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The third domain of PSD-95 (PDZ3) is a model for investigating allosteric communication in protein and ligand interactions. While motifs contributing to its binding specificity have been scrutinized, a conformational dynamical basis is yet to be established. Despite the miniscule structural changes due to point mutants, the observed significant binding affinity differences have previously been assessed with a focus on two α-helices located at the binding groove (α2) and the C-terminus (α3). Here, we employ a new computational approach to develop a generalized view on the molecular basis of PDZ3 binding selectivity and interaction communication for a set of point mutants of the protein (G330T, H372A, G330T-H372A) and its ligand (CRIPT named L1 and its T-2F variant L2) along with the wild type (WT). To analyze the dynamical aspects hidden in the conformations that are produced by molecular dynamics simulations, we utilize variations in community composition calculated based on the betweenness centrality measure from graph theory. We find that the highly charged N-terminus which is located far from the ligand has the propensity to share the same community with the ligand in the biologically functional complexes, indicating a distal segment might mediate the binding dynamics. N- and C-termini of PDZ3 share communities, and α3 acts as a hub for the whole protein by sustaining the communication with all structural segments, albeit being a trait not unique to the functional complexes. Moreover, α2 which lines the binding cavity frequently parts communities with the ligand and is not a controller of the binding but is rather a slave to the overall dynamics coordinated by the N-terminus. Thus, ligand binding fate in PDZ3 is traced to the population of community compositions extracted from dynamics despite the lack of significant conformational changes.

The journal of chemical physics 158(8), 085101 (2023). doi:10.1063/5.0136558, This study combines molecular dynamics (MD) simulations with small angle x-ray scattering (SAXS) measurements to investigate the range of conformations that can be adopted by a pH/ionic strength (IS) sensitive protein and to quantify its distinct populations in solution. To explore how the conformational distribution of proteins may be modified in the environmental niches of biological media, we focus on the periplasmic ferric binding protein A (FbpA) from Haemophilus influenzae involved in the mechanism by which bacteria capture iron from higher organisms. We examine iron-binding/release mechanisms of FbpA in varying conditions simulating its biological environment. While we show that these changes fall within the detectable range for SAXS as evidenced by differences observed in the theoretical scattering patterns calculated from the crystal structure models of apo and holo forms, detection of conformational changes due to the point mutation D52A and changes in ionic strength (IS) from SAXS scattering profiles have been challenging. Here, to reach conclusions, statistical analyses with SAXS profiles and results from different techniques were combined in a complementary fashion. The SAXS data complemented by size exclusion chromatography point to multiple and/or alternative conformations at physiological IS, whereas they are well-explained by single crystallographic structures in low IS buffers. By fitting the SAXS data with unique conformations sampled by a series of MD simulations under conditions mimicking the buffers, we quantify the populations of the occupied substates. We also find that the D52A mutant that we predicted by coarse-grained computational modeling to allosterically control the iron binding site in FbpA, responds to the environmental changes in our experiments with conformational selection scenarios that differ from those of the wild type., Published by American Institute of Physics, Melville, NY

A direct way to spot structural features that are universally shared among proteins is to find proper analogues from simpler condensed matter systems. In most cases, sphere-packing arguments provide a straightforward route for structural comparison, as they successfully characterize a wide array of materials such as close packed crystals, dense liquids, and structural glasses. In the current study, the feasibility of creating ensembles of artificial structures that can automatically reproduce a large number of geometrical and topological descriptors of globular proteins is investigated. Towards this aim, a simple cubic (SC) arrangement is shown to provide the best background lattice after a careful analysis of the residue packing trends from 210 proteins. It is shown that a minimalistic set of ground rules imposed on this lattice is sufficient to generate structures that can mimic real proteins. In the proposed method, 210 such structures are generated by randomly removing residues (beads) from clusters that have a SC lattice arrangement until a predetermined residue concentration is achieved. All generated structures are checked for residue connectivity such that a path exists between any two residues. Two additional sets are prepared from the initial structures via random relaxation and a reverse Monte Carlo simulated annealing (RMC-SA) algorithm, which targets the average radial distribution function (RDF) of 210 globular proteins. The initial and relaxed structures are compared to real proteins via RDF, bond orientational order parameters, and several descriptors of network topology. Based on these features, results indicate that the structures generated with 40% occupancy via the proposed method closely resemble real residue networks. The broad correspondence established this way indicates a non-superficial link between the residue networks and the defect laden cubic crystalline order. The presented approach of identifying a minimalistic set of operations performed on a target lattice such that each resulting cluster possess structural characteristics largely indistinguishable from that of a coarse-grained globular protein opens up new venues in structural characterization, native state recognition, and rational design of proteins.

