Biomedical subjects

Viktor Hornak

Publications and source records attributed to Viktor Hornak.

16 recordsLinked to original sources

PubMed2006

Comparison of multiple Amber force fields and development of improved protein backbone parameters.

The ff94 force field that is commonly associated with the Amber simulation package is one of the most widely used parameter sets for biomolecular simulation. After a decade of extensive use and testing, limitations in this force field, such as over-stabilization of alpha-helices, were reported by us and other researchers. This led to a number of attempts to improve these parameters, resulting in a variety of "Amber" force fields and significant difficulty in determining which should be used for a particular application. We show that several of these continue to suffer from inadequate balance between different secondary structure elements. In addition, the approach used in most of these studies neglected to account for the existence in Amber of two sets of backbone phi/psi dihedral terms. This led to parameter sets that provide unreasonable conformational preferences for glycine. We report here an effort to improve the phi/psi dihedral terms in the ff99 energy function. Dihedral term parameters are based on fitting the energies of multiple conformations of glycine and alanine tetrapeptides from high level ab initio quantum mechanical calculations. The new parameters for backbone dihedrals replace those in the existing ff99 force field. This parameter set, which we denote ff99SB, achieves a better balance of secondary structure elements as judged by improved distribution of backbone dihedrals for glycine and alanine with respect to PDB survey data. It also accomplishes improved agreement with published experimental data for conformational preferences of short alanine peptides and better accord with experimental NMR relaxation data of test protein systems.

Alanine↗

PubMed2006

The open structure of a multi-drug-resistant HIV-1 protease is stabilized by crystal packing contacts.

The introduction of HIV-1 protease (HIV-PR) inhibitors has led to a dramatic increase in patient survival; however, these gains are threatened by the emergence of multi-drug-resistant strains. Design of inhibitors that overcome resistance would be greatly facilitated by deeper insight into the mechanistic events associated with binding of substrates and inhibitors, as well as an understanding of the effects of resistance mutations on the structure and dynamic behavior of HIV-PR. We previously reported a series of simulations that provide a model for HIV-PR dynamics, with spontaneous conversions between the bound and unbound crystal forms upon addition or removal of an inhibitor. Importantly, the unbound protease transiently sampled a third fully open state that permits entry to the active site, unlike both crystallographic forms. Recently, a crystal structure of unbound HIV-PR was reported for the MDR 769 isolate (PDB: 1TW7); unlike all previous experimental structures, the binding pocket is open. It is suggested that drug resistance in this strain arises at least in part from the inability of inhibitors to induce closing. We carried out simulations of the MDR 769 HIV-PR mutant and observed that the reported structure is unstable in solution and rapidly adopts the semi-open conformation observed for the unbound wild-type protease in solution. Further analysis suggests that the wide-open structure observed for MDR 769 arises not from sequence variation, but instead is an artifact from crystal packing. Thus, despite being the first experimental structure to reveal flap opening sufficient for substrate access to the active site, this structure may not be directly relevant to studies of inhibitor entry or to the cause of HIV-PR drug resistance.

Chemistry, Pharmaceutical↗

PubMed2006

Computational analysis of the mode of binding of 8-oxoguanine to formamidopyrimidine-DNA glycosylase.

8-Oxoguanine (8OG) is the most prevalent form of oxidative DNA damage. In bacteria, 8OG is excised by formamidopyrimidine glycosylase (Fpg) as the initial step in base excision repair. To efficiently excise this lesion, Fpg must discriminate between 8OG and an excess of guanine in duplex DNA. In this study, we explore the structural basis underlying this high degree of selectivity. Two structures have been reported in which Fpg is bound to DNA, differing with respect to the position of the lesion in the active site, one structure showing 8OG bound in the syn conformation and the other in the anti conformation. Remarkably, the results of our all-atom simulations are consistent with both structures. The syn conformation observed in the crystallographic structure of Fpg obtained from Bacillus stearothermophilus is stabilized through interaction with E77, a nonconserved residue. Replacement of E77 with Ser, creating the Fpg sequence found in Escherichia coli and other bacteria, results in preferred binding of 8OG in the anti conformation. Our calculations provide novel insights into the roles of active site residues in binding and recognition of 8OG by Fpg.

Amino Acid Sequence↗

PubMed2006

The unfolded state of the villin headpiece helical subdomain: computational studies of the role of locally stabilized structure.

