Molecular dynamics of HIV-1 reverse transcriptase indicates increased flexibility upon DNA binding.

HIV-1 reverse transcriptase (RT) is one of the main targets for drugs used in the treatment of AIDS, among them, the non-nucleoside RT inhibitors (NNRTIs). The flexibility of RT unliganded and complexed to double-stranded DNA (RT/dsDNA), in water, has been studied by means of molecular dynamics. The simulations show that RT flexibility depends on its ligation state. The RT/dsDNA trajectories show larger fluctuations in the atomic positions than uncomplexed RT, particularly at the tips of the p66 fingers and thumb subdomains. This increased flexibility is consistent with the ability of the p66 fingers of the RT/dsDNA complex to close down after the binding of a deoxynucleoside triphosphate (dNTP) molecule, as observed in the crystal structures of RT/dsDNA bound to dNTP. The two complexation states present different patterns of concerted motions, indicating that the bound dsDNA alters RT flexibility. The motions of amino acid residues that form the non-nucleoside RT inhibitor binding pocket upon complexation with a NNRTI are anticorrelated with the p66 fingers (in RT/dsDNA) and correlated to the RNase H subdomain (unliganded RT). These concerted motions indicate that binding of a NNRTI could alter the flexibility of the subdomains whose motions are correlated to those of the binding pocket.

Kinetic and mechanistic studies of prolyl oligopeptidase from the hyperthermophile Pyrococcus furiosus.

Prolyl oligopeptidase (POP) is widely distributed in mammals, where it is implicated in neuropeptide processing. It is also present in some bacteria and archaea. Because POP is found in mesophilic and hyperthermophilic organisms, and is distributed among all three phylogenetic domains, studies of its function and structure could lead to new insights about the evolution of enzyme mechanisms and thermostability. Kinetic studies were conducted on the POP of the hyperthermophilic archaeon Pyrococcus furiosus (Pfu) 85 degrees C in both H(2)O and D(2)O. Pfu POP displayed many similarities to mammalian POPs, however the solvent isotope effect (k(0)/k(1)) was 2.2 at both high and low pH, indicating that general base/acid catalysis is the rate-limiting step. The pH-rate profiles indicated a three-deprotonation process with pK(a) values of 4.3, 7.2, and 9.1. The temperature dependence of these values revealed a heat of ionization of 4.7 kJ/mol for pK(es1) and 22 kJ/mol for pK(es2), suggesting the catalytic involvement of a carboxyl group and an imidazole group, respectively. Temperature dependence of the catalytic rate was assessed at pH 6.0 and 7.6. Entropy values of -119 and -143 Jmol(-1)K(-1) were calculated at the respective pH values, with a corresponding difference in enthalpy of 8.5 kJ/mol. These values suggest that two or three hydrogen bonds are broken during the transition state of the acidic enzyme form, whereas only one or two are broken during the transition state of the basic enzyme form. A model has been constructed for Pfu POP based on the crystal structure of porcine POP and the sequence alignment. The similarities demonstrated for POPs from these two organisms reflect the most highly conserved characteristics of this class of serine protease, whereas the differences between these enzymes highlights the large evolutionary distance between them. Such fundamental information is crucial to our understanding of the function of proteins at high temperature.

Docking of sulfonamides to carbonic anhydrase II and IV.

Starting with a known active site of a protein and a database of compounds, one would like to quickly identify a few compounds that "dock" into the active site and obtain "good" binding free energies. The main goal of current automated docking procedures is to predict the "best" substrate-enzyme complex while other programs such as UHBD and DelPhi can be used to compute binding free energies. In this paper, we will focus on the application of docking methods and parameters to study substrate-enzyme interactions of a metalloenzyme system. Specifically, we report on the docking of sulfonamides to carbonic anhydrase II and IV, which are of interest due to their application in glaucoma therapy. Using a standard docking protocol, it is possible to correctly predict not only the orientation of inhibitors to a specific isozyme, but also determine the qualitative affinity for a group of inhibitor for an isozyme.

Molecular recognition and binding of thermal hysteresis proteins to ice.

