes of proteins respond to changes in their environment, ligand binding, and interaction with other proteins and regulatory agents through undergoing conformational changes of various degrees and magnitudes. Membrane channels and transporters are the major classes of proteins that are responsible for mediating efficient and selective transport of materials across the cellular membrane. Similar to other proteins, they take advantage of conformational changes to make transitions between various functional states. In channels, large-scale conformational changes are mostly involved in the process of "gating", i.e., opening and closing of the pore of the channel protein in response to various signals. In transporters, conformational changes constitute various steps of the conduction process, and, thus, are more closely integrated in the transport process. Owing to significant progress in developing highly efficient parallel algorithms in molecular dynamics simulations and increased computational resources, and combined with the availability of high-resolution, atomic structures of membrane proteins, we are in an unprecedented position to use computer simulation and modeling methodologies to investigate the mechanism of function of membrane channels and transporters. While the entire transport cycle is still out of reach of current methodologies, many steps involved in the function of transport proteins have been characterized with molecular dynamics simulations. Here, we present several examples of such studies from our laboratory, in which functionally relevant conformational changes of membrane channels and transporters have been characterized using extended simulations.

Global dynamic conformational changes in the suppressor domain of IP 3 receptor by stepwise binding of the two lobes of calmodulin

The roles of calmodulin (CaM) have been key points of controversy in the regulation of inositol-1,4,5-trisphosphate receptor (IP(3)R). To address the issue, we studied the interaction between CaM and the suppressor domain of IP(3)R, a key allosteric regulatory domain. First, by means of a pulldown and a fluorescence titration experiment, we confirmed the interaction. Through subsequent NMR binding experiments, we observed dramatic peak disappearances of the suppressor domain on interaction with apo-CaM. The data indicated that apo-CaM induces large-scale dynamic conformational changes in the suppressor domain, involving partial unfolding and subdomain rearrangement. Analysis of the NMR data of CaM surprisingly revealed that its C lobe alone can cause such changes. Further binding experiments showed that calcium allows the free N lobe to bind to the suppressor domain, which induces extra conformational changes in both of the proteins. These results were also confirmed with CaM deletion mutants with either the N or C lobe. On the basis of this novel binding mechanism, we propose a model in which the partial unfolding of the suppressor domain by apo-CaM and the stepwise binding of the N lobe of CaM to the suppressor domain are important elements of calcium/CaM inhibition of IP(3)R. We believe that our working model encompasses previous regulation mechanisms of IP(3)R by calcium/CaM and provides new insights into the CaM-target interaction.

Conformational flexibility and binding interactions of the G protein βγ heterodimer

Previous NMR experiments on unbound G protein βγ heterodimer suggested that particular residues in the binding interface are mobile on the nanosecond timescale. In this work we performed nanosecond-timescale molecular dynamics simulations to investigate conformational changes and dynamics of Gβγ in the presence of several binding partners: a high-affinity peptide (SIGK), phosducin, and the GDP-bound α subunit. In these simulations, the high mobility of GβW99 was reduced by SIGK, and it appeared that a tyrosine might stabilize GβW99 by hydrophobic or aromatic stacking interactions in addition to hydrogen bonds. Simulations of the phosducin-Gβγ complex showed that the mobility of GβW99 was restricted, consistent with inferences from NMR. However, large-scale conformational changes of Gβγ due to binding, which were hypothesized in the NMR study, were not observed in the simulations, most likely due to their short (nanosecond) duration. A pocket consisting of hydrophobic amino acids on Gα appears to restrict GβW99 mobility in the crystal structure of the Gαβγ? heterotrimer. The simulation trajectories are consistent with this idea. However, local conformational changes of residues GβW63, GβW211, GβW297, GβW332, and GβW339 were detected during the MD simulations. As expected, the magnitude of atomic fluctuations observed in simulations was greater for α than for the βγ subunits, suggesting that α has greater flexibility. These observations support the notion that to maintain the high mobility of GβW99 observed by solution NMR requires that the Gβ-α interface must open up on time scale longer than can be observed in nanosecond scale simulations.