The 36 residue villin headpiece helical subdomain (HP36) is one of the fastest cooperatively folding proteins, folding on the microsecond timescale. HP36's simple three helix topology, fast folding and small size have made it an attractive model system for computational and experimental studies of protein folding. Recent experimental studies have explored the denatured state of HP36 using fragment analysis coupled with relatively low-resolution spectroscopic techniques. These studies have shown that there is apparently only a small tendency to form locally stabilized secondary structure. Here, we complement the experimental studies by using replica exchange molecular dynamics with explicit solvent to investigate the structural features of these peptide models of unfolded HP36. To ensure convergence, two sets of simulations for each fragment were performed with different initial structures, and simulations were continued until these generated very similar final ensembles. These simulations reveal low populations of native-like structure and early folding events that cannot be resolved by experiment. For each fragment, calculated J-coupling constants and helical propensities are in good agreement with experimental trends. HP-1, corresponding to residues 41 to 53 and including the first alpha-helix, contains the highest helical population. HP-3, corresponding to residues 62 through 75 and including the third alpha-helix, contains a small population of helical turn residing at the N terminus while HP-2, corresponding to residues 52 through 61 and including the second alpha-helix, formed little to no structure in isolation. Overall, HP-1 was the only fragment to adopt a native-like conformation, but the low population suggests that formation of significant structure only occurs after formation of specific tertiary interactions.

Amino Acids↗

PubMed2006

HIV-1 protease flaps spontaneously close to the correct structure in simulations following manual placement of an inhibitor into the open state.

We report unrestrained, all-atom molecular dynamics simulations of HIV-1 protease (HIV-PR) with a continuum solvent model that reproducibly sample closing of the active site flaps following manual placement of a cyclic urea inhibitor into the substrate binding site of the open protease. The open form was obtained from the unbound, semi-open HIV-PR crystal structure, which we recently reported (Hornak, V.; et al. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 915-920.) to have spontaneously opened during unrestrained dynamics. In those simulations, the transiently open flaps always returned to the semi-open form that is observed in all crystal structures of the free protease. Here, we show that manual docking of the inhibitor reproducibly induces spontaneous conversion to the closed form as seen in all inhibitor-bound HIV-PR crystal structures. These simulations reproduced not only the greater degree of flap closure, but also the striking difference in flap "handedness" between bound and free enzyme. In most of the simulations, the final structures were highly accurate. Root-mean-square deviations (RMSD) from the crystal structure of the complex were approximately 1.5 A (averaged over the last 100 ps) for the inhibitor and each flap despite initial RMSD of 2-5 A for the inhibitors and 6-11 A for the flaps. Key hydrogen bonds were formed between the flap tips and between flaps and inhibitor that match those seen in the crystal structure. The results demonstrate that all-atom simulations have the ability to significantly improve poorly docked ligand conformations and reproduce large-scale receptor conformational changes that occur upon binding.

Azepines↗

PubMed2006

HIV-1 protease flaps spontaneously open and reclose in molecular dynamics simulations.

We report unrestrained, all-atom molecular dynamics simulations of HIV-1 protease that sample large conformational changes of the active site flaps. In particular, the unliganded protease undergoes multiple conversions between the "closed" and "semiopen" forms observed in crystal structures of inhibitor-bound and unliganded protease, respectively, including reversal of flap "handedness." Simulations in the presence of a cyclic urea inhibitor yield stable closed flaps. Furthermore, we observe several events in which the flaps of the unliganded protease open to a much greater degree than observed in crystal structures and subsequently return to the semiopen state. Our data strongly support the hypothesis that the unliganded protease predominantly populates the semiopen conformation, with closed and fully open structures being a minor component of the overall ensemble. The results also provide a model for the flap opening and closing that is considered to be essential to enzyme function.

Acquired Immunodeficiency Syndrome↗

PubMed2006

Location of Trp265 in metarhodopsin II: implications for the activation mechanism of the visual receptor rhodopsin.