Protein function is constrained by the three-dimensional structure but is delineated by its dynamics. This framework must satisfy specificity of function along with adaptability to changing environments and evolvability under external constraints. The accessibility of the available regions of the energy landscape for a set of conditions and shifts in the populations upon their modulation have effects propagating across scales, from biomolecular interactions, to organisms, to populations. Developing the ability to detect and juggle protein conformations supplemented by a physics-based understanding has implications for not only in vivo problems but also for resistance impeding drug discovery and bionano-sensor design.

The antibiotic trimethoprim (TMP) is used to treat a variety of Escherichia coli infections, but its efficacy is limited by the rapid emergence of TMP-resistant bacteria. Previous laboratory evolution experiments have identified resistance-conferring mutations in the gene encoding the TMP target, bacterial dihydrofolate reductase (DHFR), in particular mutation L28R. Here, we show that 4’-desmethyltrimethoprim (4’-DTMP) inhibits both DHFR and its L28R variant, and selects against the emergence of TMP-resistant bacteria that carry the L28R mutation in laboratory experiments. Furthermore, antibiotic-sensitive E. coli populations acquire antibiotic resistance at a substantially slower rate when grown in the presence of 4’-DTMP than in the presence of TMP. We find that 4’-DTMP impedes evolution of resistance by selecting against resistant genotypes with the L28R mutation and diverting genetic trajectories to other resistance-conferring DHFR mutations with catalytic deficiencies. Our results demonstrate how a detailed characterization of resistance-conferring mutations in a target enzyme can help identify potential drugs against antibiotic-resistant bacteria, which may ultimately increase long-term efficacy of antimicrobial therapies by modulating evolutionary trajectories that lead to resistance., The efficacy of the antibiotic trimethoprim, which inhibits bacterial dihydrofolate reductase (DHFR), is limited by the rapid emergence of resistant bacteria. Here, Manna et al. show that 4’-desmethyltrimethoprim inhibits DHFR and a common TMP-resistant variant, and impedes evolution of antibiotic resistance by selecting against the emergence of this variant.

We address the problem of triggering dissociation events between proteins that have formed a complex. We have collected a set of 25 non-redundant, functionally diverse protein complexes having high-resolution three-dimensional structures in both the unbound and bound forms. We unify elastic network models with perturbation response scanning (PRS) methodology as an efficient approach for predicting residues that have the propensity to trigger dissociation of an interacting protein pair, using the three-dimensional structures of the bound and unbound proteins as input. PRS reveals that while for a group of protein pairs, residues involved in the conformational shifts are confined to regions with large motions, there are others where they originate from parts of the protein unaffected structurally by binding. Strikingly, only a few of the complexes have interface residues responsible for dissociation. We find two main modes of response: In one mode, remote control of disassociation in which disruption of the electrostatic potential distribution along protein surfaces play the major role; in the alternative mode, mechanical control of dissociation by remote residues prevail. In the former, dissociation is triggered by changes in the local environment of the protein e.g. pH or ionic strength, while in the latter, specific perturbations arriving at the controlling residues, e.g. via binding to a third interacting partner is required for decomplexation. We resolve the observations by relying on an electromechanical coupling model which reduces to the usual elastic network result in the limit of the lack of coupling. We validate the approach by illustrating the biological significance of top residues selected by PRS on select cases where we show that the residues whose perturbation leads to the observed conformational changes correspond to either functionally important or highly conserved residues in the complex. KEYWORDS Perturbation response scanning, elastic network model, protein complexes, structural motifs, electrostatic potential distribution, protein-protein disassociation, allostery, cooperative conformational change

PDZ domains constitute common models to study single domain allostery without significant structural changes. The third PDZ domain of PSD-95 (PDZ3) is known to have selective structural features that confer unique modulatory roles to this unit. In this model system two residues, H372 directly connected to the binding site and G330 holding an off-binding-site position, were designated to assess the effect of mutations on binding selectivity. It has been observed that the H372A and G330T/H372A mutations change ligand preferences from class I (T/S amino acid at position −2 of the ligand) to class II (hydrophobic amino acid at the same position). Alternatively, the G330T single mutation leads to the recognition of both ligand classes. We have performed a series of molecular dynamics (MD) simulations for wild-type, H372A, G330T single mutants and a double mutant of PDZ3 in the absence and presence of both types of ligands. With the combination of free energy difference calculations and a detailed analysis of MD trajectories, ‘class switching’ and ‘class bridging’ behavior of PDZ3 mutants, as well as their effects on ligand selection and binding affinities are explained. We show that the dynamics of the charged N-terminus plays a fundamental role in determining the binding preferences in PDZ3 by altering the electrostatic energy. These findings are corroborated by simulations on N-termini truncated versions of these systems. The dynamical allostery orchestrated by the N-terminus offers a fresh perspective to the study of communication pathways in proteins.