Engagement of Arginine Finger to ATP Triggers Large Conformational Changes in NtrC1 AAA+ ATPase for Remodeling Bacterial RNA Polymerase

The NtrC-like AAA+ ATPases control virulence and other important bacterial activities through delivering mechanical work to σ54-RNA polymerase to activate transcription from σ54-dependent genes. We report the first crystal structure for such an ATPase, NtrC1 of Aquifex aeolicus, in which the catalytic arginine engages the γ-phosphate of ATP. Comparing the new structure with those previously known for apo and ADP-bound states supports a rigid-body displacement model that is consistent with large-scale conformational changes observed by low-resolution methods. First, the arginine finger induces rigid-body roll, extending surface loops above the plane of the ATPase ring to bind σ54. Second, ATP hydrolysis permits Pi release and retraction of the arginine with a reversed roll, remodeling σ54-RNAP. This model provides a fresh perspective on how ATPase subunits interact within the ring-ensemble to promote transcription, directing attention to structural changes on the arginine-finger side of an ATP-bound interface.

Integrated prediction of one-dimensional structural features and their relationships with conformational flexibility in helical membrane proteins

BackgroundMany structural properties such as solvent accessibility, dihedral angles and helix-helix contacts can be assigned to each residue in a membrane protein. Independent studies exist on the analysis and sequence-based prediction of some of these so-called one-dimensional features. However, there is little explanation of why certain residues are predicted in a wrong structural class or with large errors in the absolute values of these features. On the other hand, membrane proteins undergo conformational changes to allow transport as well as ligand binding. These conformational changes often occur via residues that are inherently flexible and hence, predicting fluctuations in residue positions is of great significance.ResultsWe performed a statistical analysis of common patterns among selected one-dimensional equilibrium structural features (ESFs) and developed a method for simultaneously predicting all of these features using an integrated system. Our results show that the prediction performance can be improved if multiple structural features are trained in an integrated model, compared to the current practice of developing individual models. In particular, the performance of the solvent accessibility and bend-angle prediction improved in this way. The well-performing bend-angle prediction can be used to predict helical positions with severe kinks at a modest success rate. Further, we showed that single-chain conformational dynamics, measured by B-factors derived from normal mode analysis, could be predicted from observed and predicted ESFs with good accuracy. A web server was developed (http://tardis.nibio.go.jp/netasa/htmone/) for predicting the one-dimensional ESFs from sequence information and analyzing the differences between the predicted and observed values of the ESFs.ConclusionsThe prediction performance of the integrated model is significantly better than that of the models performing the task separately for each feature for the solvent accessibility and bend-angle predictions. The predictability of the features also plays a role in determining flexible positions. Although the dynamics studied here concerns local atomic fluctuations, a similar analysis in terms of global structural features will be helpful in predicting large-scale conformational changes, for which work is in progress.

Structural Basis of Local, pH-Dependent Conformational Changes in Glycoprotein B from Herpes Simplex Virus Type 1

Herpesviruses enter cells by membrane fusion either at the plasma membrane or in endosomes, depending on the cell type. Glycoprotein B (gB) is a conserved component of the multiprotein herpesvirus fusion machinery and functions as a fusion protein, with two internal fusion loops, FL1 and FL2. We determined the crystal structures of the ectodomains of two FL1 mutants of herpes simplex virus type 1 (HSV-1) gB to clarify whether their fusion-null phenotypes were due to global or local effects of the mutations on the structure of the gB ectodomain. Each mutant has a single point mutation of a hydrophobic residue in FL1 that eliminates the hydrophobic side chain. We found that neither mutation affected the conformation of FL1, although one mutation slightly altered the conformation of FL2, and we conclude that the fusion-null phenotype is due to the absence of a hydrophobic side chain at the mutated position. Because the ectodomains of the wild-type and the mutant forms of gB crystallized at both low and neutral pH, we were able to determine the effect of pH on gB conformation at the atomic level. For viruses that enter cells by endocytosis, the low pH of the endosome effects major conformational changes in their fusion proteins, thereby promoting fusion of the viral envelope with the endosomal membrane. We show here that upon exposure of gB to low pH, FL2 undergoes a major relocation, probably driven by protonation of a key histidine residue. Relocation of FL2, as well as additional small conformational changes in the gB ectodomain, helps explain previously noted changes in its antigenic and biochemical properties. However, no global pH-dependent changes in gB structure were detected in either the wild-type or the mutant forms of gB. Thus, low pH causes local conformational changes in gB that are very different from the large-scale fusogenic conformational changes in other viral fusion proteins. We propose that these conformational changes, albeit modest, play an important functional role during endocytic entry of HSV.