Isomerization of the 11-cis retinal chromophore in the visual pigment rhodopsin is coupled to motion of transmembrane helix H6 and receptor activation. We present solid-state magic angle spinning NMR measurements of rhodopsin and the metarhodopsin II intermediate that support the proposal that interaction of Trp265(6.48) with the retinal chromophore is responsible for stabilizing an inactive conformation in the dark, and that motion of the beta-ionone ring allows Trp265(6.48) and transmembrane helix H6 to adopt active conformations in the light. Two-dimensional dipolar-assisted rotational resonance NMR measurements are made between the C19 and C20-methyl groups of the retinal and uniformly 13C-labeled Trp265(6.48). The retinal C20-Trp265(6.48) contact present in the dark-state of rhodopsin is lost in metarhodopsin II, and a new contact is formed with the C19 methyl group. We have previously shown that the retinal translates 4-5 A toward H5 in metarhodopsin II. This motion, in conjunction with the Trp-C19 contact, implies that the Trp265(6.48) side-chain moves significantly upon rhodopsin activation. NMR measurements also show that a packing interaction in rhodopsin between Trp265(6.48) and Gly121(3.36) is lost in metarhodopsin II, consistent with H6 motion away from H3. However, a close contact between Gly120(3.35) on H3 and Met86(2.53) on H2 is observed in both rhodopsin and metarhodopsin II, suggesting that H3 does not change orientation significantly upon receptor activation.

Animals↗

PubMed2006

Structure of a transient intermediate for GTP hydrolysis by ras.

The flexibility of the conserved 57DTAGQ61 motif is essential for Ras proper cycling in response to growth factors. Here, we increase the flexibility of the 57DTAGQ61 motif by mutating Gln61 to Gly. The crystal structure of the RasQ61G mutant reveals a new conformation of switch 2 that bears remarkable structural homology to an intermediate for GTP hydrolysis revealed by targeted molecular dynamics simulations. The mutation increased retention of GTP and inhibited Ras binding to the catalytic site, but not to the distal site of Sos. Most importantly, the thermodynamics of RafRBD binding to Ras are altered even though the structure of switch 1 is not affected by the mutation. Our results suggest that interplay and transmission of structural information between the switch regions are important factors for Ras function. They propose that initiation of GTP hydrolysis sets off the separation of the Ras/effector complex even before the GDP conformation is reached.

Crystallography, X-Ray↗

PubMed2005

Dynamic behavior of DNA base pairs containing 8-oxoguanine.

The process by which DNA repair enzymes recognize and selectively excise damaged bases in duplex DNA is fundamental to our mechanistic understanding of these critical biological reactions. 8-Oxoguanine (8-oxoG) is the most common form of oxidative DNA damage; unrepaired, this lesion generates a G:C-->T:A mutation. Central to the recognition and repair of DNA damage is base extrusion, a process in which the damaged base lesion or, in some cases, its partner disengages from the helix and is bound to the enzyme's active site where base excision takes place. The conformation adopted by 8-oxoG in duplex DNA is affected by the base positioned opposite this lesion; conformational changes may also take place when the damaged base binds to its cognate repair enzyme. We performed unrestrained molecular dynamics simulations for several 13-mer DNA duplexes. Oligomers containing G:C and 8oxoG:C pairs adopted Watson-Crick geometries in stable B-form duplexes; 8oxoG showed increased local and global flexibility and a reduced barrier to base extrusion. Duplexes containing the G:A mismatch showed much larger structural fluctuations and failed to adopt a well-defined structure. For the 8oxoG:A mismatch that is recognized by the DNA glycosylase MutY, the damaged nucleoside underwent spontaneous and reproducible anti-->syn transitions. The syn conformation is thermodynamically preferred. Steric hindrance and unfavorable electrostatics associated with the 8oxoG O8 atom in the anti conformation were the major driving forces for this transition. Transition events follow two qualitatively different pathways. The overall anti-->syn transition rate and relative probability of the two transition paths were dependent on local sequence context. These simulations indicate that both the dynamic and equilibrium behavior of the duplex change as a result of oxidation; these differences may provide valuable new insight into the selective action of enzymes on damaged DNA.

Base Pairing↗

PubMed2005

Folding cooperativity in a three-stranded beta-sheet model.

The thermodynamic behavior of a previously designed three-stranded beta-sheet was studied via several microseconds of standard and replica exchange molecular dynamics simulations. The system is shown to populate at least four thermodynamic minima, including two partially folded states in which only a single hairpin is formed. Simulated melting curves show different profiles for the C and N-terminal hairpins, consistent with differences in secondary structure content in published NMR and CD/FTIR measurements, which probed different regions of the chain. Individual beta-hairpins that comprise the three-stranded beta-sheet are observed to form cooperatively. Partial folding cooperativity between the component hairpins is observed, and good agreement between calculated and experimental values quantifying this cooperativity is obtained when similar analysis techniques are used. However, the structural detail in the ensemble of conformations sampled in the simulations permits a more direct analysis of this cooperativity than has been performed on the basis of experimental data. The results indicate the actual folding cooperativity perpendicular to strand direction is significantly larger than the lower bound obtained previously.