Molecular recognition and binding are two very important processes in virtually all biological and chemical processes. An extremely interesting system involving recognition and binding is that of thermal hysteresis proteins at the ice-water interface. These proteins are of great scientific interest because of their antifreeze activity. Certain fish, insects and plants living in cold weather regions are known to generate these proteins for survival. A detailed molecular understanding of how these proteins work could assist in developing synthetic analogs for use in industry. Although the shapes of these proteins vary from completely alpha-helical to globular, they perform the same function. It is the shapes of these proteins that control their recognition and binding to a specific face of ice. Thermal hysteresis proteins modify the morphology of the ice crystal, thereby depressing the freezing point. Currently there are three hypotheses proposed with respect to the antifreeze activity of thermal hysteresis proteins. From structure-function experiments, ice etching experiments, X-ray structures and computer modeling at the ice-vacuum interface, the first recognition and binding hypothesis was proposed and stated that a lattice match of the ice oxygens with hydrogen-bonding groups on the proteins was important. Additional mutagenesis experiments and computer simulations have lead to the second hypothesis, which asserted that the hydrophobic portion of the amphiphilic helix of the type I thermal hysteresis proteins accumulates at the ice-water interface. A third hypothesis, also based on mutagenesis experiments and computer simulations, suggests that the thermal hysteresis proteins accumulate in the ice-water interface and actually influence the specific ice plane to which the thermal hysteresis protein ultimately binds. The first two hypotheses emphasize the aspect of the protein 'binding or accumulating' to specific faces of ice, while the third suggests that the protein assists in the development of the binding site. Our modeling and analysis supports the third hypothesis, however, the first two cannot be completely ruled out at this time. The objective of this paper is to review the computational and experimental efforts during the past 20 years to elucidate the recognition and binding of thermal hysteresis proteins at the ice-vacuum and ice-water interface.

Homology modeling of glycosyl hydrolase family 18 enzymes and proteins.

Using state-of-the-art homology modeling methods, three-dimensional coordinates for three family 18 glycosyl hydrolases were determined. The structures for Gp39, Brp39, and chitotriosidase were computer determined using the X-ray coordinates from SmChiA. The results of the modeling efforts are assessed, and comparison of the modeled structures to other known family 18 members is made.

Modeling studies of binding of sea raven type II antifreeze protein to ice.

Certain plants, insects, and fish living in cold environments prevent tissue damage due to freezing by producing antifreeze proteins or antifreeze glycoproteins that inhibit ice growth below the normal equilibrium freezing point of water in a noncolligative fashion. In polar fish these macromolecules, taking into account their structural characteristics, are grouped into three broad classes, namely Type I, Type II, and Type III. In this paper we report the results of our studies on the stereospecific binding of sea raven, a Type II antifreeze protein (AFP) to (111) hexagonal bipyramidal faces of ice. Earlier studies of Type I and Type III AFPs have shown that stereospecific binding of these proteins, recognizing specific planes of ice, is essential for their noncolligative antifreeze point depression. Moreover, as it has been shown for the AFT of Type I, this binding also occurs along specific vectors on these planes and also is enantioselective, distinguishing between the mirror related directions. In this study we will show, by using molecular modeling, that the fold of Type II AFP could facilitate a stereospecific mode of interaction with (111) planes of ice. Similar to Type I AFP, preferential directionality of binding was also observed in the simulations.

Analysis of shorthorn sculpin antifreeze protein stereospecific binding to (2-1 0) faces of ice.

In this paper we report the results of our studies on the stereospecific binding of shorthorn sculpin antifreeze protein (AFP) to (2 -1 0) secondary prism faces of ice. Using ice crystal growth and etching techniques together with molecular modeling, molecular dynamics, and energy minimization, we explain the nature of preferential binding of shorthorn sculpin AFP along the [1 2 2] direction on (2- 1 0) planes. In agreement with ice etching studies, the mechanism of preferential binding suggested by molecular modeling explains why the binding of shorthorn sculpin AFP occurs along [1 2 2] and not along its mirror symmetry-related direction [-1 -2 2] on (2 -1 0). This binding mechanism is based on the protein-crystal surface enantioselective recognition that utilizes both alpha-helical protein backbone matching to the (2 -1 0) surface topography and matching of side chains of polar/charged residues with specific water molecule positions in the ice surface. The mechanisms of winter flounder and shorthorn sculpin antifreeze binding to ice are compared.

Scanning electron microscopy and molecular modeling of inhibition of calcium oxalate monohydrate crystal growth by citrate and phosphocitrate.

Binding of citrate and phosphocitrate to calcium oxalate monohydrate crystals has been studied using scanning electron microscopy (SEM) and molecular modeling. Phosphocitrate structure has been resolved using low temperature X-ray analysis and ab initio computational methods. The (-1 0 1) crystal surface of calcium oxalate monohydrate is involved in binding of citrate and phosphocitrate, as shown by SEM and molecular modeling. Citrate and phosphocitrate conformations and binding energies to (-1 0 1) faces have been obtained and compared to binding to another set of calcium-rich planes (0 1 0). Difference in inhibitory properties of these compounds has been attributed to better coordination of functional groups of phosphocitrate with calcium ions in (-1 0 1). Relevance of this study to design of new calcium oxalate monohydrate inhibitors is discussed.