Interfacial properties and structure stability of the gp41 tryptophan-rich peptide from HIV-1

The HIV-1 envelope glycoprotein 41 (gp41) undergoes large-scale conformational changes in order to induce the fusion of the virus and cell membranes. Thus, we investigated a possible structure transit at the air-water interface for the tryptophan-rich peptide of gp41 (gp41W). The synthetic peptide (KWASLWNWFNITNWLWYIK), corresponding to gp41W, shows interfacial properties on pure water and Tris buffer at pH 8.5. Isotherm measurements and Brewster angle microscopy (BAM) imaging showed that the behavior of the peptide monolayer was dependent on the subphase composition. A homogenous film was formed on buffer during the peptide monolayer compression, while the appearance of condensed domains on pure water could indicate the oligomerization of gp41W during the surface pressure increase. Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) showed that, whatever the subphase, gp41W adopts an α-helix structure at the air-water interface and does not transit for any other structure even at high surface pressures.

Induced-fit Mechanism for Prolyl Endopeptidase

Prolyl peptidases cleave proteins at proline residues and are of importance for cancer, neurological function, and type II diabetes. Prolyl endopeptidase (PEP) cleaves neuropeptides and is a drug target for neuropsychiatric diseases such as post-traumatic stress disorder, depression, and schizophrenia. Previous structural analyses showing little differences between native and substrate-bound structures have suggested a lock-and-key catalytic mechanism. We now directly demonstrate from seven structures of Aeromonus punctata PEP that the mechanism is instead induced fit: the native enzyme exists in a conformationally flexible opened state with a large interdomain opening between the β-propeller and α/β-hydrolase domains; addition of substrate to preformed native crystals induces a large scale conformational change into a closed state with induced-fit adjustments of the active site, and inhibition of this conformational change prevents substrate binding. Absolute sequence conservation among 28 orthologs of residues at the active site and critical residues at the interdomain interface indicates that this mechanism is conserved in all PEPs. This finding has immediate implications for the use of conformationally targeted drug design to improve specificity of inhibition against this family of proline-specific serine proteases.

Incorporation of 2,3‐Diaminopropionic Acid into Linear Cationic Amphipathic Peptides Produces pH‐Sensitive Vectors

Nonviral vectors that harness the change in pH in endosomes, are increasingly being used to deliver cargoes, including nucleic acids, into mammalian cells. Here we present evidence that the pK(a) of the beta-NH(2) in 2,3-diaminopropionic acid (Dap) is sufficiently lowered, when Dap is incorporated into peptides, that its protonation state is sensitive to the pH changes that occur during endosomal acidification. The lowered pK(a) of around 6.3 is stabilized by the increased electron-withdrawing effect of the peptide bonds, by intermolecular hydrogen bonding and from contributions arising from the peptide conformation. These include mixed polar/apolar environments, Coulombic interactions and intermolecular hydrogen bonding. Changes in the charged state are therefore expected between pH 5 and 7, and large-scale conformational changes are observed in Dap-rich peptides, in contrast to analogues containing lysine or ornithine, when the pH is altered through this range. These physical properties confer a robust gene-delivery capability on designed cationic amphipathic peptides that incorporate Dap.

A Large-Scale Conformational Change Couples Membrane Recruitment to Cargo Binding in the AP2 Clathrin Adaptor Complex

SummaryThe AP2 adaptor complex (α, β2, σ2, and μ2 subunits) crosslinks the endocytic clathrin scaffold to PtdIns4,5P2-containing membranes and transmembrane protein cargo. In the “locked” cytosolic form, AP2's binding sites for the two endocytic motifs, YxxΦ on the C-terminal domain of μ2 (C-μ2) and [ED]xxxL[LI] on σ2, are blocked by parts of β2. Using protein crystallography, we show that AP2 undergoes a large conformational change in which C-μ2 relocates to an orthogonal face of the complex, simultaneously unblocking both cargo-binding sites; the previously unstructured μ2 linker becomes helical and binds back onto the complex. This structural rearrangement results in AP2's four PtdIns4,5P2- and two endocytic motif-binding sites becoming coplanar, facilitating their simultaneous interaction with PtdIns4,5P2/cargo-containing membranes. Using a range of biophysical techniques, we show that the endocytic cargo binding of AP2 is driven by its interaction with PtdIns4,5P2-containing membranes.