Computer Simulation↗

PubMed2005

Comparison of class A and D G protein-coupled receptors: common features in structure and activation.

All G protein-coupled receptors (GPCRs) share a common seven TM helix architecture and the ability to activate heterotrimeric G proteins. Nevertheless, these receptors have widely divergent sequences with no significant homology. We present a detailed structure-function comparison of the very divergent Class A and D receptors to address whether there is a common activation mechanism across the GPCR superfamily. The Class A and D receptors are represented by the vertebrate visual pigment rhodopsin and the yeast alpha-factor pheromone receptor Ste2, respectively. Conserved amino acids within each specific receptor class and amino acids where mutation alters receptor function were located in the structures of rhodopsin and Ste2 to assess whether there are functionally equivalent positions or regions within these receptors. We find several general similarities that are quite striking. First, strongly polar amino acids mediate helix interactions. Their mutation generally leads to loss of function or constitutive activity. Second, small and weakly polar amino acids facilitate tight helix packing. Third, proline is essential at similar positions in transmembrane helices 6 and 7 of both receptors. Mapping the specific location of the conserved amino acids and sites of constitutively active mutations identified conserved microdomains on transmembrane helices H3, H6, and H7, suggesting that there are underlying similarities in the mechanism of the widely divergent Class A and Class D receptors.

Amino Acid Sequence↗

PubMed2005

Modified replica exchange simulation methods for local structure refinement.

Parallel tempering, also known as replica exchange molecular dynamics (REMD), has recently been successfully used to study the structure and thermodynamic properties of biomolecules such as peptides and small proteins. For large systems, however, applying REMD can be costly since the number of replicas needed increases as the square root of the number of degrees of freedom in the system. Often, enhanced sampling is only needed for a subset of atoms, such as a loop region of a large protein or a small ligand binding to a receptor. In such applications, it is often reasonable to assume a weak dependence of the structure of the larger region on the instantaneous conformation of the smaller region of interest. For these cases, we derived two variant replica exchange methods, partial replica exchange molecular dynamics (PREMD) and local replica exchange molecular dynamics (LREMD). The Hamiltonian for the system is separated, with replica exchange carried out only for terms involving the subsystem of interest while the remainder of the system is maintained at a single temperature. The number of replicas required for efficient exchange thus depends on the number of degrees of freedom in the fragment needing refinement rather than on the size of the full system. The method can be applied to much larger systems than was previously practical. This also provides a means to preserve the integrity of the structure outside the refinement region without introduction of restraints. LREMD takes this weak coupling approximation a step further, employing only a single representation of the large fragment that simultaneously interacts with all of the replicas of the subsystem of interest. This is obtained by combining replica exchange with the locally enhanced sampling approximation (LES), reducing the computational expense of replica exchange simulations to near that of a single standard molecular dynamics (MD) simulation. Use of LREMD also permits the use of LES without requiring the specification of a single temperature, a known difficulty for standard LES simulations. We tested these two methods on the loop region of an RNA hairpin model system and find significant advantages over standard MD and REMD simulations.

Algorithms↗

PubMed2004

Development of softcore potential functions for overcoming steric barriers in molecular dynamics simulations.

In this work, we describe the development of softcore potential functions that permit occasional "tunneling" through the regions of conformational space during molecular dynamics (MD) simulations, which would otherwise be sterically prohibited. The modification consists of a truncation of the nonbonded interaction before the steeply repulsive region encountered at short interatomic distances. This modification affects both Lennard-Jones and Coulomb parts of the nonbonded potential. Critical to success is the choice of appropriate pairwise switching distances at which this modification should be made. In the present work, these are calculated based on potential of mean force functions extracted from model system molecular dynamics simulations. We believe that these functions describe the dynamic short-range interactions much better than mean force potentials derived from an ensemble of static structures (e.g. protein data bank (PDB)). Once a set of mean force potentials is obtained, a single empirical parameter, effective barrier height, is employed to determine switching distances for all pairwise atomic interactions. Changing this single parameter allows adjustment of the "softness" of the whole system. We tested the applicability of the new softcore potentials in a loop structure optimization study. The H1 loop in the antibody 17/9 was selected as our test case because substantial repacking of loop residues in the dense protein environment is necessary for successful relaxation of random initial conformations. Softcore simulations converted to correct loop conformations, in contrast to standard simulations which never sampled this structure even after 10 ns. The resulting root mean square deviation (RMSD) values (below 1.3 A for all heavy atoms of the loop) demonstrate the usefulness of the approach based on mean force derived softcore functions.