Enterovirus 71 can be a severe pathogen in small children and immunocompromised adults. Virus uncoating is a critical step in the infection of the host cell; however, the mechanisms that control this process remain poorly understood. We applied normal mode analysis and perturbation response scanning to several complexes of the virus capsid and present a coarse-graining approach to analyze the full capsid. We show that our method offers an alternative to expressing the system as a set of rigid blocks and accounts for the interconnection between nodes within each subunit and protein interfaces across the capsid. In our coarse-grained approach, the modes associated with capsid expansion are captured in the first three nondegenerate modes and correspond to the changes observed in structural studies of the virus. We show that the resolution of the analysis may be modified without losing information on the global motions leading to uncoating. Perturbation response scanning revealed that a protomer cannot serve as a functional unit to explain deformations of the capsid. Instead, we define a pentamer as the minimum functional unit to investigate changes within the capsid. From the modal analysis and perturbation response scanning, we locate a hotspot region surrounding the fivefold axis. The range of the effect of these single, hotspot residues extend to 140 A. The perturbation of internal capsid residues in this region displayed greatest propensity to capsid expansion, thus indicating the significant role that the RNA genome may play in triggering uncoating.

With the advances in three-dimensional structure determination techniques, high quality structures of the iron transport proteins transferrin and the bacterial ferric binding protein (FbpA) have been deposited in the past decade. These are proteins of relatively large size, and developments in hardware and software have only recently made it possible to study their dynamics using standard computational resources. We review computational techniques towards understanding the equilibrium and kinetic properties of iron transport proteins under different environmental conditions. At the level of detail that requires quantum chemical treatments, the octahedral geometry around iron has been scrutinized and it has been established that the iron coordinating tyrosines are in an unusual deprotonated state. At the atomistic level, both the N-lobe and the full bilobal structure of transferrin have been studied under varying conditions of pH, ionic strength and binding of other metal ions by molecular dynamics (MD) simulations. These studies have allowed questions to be answered, among others, on the function of second shell residues in iron release, the role of synergistic anions in preparing the active site for iron binding, and the differences between the kinetics of the N- and the C-lobe. MD simulations on FbpA have led to the detailed observation of the binding kinetics of phosphate to the apo form, and to the conformational preferences of the holo form under conditions mimicking the environmental niches provided by the periplasmic space. To study the dynamics of these proteins with their receptors, one must resort to coarse-grained methodologies, since these systems are prohibitively large for atomistic simulations. A study of the complex of human transferrin (hTf) with its pathogenic receptor by such methods has revealed a potential mechanistic explanation for the defense mechanism that arises in evolutionary warfare. Meanwhile, the motions in the transferrin receptor bound hTf have been shown to disfavor apo hTf dissociation, explaining why the two proteins remain in complex during the recycling process from the endosome to the cell surface. Open problems and possible technological applications related to metal ion binding-release in iron transport proteins that may be handled by hybrid use of quantum mechanical, MD and coarse-grained approaches are discussed.

Evolutionary fitness landscapes of certain antibiotic target enzymes have been comprehensively mapped showing strong high order epistasis between mutations, but understanding these effects at the biochemical and molecular levels remained open. Here, we carried out an extensive experimental and computational study to quantitatively understand the evolutionary dynamics of Escherichia coli dihydrofolate reductase (DHFR) enzyme in the presence of trimethoprim induced selection. Biochemical and structural characterization of resistance-conferring mutations targeting a total of ten residues spanning the substrate binding pocket of DHFR revealed distinct resistance mechanisms. Next, we experimentally measured biochemical parameters (Km, Ki, and kcat) for a mutant library carrying all possible combinations of six resistance-conferring DHFR mutations and quantified epistatic interactions between them. We found that the epistasis between DHFR mutations is high-order for catalytic power of DHFR (kcat and Km), but less prevalent for trimethoprim affinity (Ki). Taken together our data provide a concrete illustration of how epistatic coupling at the level of biochemical parameters can give rise to complex fitness landscapes, and suggest new strategies for developing mutant specific inhibitors.

Summary MODE-TASK, a novel and versatile software suite, comprises Principal Component Analysis, Multidimensional Scaling, and t-Distributed Stochastic Neighbor Embedding techniques using Molecular Dynamics trajectories. MODE-TASK also includes a Normal Mode Analysis tool based on Anisotropic Network Model so as to provide a variety of ways to analyse and compare large-scale motions of protein complexes for which long MD simulations are prohibitive. Beside the command line function, a GUI has been developed as a PyMOL plugin. Availability and implementation MODE-TASK is open source, and available for download from https://github.com/RUBi-ZA/MODE-TASK. It is implemented in Python and C++. It is compatible with Python 2.x and Python 3.x and can be installed by Conda. Supplementary information Supplementary data are available at Bioinformatics online.