Mechanisms underlying taurine-mediated alterations in membrane function.

Taurine mediates a plethora of membrane-linked effects in excitable tissues. To account for these multiple actions, four hypotheses have been proposed. One theory is based on the observation that taurine diminishes the inflammatory response of several cytotoxic oxidants. It is proposed that a reduction in the extent of membrane oxidative injury contributes to these cytoprotective actions. The second theory maintains that alterations in protein phosphorylation may underlie certain effects of taurine, particularly its effect on calcium transport. The third hypothesis assumes that the interaction of taurine with the neutral phospholipids leads to altered membrane calcium binding and function. The final theory ties the actions of taurine to inhibition of phospholipid N-methylation and the resulting changes in membrane composition and structure. While each of these hypotheses has merit, none of them can fully explain the membrane actions of taurine. Further studies are required to ascertain the importance of each theory.

Atomic force microscopy and molecular modeling of protein and peptide binding to calcite.

Oyster shell protein and polyaspartate bound to calcite have been visualized at the atomic and molecular levels by atomic force microscopy. The identities of potential binding sites have been suggested from atomic force microscopy (AFM) images and have been evaluated by molecular modeling. Energies and conformations of binding to (110) and (110) prism faces, (001) basal calcium planes, and (104) cleavage planes are considered. The interaction with the basal plane is strongest and is essentially irreversible. Binding to (110) prism surfaces is also energetically favored and selective for orientations parallel or perpendicular to the c-axis. Binding to (110) faces is significantly weaker and orientation nonspecific. If carboxyl groups of the protein or peptide replace select carbonate ions of the (110) face, the binding energy increases significantly, favoring binding in the parallel direction. Binding to (104) cleavage surfaces is weak and probably reversible. Specific alignment of oyster shell protein molecules on calcite surfaces is shown by AFM, and the relevance to the binding model is discussed.

Simulation of enzyme-substrate encounter with gated active sites.

We describe a brownian dynamics simulation method that allows investigation of the effects of receptor flexibility on ligand binding rates. The method is applied to the encounter of substrate, glyceraldehyde 3-phosphate, with triose phosphate isomerase, a diffusion-controlled enzyme with flexible peptide loops at its active sites. The simulations show that while the electrostatic field surrounding the enzyme steers the substrate into its active sites, the flexible loops appear to have little influence on the substrate binding rate. The dynamics of the loops may therefore have been optimized during evolution to minimize their interference with the substrate's access to the active sites. The calculated and experimental rate constants are in good agreement.

Gating of the active site of triose phosphate isomerase: Brownian dynamics simulations of flexible peptide loops in the enzyme.

The enzyme triose phosphate isomerase has flexible peptide loops at its active sites. The loops close over these sites upon substrate binding, suggesting that the dynamics of the loops could be of mechanistic and kinetic importance. To investigate these issues, the loop motions in the dimeric enzyme were simulated by Brownian dynamics. The two loops, one on each monomer, were represented by linear chains of appropriately parameterized spheres, each sphere corresponding to an amino acid residue. The loops moved in the electrostatic field of the rest of the enzyme, which was held rigid in its crystallographically observed conformation. In the absence of substrate, the loops exhibited gating of the active site with a period of about 1 ns and occupied "closed" conformations for about half of the time. As the period of gating is much shorter than the enzyme-substrate relaxation time, the motion of the loops does not reduce the rate constant for the approach of substrate from its simple diffusion-controlled value. This suggests that the flexible loops may have evolved to create the appropriate environment for catalysis while, at the same time, minimizing the kinetic penalty for gating the active site.

Computer simulations of organic reactions in solution.

Quantum and statistical mechanics have been used to determine energy profiles for the SN2 reaction of Cl- + CH3Cl in the gas phase, in aqueous solution, and in liquid DMF. The energy profile in the gas phase has the characteristic double-well form featuring unsymmetrical ion-dipole complexes as minima and a symmetrical transition state. Hydration causes the reaction surface to become almost unimodal and increases the barrier significantly. The reaction profile in DMF is intermediate between those for the gas phase and aqueous solution. The ion-dipole complexes are still free energy minima in DMF. Thus, the reaction in DMF involves initial formation of the complex before the rate-determining step. The computed results are shown to be in good accord with experimental free energies of activation. The same technique has been applied to the addition reaction of OH- + H2C = O in the gas phase and aqueous solution. Ab initio 6-31 + G* calculations indicate that the reaction proceeds essentially without activation in the gas phase. Hydration introduces a substantial energy barrier. The transition state in water has been located at a C-O separation of roughly 2 A. A key finding for both reactions is that the activation barriers induced by hydration result primarily from change in strengths rather than in numbers of solute-water hydrogen bonds along the reaction paths.