Freddie R. Salsbury

New analytic approximation to the standard molecular volume definition and its application to generalized Born calculations

In a recent article (Lee, M. S.; Salsbury, F. R. Jr.; Brooks, C. L., III. J Chem Phys 2002, 116, 10606), we demonstrated that generalized Born (GB) theory provides a good approximation to Poisson electrostatic solvation energy calculations if one uses the same definitions of molecular volume for each. In this work, we present a new and improved analytic method for reproducing the Lee–Richards molecular volume, which is the most common volume definition for Poisson calculations. Overall, 1% errors are achieved for absolute solvation energies of a large set of proteins and relative solvation energies of protein conformations. We also introduce an accurate SASA approximation that uses the same machinery employed by our GB method and requires a small addition of computational cost. The combined methodology is shown to yield an efficient and accurate implicit solvent representation for simulations of biopolymers.

Constant-pH Molecular Dynamics using Continuous Titration Coordinates

In this work, we explore the question of whether pKa calculations based on a microscopic description of the protein and a macroscopic description of the solvent can be implemented to examine conformationally dependent proton shifts in proteins. To this end, we introduce a new method for performing constant‐pH molecular dynamics (PHMD) simulations utilizing the generalized Born implicit solvent model. This approach employs an extended Hamiltonian in which continuous titration coordinates propagate simultaneously with the atomic motions of the system. The values adopted by these coordinates are modulated by potentials of mean force of isolated titratable model groups and the pH to control the proton occupation at particular sites in the polypeptide. Our results for four different proteins yield an absolute average error of ∼1.6 pK units, and point to the role that thermally driven relaxation of the protein environment in the vicinity of titrating groups plays in modulating the local pKa, thereby influencing the observed pK1/2 values. While the accuracy of our method is not yet equivalent to methods that obtain pK1/2 values through the ad hoc scaling of electrostatics, the present approach and constant pH methods in general provide a useful framework for studying pH‐dependent phenomena. Further work to improve our model to approach quantitative agreement with experiment is outlined. Proteins 2004.

Functional site profiling and electrostatic analysis of cysteines modifiable to cysteine sulfenic acid.

Cysteine sulfenic acid (Cys‐SOH), a reversible modification, is a catalytic intermediate at enzyme active sites, a sensor for oxidative stress, a regulator of some transcription factors, and a redox‐signaling intermediate. This post‐translational modification is not random: specific features near the cysteine control its reactivity. To identify features responsible for the propensity of cysteines to be modified to sulfenic acid, a list of 47 proteins (containing 49 known Cys‐SOH sites) was compiled. Modifiable cysteines are found in proteins from most structural classes and many functional classes, but have no propensity for any one type of protein secondary structure. To identify features affecting cysteine reactivity, these sites were analyzed using both functional site profiling and electrostatic analysis. Overall, the solvent exposure of modifiable cysteines is not different from the average cysteine. The combined sequence, structure, and electrostatic approaches reveal mechanistic determinants not obvious from overall sequence comparison, including: (1) pKas of some modifiable cysteines are affected by backbone features only; (2) charged residues are underrepresented in the structure near modifiable sites; (3) threonine and other polar residues can exert a large influence on the cysteine pKa; and (4) hydrogen bonding patterns are suggested to be important. This compilation of Cys‐SOH modification sites and their features provides a quantitative assessment of previous observations and a basis for further analysis and prediction of these sites. Agreement with known experimental data indicates the utility of this combined approach for identifying mechanistic determinants at protein functional sites.

An efficient hybrid explicit/implicit solvent method for biomolecular simulations†

We present a new hybrid explicit/implicit solvent method for dynamics simulations of macromolecular systems. The method models explicitly the hydration of the solute by either a layer or sphere of water molecules, and the generalized Born (GB) theory is used to treat the bulk continuum solvent outside the explicit simulation volume. To reduce the computational cost, we implemented a multigrid method for evaluating the pairwise electrostatic and GB terms. It is shown that for typical ion and protein simulations our method achieves similar equilibrium and dynamical observables as the conventional particle mesh Ewald (PME) method. Simulation timings are reported, which indicate that the hybrid method is much faster than PME, primarily due to a significant reduction in the number of explicit water molecules required to model hydration effects.

Molecular dynamics simulations of protein dynamics and their relevance to drug discovery.

Molecular dynamics simulations have become increasingly useful in studying biological systems of biomedical interest, and not just in the study of model or toy systems. In this article, the methods and principles of all-atom molecular dynamics will be elucidated with several examples provided of their utility to investigators interested on drug discovery.

Modeling of the metallo-β-lactamase from B. fragilis: Structural and dynamic effects of inhibitor binding†

The structure and dynamics of an inhibitor‐bound complex of the metallo‐β‐lactamase from Bacteroides fragilis are studied by using molecular dynamics. A search of the conformational space was performed to obtain three distinct models of the complex, which were then subjected to solvated molecular dynamics. A solvated molecular dynamics study of the apo protein was performed to serve as a baseline for comparison with the bound simulations. We find loop conformation changes due to binding as well as a decrease in flexibility of the protein as a whole and especially in the major loop of the β‐lactamase. We report the structural and dynamical features of the inhibitor‐bound and apo models, as well as experimentally measurable quantities, which should be capable of distinguishing the two binding modes we have determined. Proteins 2001;44:448–459.