The Mutation-Minimization Method (MuMi) to study the local response of proteins to point mutations has been introduced here. The heat shock protein Hsp70 as the test system since it displays features that have been studied in great detail has been used here. It has many conserved residues, serves several different functions on each of its domains, and displays interdomain allostery. For the analysis of spatial arrangement of residues within the protein, the network properties of the wild type (WT) protein as well as its all single alanine residue mutants using MuMi has been investigated. The measures to express the amount of change from the WT structure upon mutation and compare these deviations to find potential critical sites have been proposed. The functional significance of the potential sites to the parameter that uncovers them has been mapped. It was found that sites directly involved in binding were sensitive to mutations and were characterized by large displacements. On the other hand, sites that steer large conformational changes typically had increased reachability upon alanine mutations occurring elsewhere in the protein. Finally, residues that control communication within and between domains reside on the largest number of paths connecting pairs of residues in the protein.

The dynamical and thermodynamic behavior of human transferrin (hTf) protein in saline aqueous solution of various concentrations is studied. hTf is an essential transport protein circulating iron in the blood and delivering it to tissues. It displays highly pH dependent cooperativity between the two lobes, each carrying an iron, and forms a tight complex with the receptor during endocytosis, eventually recycled to the serum after iron release. Molecular dynamics simulations are used to investigate the effects of the amount of salt on protein conformation and dynamics to analyze the structure-function relationship in free hTf at serum pH. To monitor the ionic strength dependence, four different ionic concentrations, 0, 50, 130, and 210 mM NaCl for two protonation states of the iron coordination site is considered. Two mechanisms by which salt affects hTf are disclosed. In the totally closed state where iron coordinating tyrosines are deprotonated, the addition of even 50 mM of salt alters the electrostatic potential distribution around the protein; opening energetic pathways for tyrosine protonation from nearby charged residues as a required first step for iron release. Once domain opening is observed, conformational plasticity renders the iron binding site more accessible by the solvent. At this second stage of iron release, R124 in the N-lobe is identified as kinetically significant anion binding site that accommodates chloride ions and: allosterically communicates with the iron binding residues. Opening motions are maximized at 150 mM IS in the N-lobe, and at 210 mM in the C-lobe. The extra mobility in the latter is thought to preclude binding of hTf to its receptor. Thus, the physiological IS is Optimal for exposing iron for release from hTf. However, the calculated binding affinities of iron show that even in the most open conforinations, iron dissociation needs to be accompanied by chelators.

Dihydrofolate reductase (DHFR) is a ubiquitous enzyme with an essential role in cell metabolism. DHFR catalyzes the reduction of dihydrofolate to tetrahydrofolate, which is a precursor for purine and thymidylate synthesis. Several DHFR targeting antifolate drugs including trimethoprim, a competitive antibacterial inhibitor, have therefore been developed and are clinically used. Evolution of resistance against antifolates is a common public health problem rendering these drugs ineffective. To combat the resistance problem, it is important to understand resistance-conferring changes in the DHFR structure and accordingly develop alternative strategies. Here, we structurally and dynamically characterize Escherichia coli DHFR in its wild type (WT) and trimethoprim resistant L28R mutant forms in the presence of the substrate and its inhibitor trimethoprim. We use molecular dynamics simulations to determine the conformational space, loop dynamics and hydrogen bond distributions at the active site of DHFR for the WT and the L28R mutant. We also report their experimental kcat, Km, and Ki values, accompanied by isothermal titration calorimetry measurements of DHFR that distinguish enthalpic and entropic contributions to trimethoprim binding. Although mutations that confer resistance to competitive inhibitors typically make enzymes more promiscuous and decrease affinity to both the substrate and the inhibitor, strikingly, we find that the L28R mutant has a unique resistance mechanism. While the binding affinity differences between the WT and the mutant for the inhibitor and the substrate are small, the newly formed extra hydrogen bonds with the aminobenzoyl glutamate tail of DHF in the L28R mutant leads to increased barriers for the dissociation of the substrate and the product. Therefore, the L28R mutant indirectly gains resistance by enjoying prolonged binding times in the enzyme-substrate complex. While this also leads to slower product release and decreases the catalytic rate of the L28R mutant, the overall effect is the maintenance of a sufficient product formation rate. Finally, the experimental and computational analyses together reveal the changes that occur in the energetic landscape of DHFR upon the resistance-conferring L28R mutation. We show that the negative entropy associated with the binding of trimethoprim in WT DHFR is due to water organization at the binding interface. Our study lays the framework to study structural changes in other trimethoprim resistant DHFR mutants.