Probing DNA Binding, DNA Opening, and Assembly of a Downstream Clamp/Jaw in Escherichia coli RNA Polymerase−λPR Promoter Complexes Using Salt and the Physiological Anion Glutamate

Transcription by all RNA polymerases (RNAPs) requires a series of large-scale conformational changes to form the transcriptionally competent open complex RP(o). At the lambdaP(R) promoter, Escherichia coli sigma(70) RNAP first forms a wrapped, closed 100 bp complex I(1). The subsequent step opens the entire DNA bubble, creating the relatively unstable (open) complex I(2). Additional conformational changes convert I(2) to the stable RP(o). Here we probe these events by dissecting the effects of Na(+) salts of Glu(-), F(-), and Cl(-) on each step in this critical process. Rapid mixing and nitrocellulose filter binding reveal that the binding constant for I(1) at 25 degrees C is approximately 30-fold larger in Glu(-) than in Cl(-) at the same Na(+) concentration, with the same log-log salt concentration dependence for both anions. In contrast, both the rate constant and equilibrium constant for DNA opening (I(1) to I(2)) are only weakly dependent on salt concentration, and the opening rate constant is insensitive to replacement of Cl(-) with Glu(-). These very small effects of salt concentration on a process (DNA opening) that is strongly dependent on salt concentration in solution may indicate that the backbones of both DNA strands interact with polymerase throughout the process and/or that compensation is present between ion uptake and release. Replacement of Cl(-) with Glu(-) or F(-) at 25 degrees C greatly increases the lifetime of RP(o) and greatly reduces its salt concentration dependence. By analogy to Hofmeister salt effects on protein folding, we propose that the excluded anions Glu(-) and F(-) drive the folding and assembly of the RNAP clamp/jaw domains in the conversion of I(2) to RP(o), while Cl(-) does not. Because the Hofmeister effect of Glu(-) or F(-) largely compensates for the destabilizing Coulombic effect of any salt on the binding of this assembly to downstream promoter DNA, RP(o) remains long-lived even at 0.5 M Na(+) in Glu(-) or F(-) salts. The observation that Esigma(70) RP(o) complexes are exceedingly long-lived at moderate to high Glu(-) concentrations argues that Esigma(70) RNAP does not dissociate from strong promoters in vivo when the cytoplasmic glutamate concentration increases during osmotic stress.

A Molecular Model of the Human UDP-Glucuronosyltransferase 1A1, Its Membrane Orientation, and the Interactions between Different Parts of the Enzyme

The vertebrate UDP-glucuronosyltransferases (UGTs) are membrane-bound enzymes of the endoplasmic reticulum that process both endogenous and exogenous substrates. The human UGTs are well known biologically, but biophysical understanding is scarce, largely because of problems in purification. The one resolved crystal structure covers the C-terminal domain of the human UGT2B7. Here, we present a homology model of the complete monomeric human UGT1A1, the enzyme that catalyzes bilirubin glucuronidation. The enzyme can be seen as composed of four different domains: two large ones, the N- and C-terminal domains, and two small ones, the "envelope" helices and the transmembrane segment that includes the cytoplasmic tail. The hydrophobic core of the N-terminal domain and the two envelope helices that connect the large domains are shown to be structurally well conserved even among distant homologs and can thus be modeled with good certainty according to plant and bacterial structures. We consider alternative solutions for the highly variable N-terminal regions that probably contribute to substrate binding. The bilirubin binding site, known pathological mutations in UGT1A1, and other specific residues have been examined in the context of the model with regard to available experimental data. A putative orientation of the protein relative to the membrane has been derived from the location of predicted N-glycosylation sites. The model presents extensive interactions between the N- and C-terminal domains, the two envelope helices, and the membrane. Together, these interactions could allow for a concerted large-scale conformational change during catalysis.

Global Conformational Change Associated with the Two-step Reaction Catalyzed by Escherichia coli Lipoate-Protein Ligase A

Lipoate-protein ligase A (LplA) catalyzes the attachment of lipoic acid to lipoate-dependent enzymes by a two-step reaction: first the lipoate adenylation reaction and, second, the lipoate transfer reaction. We previously determined the crystal structure of Escherichia coli LplA in its unliganded form and a binary complex with lipoic acid (Fujiwara, K., Toma, S., Okamura-Ikeda, K., Motokawa, Y., Nakagawa, A., and Taniguchi, H. (2005) J Biol. Chem. 280, 33645-33651). Here, we report two new LplA structures, LplA.lipoyl-5'-AMP and LplA.octyl-5'-AMP.apoH-protein complexes, which represent the post-lipoate adenylation intermediate state and the pre-lipoate transfer intermediate state, respectively. These structures demonstrate three large scale conformational changes upon completion of the lipoate adenylation reaction: movements of the adenylate-binding and lipoate-binding loops to maintain the lipoyl-5'-AMP reaction intermediate and rotation of the C-terminal domain by about 180 degrees . These changes are prerequisites for LplA to accommodate apoprotein for the second reaction. The Lys(133) residue plays essential roles in both lipoate adenylation and lipoate transfer reactions. Based on structural and kinetic data, we propose a reaction mechanism driven by conformational changes.