Amino Acids↗

PubMed2003

Generation of accurate protein loop conformations through low-barrier molecular dynamics.

Prediction and refinement of protein loop structures are important and challenging tasks for which no general solution has been found. In addition to the accuracy of scoring functions, the main problems reside in (1) insufficient statistical sampling and (2) crossing energy barriers that impede conformational rearrangements of the loop. We approach these two issues by using "low-barrier molecular dynamics," a combination of energy smoothing techniques. To address statistical sampling, locally enhanced sampling (LES) is used to produce multiple copies of the loop, thus improving statistics and reducing energy barriers. We introduce a novel extension of LES that can improve local sampling even further through hierarchical subdivision of copies. Even though LES reduces energy barriers, it cannot provide for crossing infinite barriers, which can be problematic when substantial rearrangement of residues is necessary. To permit this kind of loop residue repacking, a "soft-core" potential energy function is introduced, so that atomic overlaps are temporarily allowed. We tested this new combined methodology to a loop in anti-influenza antibody Fab 17/9 (7 residues long) and to another loop in the antiprogesterone antibody DB3 (8 residues). In both cases, starting from random conformations, we were able to locate correct loop structures (including sidechain orientations) with heavy-atom root-mean-square deviation (fit to the nonloop region) of approximately 1.1 A in Fab 17/9 and approximately 1.8 A in DB3. We show that the combination of LES and soft-core potential substantially improves sampling compared to regular molecular dynamics. Moreover, the sampling improvement obtained with this combined approach is significantly better than that provided by either of the two methods alone.

Algorithms↗

PubMed2003

Using PC clusters to evaluate the transferability of molecular mechanics force fields for proteins.

The transferability of molecular mechanics parameters derived for small model systems to larger biopolymers such as proteins can be difficult to assess. Even for small peptides, molecular dynamics simulations are typically too short to sample structures significantly different than initial conformations, making comparison to experimental data questionable. We employed a PC cluster to generate large numbers of native and non-native conformations for peptides with experimentally measured structural data, one predominantly helical and the other forming a beta-hairpin. These atomic-detail sets do not suffer from slow convergence, and can be used to rapidly evaluate important force field properties. In this case a suspected bias toward alpha-helical conformations in the ff94 and ff99 force fields distributed with the AMBER package was verified. The sets provide critical feedback not only on force field transferability, but may also predict modifications for improvement. Such predictions were used to modify the ff99 parameter set, and the resulting force field was used to test stability and folding of model peptides. Structural behavior during molecular dynamics with the modified force field is found to be very similar to expectations, suggesting that these basis sets of conformations may themselves have significant transferability among force fields. We continue to improve and expand this data set and plan to make it publicly accessible. The calculations involved in this process are trivially parallel and can be performed using inexpensive personal computers with commodity components.

Computer Simulation↗

PubMed2002

Crystallographic structure of the K intermediate of bacteriorhodopsin: conservation of free energy after photoisomerization of the retinal.

The K state, an early intermediate of the bacteriorhodopsin photocycle, contains the excess free energy used for light-driven proton transport. The energy gain must reside in or near the photoisomerized retinal, but in what form has long been an open question. We produced the K intermediate in bacteriorhodopsin crystals in a photostationary state at 100K, with 40% yield, and determined its X-ray diffraction structure to 1.43 A resolution. In independent refinements of data from four crystals, the changes are confined mainly to the photoisomerized retinal. The retinal is 13-cis,15-anti, as known from vibrational spectroscopy. The C13=C14 bond is rotated nearly fully to cis from the initial trans configuration, but the C14-C15 and C15=NZ bonds are partially counter-rotated. This strained geometry keeps the direction of the Schiff base N-H bond vector roughly in the extracellular direction, but the angle of its hydrogen bond with water 402, that connects it to the anionic Asp85 and Asp212, is not optimal. Weakening of this hydrogen bond may account for many of the reported features of the infrared spectrum of K, and for its photoelectric signal, as well as the deprotonation of the Schiff base later in the cycle. Importantly, although 13-cis, the retinal does not assume the expected bent shape of this configuration. Comparison of the calculated energy of the increased angle of C12-C13=C14, that allows this distortion, with the earlier reported calorimetric measurement of the enthalpy gain of the K state indicates that a significant part of the excess energy is conserved in the bond strain at C13.

Bacteriorhodopsins↗