Summary Molecular dynamics (MD) determines the physical motions of atoms of a biological macromolecule in a cell-like environment and is an important method in structural bioinformatics. Traditionally, measurements such as root mean square deviation, root mean square fluctuation, radius of gyration, and various energy measures have been used to analyze MD simulations. Here, we present MD-TASK, a novel software suite that employs graph theory techniques, perturbation response scanning, and dynamic cross-correlation to provide unique ways for analyzing MD trajectories. Availability and implementation MD-TASK has been open-sourced and is available for download from https://github.com/RUBi-ZA/MD-TASK, implemented in Python and supported on Linux/Unix.

WOS: 000395869500041 PubMed ID: 28191562 Ferric binding protein (FbpA) is part of an elaborate iron piracy mechanism evolved in Gram-negative bacteria, shuttling iron in the periplasmic space, from the outer to the cytoplasmic membrane side. We address how the dissociation process of iron is facilitated, since the binding constant of iron is on the order of 10(18) M-1 at 6.5 pH and 200 mM ionic strength (IS). We monitor the conformational preferences of FbpA by extensive molecular dynamics (MD) simulations under conditions where IS, charge states of iron coordinating tyrosines and pH are varied, as well as when a mutation is introduced at an allosteric site. Steered MD is utilized to predict the binding affinity of iron. After triggering lobe opening by changing the charge states of tyrosines, the conformations adopted and the iron binding affinity still depend on pH, IS and allosteric interactions. To relate the observed conformational changes to the environmental conditions that might be encountered in the periplasmic space, we offer a plausible model that couples electrostatic potential distribution to the mechanical motions invoked. Although low pH/IS and allosteric perturbations decrease the affinity of iron, it remains high for spontaneous dissociation. However, the conformational changes modulated by the environmental conditions expose iron for chelation. Our study provides a quantitative dimension and molecular details to interpret the contribution of possible environmental conditions present in the periplasmic space to iron dissociation from FbpA, opening up the opportunity of modulating function via allosteric mutations or altering environmental conditions, thus offering a new route to developing strategies towards antibiotic resistance by targeting nutritional requirements. Scientific and Technological Research Council of Turkey Projects [113Z408] This work was supported by the Scientific and Technological Research Council of Turkey Projects, grant no. 113Z408. We thank Gokce Guven for doing the initial MD calculations.

We characterize structural and dynamical changes induced on dihydrofolate reductase(DHFR). We investigate the structural features of the mutant protein that allow it to survive natural selection. Single/double/triple mutants detected via the systematic experimental approach carried out in the morbidostat1 under the selection pressure induced by the antibiotic trimethoprim(TMP), a competitive inhibitor of dihydrofolate, are studied. We investigate the structural features of DHFR that lead to catalytic activity while simultaneously casting out TMP. Binding constant and catalytic activity measurements on different mutants provide conflicting results, implying alternative routes towards conferring drug resistance. We execute extensive molecular dynamics(MD) simulations for the mutants in their folate or TMP bound conformations. We evaluate the experimental findings regarding DHFR drug resistance through structural changes in the enzyme. To understand if the molecules displaying competitive binding to the same region are discriminated by free energy changes affecting binding probabilities, we perform thermodynamic analyses via alchemical free energy perturbation calculations. Another effective factor in DHFR function is the dynamics of the Met20 loop direct contacting folate. These dynamics include an isomerization that is too slow to be directly observed by conventional MD.2 We employ perturbation-response scanning method3 to quantify the interconversion propensity of the conformers. The competing(or synergistic) effects of dynamics and thermodynamics in the mutants are assessed through a combination of these computational approaches. We reveal a trade-off between stability and enzymatic efficiency: Those mutants that have increased affinity for TMP also have larger k_cat values, albeit always below that of the wild-type. Furthermore, mutants disrupt the correlated motions observed in the TMP-bound wild-type enzyme, while that of the DHF-bound remain unaltered.1. Toprak et al., Nature Genet. 44, 101(2012).2. Schnell et al., Annu. Rev. Biophys. Biomol. Struct. 33, 119(2004).3. Atilgan&Atilgan, PLoS Comput. Biol. 5, e1000544(2009).