Predicting the reaction coordinates of millisecond light-induced conformational changes in photoactive yellow protein.

Understanding the dynamics of large-scale conformational changes in proteins still poses a challenge for molecular simulations. We employ transition path sampling of explicit solvent molecular dynamics trajectories to obtain atomistic insight in the reaction network of the millisecond timescale partial unfolding transition in the photocycle of the bacterial sensor photoactive yellow protein. Likelihood maximization analysis predicts the best model for the reaction coordinates of each substep as well as tentative transition states, without further simulation. We find that the unfolding of the alpha-helical region 43-51 is followed by sequential solvent exposure of both Glu46 and the chromophore. Which of these two residues is exposed first is correlated with the presence of a salt bridge that is part of the N-terminal domain. Additional molecular dynamics simulations indicate that the exposure of the chromophore does not result in a productive pathway. We discuss several possibilities for experimental validation of these predictions. Our results open the way for studying millisecond conformational changes in other medium-sized (signaling) proteins.

FiberDock: Flexible induced-fit backbone refinement in molecular docking.

Upon binding, proteins undergo conformational changes. These changes often prevent rigid-body docking methods from predicting the 3D structure of a complex from the unbound conformations of its proteins. Handling protein backbone flexibility is a major challenge for docking methodologies, as backbone flexibility adds a huge number of degrees of freedom to the search space, and therefore considerably increases the running time of docking algorithms. Normal mode analysis permits description of protein flexibility as a linear combination of discrete movements (modes). Low-frequency modes usually describe the large-scale conformational changes of the protein. Therefore, many docking methods model backbone flexibility by using only few modes, which have the lowest frequencies. However, studies show that due to molecular interactions, many proteins also undergo local and small-scale conformational changes, which are described by high-frequency normal modes. Here we present a new method, FiberDock, for docking refinement which models backbone flexibility by an unlimited number of normal modes. The method iteratively minimizes the structure of the flexible protein along the most relevant modes. The relevance of a mode is calculated according to the correlation between the chemical forces, applied on each atom, and the translation vector of each atom, according to the normal mode. The results show that the method successfully models backbone movements that occur during molecular interactions and considerably improves the accuracy and the ranking of rigid-docking models of protein-protein complexes. A web server for the FiberDock method is available at: http://bioinfo3d.cs.tau.ac.il/FiberDock.

Shape-dependent global deformation modes of large protein structures

Conformational changes are central to the functioning of pore-forming proteins that open and close their molecular gates in response to external stimuli such as pH, ionic strength, membrane voltage or ligand binding. Normal mode analysis (NMA) is used to identify and characterize the slowest motions in the gA, KcsA, ClC-ec1, LacY and LeuT Aa proteins at the onset of gating. Global deformation modes of the essentially cylindrical gA, KcsA, LacY and LeuT Aa biomolecules are reminiscent of global twisting, transverse and longitudinal motions in a homogeneous elastic rod. The ClC-ec1 protein executes a splaying motion in the plane perpendicular to the lipid bilayer. These global collective deformations are determined by protein shape. New methods, all-atom Monte Carlo Normal Mode Following and its simplification using a rotation–translation of protein blocks (RTB), are described and applied to gain insight into the nature of gating transitions in gA and KcsA. These studies demonstrate the severe limitations of standard NMA in characterizing the structural rearrangements associated with gating transitions. Comparison of all-atom and RTB transition pathways in gA clearly illustrates the impact of the rigid protein block approximation and the need to include all degrees of freedom and their relaxation in computational studies of protein gating. The effects of atomic level structure, pH, hydrogen bonding and charged residues on the large-scale conformational changes associated with gating transitions are discussed.