Parameters of Reserpine Analogs That Induce MSH2/MSH6-Dependent Cytotoxic Response

Mismatch repair proteins modulate the cytotoxicity of several chemotherapeutic agents. We have recently proposed a “death conformation” of the MutS homologous proteins that is distinguishable from their “repair conformation.” This conformation can be induced by a small molecule, reserpine, leading to DNA-independent cell death. We investigated the parameters for a small reserpine-like molecule that are required to interact with MSH2/MSH6 to induce MSH2/MSH6-dependent cytotoxic response. A multidisciplinary approach involving structural modeling, chemical synthesis, and cell biology analyzed reserpine analogs and modifications. We demonstrate that the parameters controlling the induction of MSH2/MSH6-dependent cytotoxicity for reserpine-analogous molecules reside in the specific requirements for methoxy groups, the size of the molecule, and the orientation of molecules within the protein-binding pocket. Reserpine analog rescinnamine showed improved MSH2-dependent cytotoxicity. These results have important implications for the identification of compounds that require functional MMR proteins to exhibit their full cytotoxicity, which will avoid resistance in MMR-deficient cells.

Molecular dynamic simulations of the metallo-beta-lactamase from Bacteroides fragilis in the presence and absence of a tight-binding inhibitor

The beta-lactam-based antibiotics are among the most prescribed and effective antibacterial agents. Widespread use of these antibiotics, however, has created tremendous pressure for the emergence of resistance mechanisms in bacteria. The most common cause of antibiotic resistance is bacterial production of actamases that efficiently degrade antibiotics. The metallo-beta-lactamases are of particular clinical concern due to their transference between bacterial strains. We used molecular dynamics (MD) simulations to further study the conformational changes that occur due to binding of an inhibitor to the dicanzinc metallo-beta-lactamase from Bacteroides fragilis. Our studies confirm previous findings that the major flap is a major source of plasticity within the active site, therefore its dynamic response should be considered in drug development. However, our results also suggest the need for care in using MD simulations in evaluating loop mobility, both due to relaxation times and to the need to accurately model the zinc active site. Our study also reveals two new robust responses to ligand binding. First, there are specific localized changes in the zinc active site—a local loop flip—due to ligand intercalation that may be critical to the function of this enzyme. Second, inhibitor binding perturbs the dynamics throughout the protein, without otherwise perturbing the enzyme structure. These dynamic perturbations radiate outward from the active site and their existence suggests that long-range communication and dynamics may be important in the activity of this enzyme.

Insights into Protein—DNA Interactions, Stability and Allosteric Communications: A Computational Study of Mutsα-DNA Recognition Complexes

Abstract DNA mismatch repair proteins (MMR) maintain genetic stability by recognizing and repairing mismatched bases and insertion/deletion loops mistakenly incorporated during DNA replication, and initiate cellular response to certain types of DNA damage. Loss of MMR in mammalian cells has been linked to resistance to certain DNA damaging chemotherapeutic agents, as well as to increase risk of cancer. Mismatch repair pathway is considered to involve the concerted action of at least 20 proteins. The most abundant MMR mismatch-binding factor in eukaryotes, MutSα, recognizes and initiates the repair of base-base mismatches and small insertion/deletion. We performed molecular dynamics simulations on mismatched and damaged MutSα-DNA complexes. A comprehensive DNA binding site analysis of relevant conformations shows that MutSα proteins recognize the mismatched and platinum crosslinked DNA substrates in significantly different modes. Distinctive conformational changes associated with MutSα binding to mismatched and damaged DNA have been identified and they provide insight into the involvement of MMR proteins in DNA-repair and DNA-damage pathways. Stability and allosteric interactions at the heterodimer interface associated with the mismatch and damage recognition step allow for prediction of key residues in MMR cancer-causing mutations. A rigorous hydrogen bonding analysis for ADP molecules at the ATPase binding sites is also presented. Due to extended number of known MMR cancer causing mutations among the residues proved to make specific contacts with ADP molecules, recommendations for further studies on similar mutagenic effects were made.

Zn2+ selectively stabilizes FdU-substituted DNA through a unique major groove binding motif

We report, based on semi-empirical calculations, that Zn2+ binds duplex DNA containing consecutive FdU–dA base pairs in the major groove with distorted trigonal bipyramidal geometry. In this previously uncharacterized binding motif, O4 and F5 on consecutive FdU are axial ligands while three water molecules complete the coordination sphere. NMR spectroscopy confirmed Zn2+ complexation occurred with maintenance of base pairing while a slight hypsochromic shift in circular dichroism (CD) spectra indicated moderate structural distortion relative to B-form DNA. Zn2+ complexation inhibited ethidium bromide (EtBr) intercalation and stabilized FdU-substituted duplex DNA (ΔTm > 15°C). Mg2+ neither inhibited EtBr complexation nor had as strong of a stabilizing effect. DNA sequences that did not contain consecutive FdU were not stabilized by Zn2+. A lipofectamine preparation of the Zn2+–DNA complex displayed enhanced cytotoxicity toward prostate cancer cells relative to the individual components prepared as lipofectamine complexes indicating the potential utility of Zn2+–DNA complexes for cancer treatment.