We study geometric and energetic factors that partake in modifying properties of polymeric melts via inserting well-dispersed nanoscopic particles (NP). Model systems are polybutadiene melts including 10-150 atom atomic clusters (0.1-1.5% v/v). We tune interactions between chains and particle by van der Waals terms. Using molecular dynamics we study equilibrium fluctuations and dynamical properties at the interface. Effect of bead size and interaction strength both on volume and volumetric fluctuations is manifested in mechanical properties, quantified here by bulk modulus, K. Tuning NP size and non-bonded interactions results in ~15% enhancement in K by addition of a maximum of 1.5% v/v NP., 25 pages, 7 figures

We investigate protonation as a possible route for triggering conformational change in proteins by focusing on the calmodulin (CaM) example. Two hundred nanosecond molecular dynamics (MD) simulations are performed on both the extended and compact forms of calcium loaded CaM. The stability of both structures is confirmed under prevailing conditions. Protonation of nine acidic residues with upshifted pK(a) values leads to a large conformational change in less than 100 ns. The structure attained is consistent with fluorescence resonance energy transfer experimental results as well as structures from an ensemble compatible with NMR data. Analysis of the MD trajectories summing up to one microsecond implies that the key events leading to the completion of the conformational change begins with an initial formation of a salt bridge between the N-lobe and the linker, followed by the bending of the C-lobe and the organization of a stabilizing hydrophobic patch between the lobes. We find that CaM utilizes its Ca(2+) ions to harden/soften different regions so as to achieve various conformations. Thus, barrier crossing between extended and compact forms of CaM which is normally a rare event due to the repulsive electrostatic interactions between the two lobes is facilitated by protonation of high pK(a) residues. The results delineate how pH changes might be utilized in the cell to achieve different conformation-related functions.

The Mutation-Minimization Method (MuMi) to study the local response of proteins to point mutations has been introduced here. The heat shock protein Hsp70 as the test system since it displays features that have been studied in great detail has been used here. It has many conserved residues, serves several different functions on each of its domains, and displays interdomain allostery. For the analysis of spatial arrangement of residues within the protein, the network properties of the wild type (WT) protein as well as its all single alanine residue mutants using MuMi has been investigated. The measures to express the amount of change from the WT structure upon mutation and compare these deviations to find potential critical sites have been proposed. The functional significance of the potential sites to the parameter that uncovers them has been mapped. It was found that sites directly involved in binding were sensitive to mutations and were characterized by large displacements. On the other hand, sites that steer large conformational changes typically had increased reachability upon alanine mutations occurring elsewhere in the protein. Finally, residues that control communication within and between domains reside on the largest number of paths connecting pairs of residues in the protein.

Human serum transferrin (hTf) transports ferric ions in the blood stream and delivers them to cells via receptor-mediated endocytosis. hTf is folded into two homologous lobes; we utilize three of the available crystal structures delineating large conformational changes involved in iron binding/dissociation. We address the problems of whether the release process follows the same trend at serum (~7.4) and endosomal (~5.6) pH, and if there is communication between the lobes. In the absence of the transferrin receptor, we study the dynamics of the full structure as well as the separate lobes in different closed, partially open, and open conformations under neutral and endosomal pH conditions. Results corroborate those experimental observations underscoring the distinguishing effect of pH on the dynamics of hTf. Furthermore, in a total of 2 μs molecular dynamics simulations, residue fluctuations elucidate the cross talk between the lobes correlated by the peptide linker bridging them at serum pH, while their correlations are lost under endosomal conditions. At serum pH, interplay between relative mobility of the lobes is correlated with iron release rates, rendering the initial conformational change an important contributor to the dynamics under these conditions. Interestingly, C-lobe opening lags behind that of the N-lobe as long as there is at least one iron bound, making the more stable C-lobe an attractive target for recognition by receptors. At endosomal pH, both lobes readily open, making irons available for delivery.

Can one infer the amino acids of the enzymes that are responsible for the stability or the level of the catalytic activity by computationally experimenting on the inhibited enzyme in the enzyme-inhibitor complex? In this article, we answer this question positively both by designing molecular dynamics simulations and by devising coarse-grained methodologies on the subtilisin serine protease. Both methodologies are based on the cross-correlations of the fluctuations of the residues, obtained either by monitoring the trajectories from the simulation or by constructing the inverse Laplacian of the elastic network model, of the complex. A perturbation scanning is applied to the complex using these correlations. The results indicate that the two methods almost point out the same regions on the flexible of the enzyme. These regions are: (i) 50-61, (ii) 155-164 and (iii) 192-194, all of which are designated to be important by experimental studies in the literature.