The Conformational Transition Pathway of ATP Binding Cassette Transporter MsbA Revealed by Atomistic Simulations

ATP binding cassette transporters are integral membrane proteins that use the energy released from ATP hydrolysis at the two nucleotide binding domains (NBDs) to translocate a wide variety of substrates through a channel at the two transmembrane domains (TMDs) across the cell membranes. MsbA from Gram-negative bacteria is a lipid and multidrug resistance ATP binding cassette exporter that can undergo large scale conformational changes between the outward-facing and the inward-facing conformations revealed by crystal structures in different states. Here, we use targeted molecular dynamics simulation methods to explore the atomic details of the conformational transition from the outward-facing to the inward-facing states of MsbA. The molecular dynamics trajectories revealed a clear spatiotemporal order of the conformational movements. The disruption of the nucleotide binding sites at the NBD dimer interface is the very first event that initiates the following conformational changes, verifying the assumption that the conformational conversion is triggered by ATP hydrolysis. The conserved x-loops of the NBDs were identified to participate in the interaction network that stabilizes the cytoplasmic tetrahelix bundle of the TMDs and play an important role in mediating the cross-talk between the NBD and TMD. The movement of the NBD dimer is transmitted through x-loops to break the tetrahelix bundle, inducing the packing rearrangements of the transmembrane helices at the cytoplasmic side and the periplasmic side sequentially. The packing rearrangement within each periplasmic wing of TMD that results in exposure of the substrate binding sites occurred at the end stage of the trajectory, preventing the wrong timing of the binding site accessibility.

The Small Angle Scattering Toolbox

Small Angle Scattering (SAS) is a technique used to investigate structure and dynamics of macromolecules in solution. Proteins in buffer conditions close to their physiological environment, are subject to Xray or Neutron scattering experiments. The resulting one-dimensional scattering curves are directly related to their three-dimensional structure. The SAS technique is routinely used to determining the low resolution shape of protein and map specific large scale conformation changes in protein structures.We present a recently developed computational platform for SAS data analysis and model construction/refinement.The Small Angle Scattering Toolbox (SASTBX) has tools four major modules: (1) Raw data reduction; (2) theoretical scattering profile calculation based on PDB structures; (3) Pair distance distribution function (PDDF) estimation; and (4) 3D model construction and structure refinement.The toolbox can be utilized to read raw scattering images obtained from the detector to generate an intensity profile. The basic analyses, such as Guiner and Kratky plots can be carried out in real time to assess the sample and data quality while collecting data. The PDDF estimation is a fully automated procedure, linked with a database a known PDDF's allowing for a rough initial classification of the shape of the protein. Model data can be calculated on the basis of a spherical harmonics expansions. Initial structures can be further refined with normal mode movements or rigid-body motions.The sastbx is built on the open source Computational Crystallography Toolbox (CCTBX). The toolbox is implemented by using Python/C++ hybrid approach: the computing intensive jobs are handled in C++, and the python allows easy integration between other components. The source code will be distributed as open source project.

Structural Transition Between the Ion-Releasing and Ion-Binding States of a Secondary Membrane Transporter

The crystal structure of Na+-coupled galactose symporter (vSGLT) reports the transporter in its substrate-bound state, with a Na+ ion modeled in a binding site corresponding to that of a homologous protein, leucine transporter (LeuT). In molecular dynamics simulations, however, we find the Na+ ion instable, invariably and spontaneously diffusing out of the transporter through a pathway lined by D189, which appears to facilitate the diffusion of the ion toward the cytoplasm. Further analysis of the trajectories and close structural examination, in particular comparison of the Na+ binding sites of vSGLT and LeuT, strongly indicates that the crystal structure of vSGLT actually represents an ion-releasing state of the transporter. The observed dynamics of the Na+ ion, in contrast to the substrate, in a 200 ns equilibrium simulation, also suggests that the cytoplasmic release of the Na+ ion precedes that of the substrate.Through comparison of the “open” conformation of the Na+ binding site in vSGLT and the “close” conformation in LeuT, we used constrained simulation to develop a model for the ion-binding state in vSGLT. SMD simulations were then used to pull out substrate from the substrate-binding site both in ion-releasing state and the modeled ion-binding state, confirming that it is more difficult to release the substrate in the presence of the Na+ ion in its binding site.Furthermore, local transition of the Na+ binding site from an ion-releasing state to an ion-binding state in our constrained simulation induced significantly global conformational change in the protein, specifically, partial opening of the periplasmic side and closing of the cytoplasmic side, thus, capturing for the first time large-scale conformational changes between the inward-facing and outward-facing states in the transport cycle of this secondary transporter.