The Drosophila gene Spatzle encodes the activating ligand for the Toll receptor. This signaling pathway is required for dorso-ventral patterning in the early embryo and an antifungal immune response in larvae and adults. The genome sequence of Drosophila shows that there are a total of eight Toll-like receptors and these may function in other aspects of embryonic development and innate immunity. Here we describe five Drosophila homologues of Spatzle (Spz2-6) found using an iterative searching method. All five appear to encode proteins containing neurotrophin-like cystine-knot domains. In addition, most retain a characteristic intron-exon structure shared with the prototype Spatzle gene. This provides evidence that the family arose by ancient gene duplication events and indicates that the gene products may represent activating ligands for corresponding Toll receptors. Expression studies show that only Spz4 is expressed strongly in larvae and adults and thus may be involved in an ancillary antifungal response mediated by Toll-5. By contrast, Spz6 shows a complex spatial and temporally regulated expression pattern in the late embryo. Thus the new Toll/Spatzle families of signaling molecules may have important roles in other aspects of development and immunity.

We demonstrate that the stabilization of the binding region is accomplished at the expense of a loss in the stability of the rest of the protein. A novel molecular mechanics (MM) approach is introduced to distinguish residue stabilities of proteins in a given conformation. As an example, the relative stabilities of folded chymotrypsin inhibitor 2 (CI2) in unbound form, and CI2 in complex with subtilisin novo is investigated. The conformation of the molecule in the two states is almost identical, with an approximately 0.6-A root-mean-square deviation (RMSD) of the Calpha atoms. On binding, the packing density changes only at the binding loop. However, residue fluctuations in the rest of the protein are greatly altered solely due to those contacts, indicating the effective propagation of perturbation and the presence of remotely controlling residues. To quantify the interplay between packing density, packing order, residue fluctuations, and residue stability, we adopt an MM approach whereby small displacements are inserted at selected residues, followed by energy minimization; the displacement of each residue in response to such perturbations are organized in a perturbation-response matrix L. We define residue stability lambda(i) = summation operator((j)L(ij))/ summation operator((j) L(ji)) as the ratio of the amount of change to which the residue is amenable, to the ability of a given residue to induce change. We then define the free energy associated with residue stability, DeltaG(lambda) = -RT ln lambda. DeltaG(lambda) intrinsically selects the residues that are in the folding core. Upon complexation, the binding loop becomes more resistant to perturbation, in contrast to the alpha-helix that favors change. Although the two forms of CI2 are structurally similar, residue fluctuations differ vastly, and the stability of many residues is altered upon binding. The decrease in entropy introduced by binding is thus compensated by these changes.

Chemokines are a family of proteins involved in inflammatory and immune response. They share a common fold, made up of a three-stranded beta -sheet, and an overlaying alpha -helix, Chemokines are mainly categorized into two subfamilies distinguished by the presence or absence of a residue between two conserved cysteines in the N-terminus, Although dimers and higher-order quaternary structures are common in chemokines, they are known to function as monomers, Yet, there is quite a bit of controversy on how the actual function takes place. The mechanisms of binding and activation in the chemokine family are investigated using the gaussian network model of proteins, a low-resolution model that monitors the collective motions in proteins. It is particularly suitable for elucidating the global dynamic characteristics of large proteins or the common properties of a group of related proteins such as the chemokine family presently investigated. A sample of 16 proteins that belong to the CC, CXC, or CX3C subfamilies are inspected. Local packing density and packing order of residues are used to determine the type and range of motions on a global scale, such as those occurring between various loop regions, The 30s-loop, although not directly involved in the binding interface like the N-terminus and the N-loop, is identified as having a prominent role in both binding/activation and dimerization. Two mechanisms are distinguished based on the communication among the three flexible regions, In these two-step mechanisms, the 30s-loop assists either the N-loop or the N-terminus during binding and activation, The findings are verified by molecular mechanics and molecular dynamics simulations carried out on the detailed structure of representative proteins from each mechanism type. A basis for the construction of hybrids of chemokines to bind and/or activate various chemokine receptors is presented.

A low-resolution structural model based on the packing geometry of α-carbons is utilized to establish a connection between the flexible and rigid parts of a folded protein. The former commonly recognizes a complementing molecule for making a complex, while the latter manipulates the necessary conformational change for binding. We attempt analytically to distinguish this control architecture that intrinsically exists in globular proteins. First with two-dimensional simple models, then for a native protein, bovine pancreatic trypsin inhibitor, we explicitly demonstrate that inserting fluctuations in tertiary contacts supported by the stable core, one can regulate the displacement of residues on loop regions. The positional fluctuations of the flexible regions are annihilated by the rest of the protein in conformity with the Le Chatelier–Braun principle. The results indicate that the distortion of the principal nonbonded contacts between highly packed residues is accompanied by that of the slavery fluctuatio...

The dynamics of a-amylase inhibi- tors has been investigated using molecular dynam- ics (MD) simulations and two analytical approaches, the Gaussian network model (GNM) and anisotropic network model (ANM). MD simulations use a full atomic approach with empirical force fields, while the analytical approaches are based on a coarse- grained single-site-per-residue model with a single- parameter harmonic potential between sufficiently close (r < 7 A) residue pairs. The major difference between the GNM and the ANM is that no direc- tional preferences can be obtained in the GNM, all residue fluctuations being theoretically isotropic, while ANM does incorporate directional prefer- ences. The dominant modes of motions are identi- fied by (i) the singular value decomposition (SVD) of the MD trajectory matrices, and (ii) the similarity transformation of the Kirchhoff matrices of inter- residue contacts in the GNM or ANM. The mean- square fluctuations of individual residues and the cross-correlations between domain movements re- tain the same characteristics, in all approaches— although the dispersion of modes and detailed ampli- tudes of motion obtained in the ANM conform more closely with MD results. The major weakness of the analytical approaches appears, on the other hand, to be their inadequacy to account for the anhar- monic motions or multimeric transitions driven by the slowest collective mode observed in MD. Such motions usually suffer, however, from MD sampling inefficiencies, and multiple independent runs should be tested before making conclusions about their validity and detailed mechanisms. Overall this study invites attention to (i) the robustness of the average properties (mean-square fluctuations, cross-correla- tions) controlled by the low frequency motions, which are invariably reproduced in all approaches, and (ii) the utility and efficiency of the ANM, the computational time cost of which is of the order of "minutes" (real time), as opposed to "days" for MD simulations. Proteins 2000;40:512-524.

By treating an enzyme as a coarse-grained uniform block of material, utilizing only the α-Carbon positions, the normal modes of motion can be obtained. For reverse transcriptase the slower of these motions are suggestive of being involved in the processing step, where the RNA or DNA strand is copied onto a new DNA strand at a polymerase site, and the RNA strand is subsequently cut up at the distant Ribonuclease H site. The slowest mode of motion involves hinge bending about a site midway between the polymerase and Ribonuclease H sites, suggesting that it can push or pull the RNA strand between these two sites. Pulling the nucleic acid strand would require tight binding to the RNase H site. The next slowest mode involves a hinge that opens and closes the protein like a clamp, which could facilitate the release of the nucleic acids for their step-wise progression. The third mode could rotate the substrate. An overall description of the step-wise processing step would involve close coordination among these steps. Results suggest that the smaller p51 subunit serves only as ballast to support the various modes of motion involving the different parts of the p66 subunit.

In order to study the inferences of structure for mechanism, the collective motions of the retroviral reverse transcriptase HIV-1 RT (RT) are examined using the Gaussian network model (GNM) of proteins. This model is particularly suitable for elucidating the global dynamic characteristics of large proteins such as the presently investigated heterodimeric RT comprising a total of 982 residues. Local packing density and coordination order of amino acid residues is inspected by the GNM to determine the type and range of motions, both at the residue level and on a global scale, such as the correlated movements of entire subdomains. Of the two subunits, p66 and p51, forming the RT, only p66 has a DNA-binding cleft and a functional polymerase active site. This difference in the structure of the two subunits is shown here to be reflected in their dynamic characteristics: only p66 has the potential to undergo large-scale cooperative motions in the heterodimer, while p51 is essentially rigid. Taken together, the global motion of the RT heterodimer is comprised of movements of the p66 thumb subdomain perpendicular to those of the p66 fingers, accompanied by anticorrelated fluctuations of the RNase H domain and p51 thumb, thus providing information about the details of one processivity mechanism. A few clusters of residues, generally distant in sequence but close in space, are identified in the p66 palm and connection subdomains, which form the hinge-bending regions that control the highly concerted motion of the subdomains. These regions include the catalytically active site and the non-nucleoside inhibitor binding pocket of p66 polymerase, as well as sites whose mutations have been shown to impair enzyme activity. It is easily conceivable that this hinge region, indicated by GNM analysis to play a critical role in modulating the global motion, is locked into an inactive conformation upon binding of an inhibitor. Comparative analysis of the dynamic characteristics of the unliganded and liganded dimers indicates severe repression of the mobility of the p66 thumb in RT's global mode, upon binding of non-nucleoside inhibitors.

A simple method for predicting the continuation of scalar chaotic time series ahead in time is proposed. The false nearest neighbors technique in connection with the time-delayed embedding is employed so as to reconstruct the state space. A local forecasting model based upon the time evolution of the topological neighboring in the reconstructed phase space is suggested. A moving root-mean-square error is utilized in order to monitor the error along the prediction horizon. The model is tested for the convection amplitude of the Lorenz model; The results indicate that for approximately 100 cycles of the training data, the prediction follows the actual continuation very closely about six cycles. The proposed model, like other state-space forecasting models, captures the long-term behavior of the system due to the use of spatial neighbors in the state space.