Biomedical subjects

Kenneth M Merz

Publications and source records attributed to Kenneth M Merz.

At least 19 recordsLinked to original sources

PubMed2006

Exploring the role of the active site cysteine in human muscle creatine kinase.

All known guanidino kinases contain a conserved cysteine residue that interacts with the non-nucleophilic eta1-nitrogen of the guanidino substrate. Site-directed mutagenesis studies have shown that this cysteine is important, but not essential for activity. In human muscle creatine kinase (HMCK) this residue, Cys283, forms part of a conserved cysteine-proline-serine (CPS) motif and has a pKa about 3 pH units below that of a regular cysteine residue. Here we employ a computational approach to predict the contribution of residues in this motif to the unusually low cysteine pKa. We calculate that hydrogen bonds to the hydroxyl and to the backbone amide of Ser285 would both contribute approximately 1 pH unit, while the presence of Pro284 in the motif lowers the pKa of Cys283 by a further 1.2 pH units. Using UV difference spectroscopy the pKa of the active site cysteine in WT HMCK and in the P284A, S285A, and C283S/S285C mutants was determined experimentally. The pKa values, although consistently about 0.5 pH unit lower, were in broad agreement with those predicted. The effect of each of these mutations on the pH-rate profile was also examined. The results show conclusively that, contrary to a previous report (Wang et al. (2001) Biochemistry 40, 11698-11705), Cys283 is not responsible for the pKa of 5.4 observed in the WT V/K(creatine) pH profile. Finally we use molecular dynamics simulations to demonstrate that, in order to maintain the linear alignment necessary for associative inline transfer of a phosphoryl group, Cys283 needs to be ionized.

Adenosine Diphosphate↗

PubMed2006

Role of solvation in the energy stabilization inside the hydrophobic core of the protein rubredoxin.

There are many forces that contribute to the stability of a protein; among these are dispersion interactions, hydrogen bonding, and solvation effects. In a recent work, Vondrasek et al. estimated the in vacuo stabilization energy of the hydrophobic core of the protein rubredoxin using high level ab initio methods (Vondrasek, J.; et al. J. Am. Chem. Soc. 2005, 127, 2615). In this work, we evaluate the effects of solvation on the stability of the hydrophobic core of this protein. Solvation calculations are made using the polarizable continuum method at the MP2/aug-cc-pVDZ level of theory. It is found that, in a protein-like environment (mimicked by a continuum solvent with a dielectric constant of approximately 4), the stability of rubredoxin's hydrophobic core is decreased by 40-50%. We also observed that the stabilization energy of the hydrophobic core is only slightly lower in a protein-like medium than in an aqueous one (DeltaGether-DeltaGwater approximately 1.0-3.5 kcal/mol).

Hydrophobic and Hydrophilic Interactions↗

PubMed2006

Interaction energy decomposition in protein-protein association: a quantum mechanical study of barnase-barstar complex.

Protein-protein interactions are very important in the function of a cell. Computational studies of these interactions have been of interest, but often they have utilized classical modelling techniques. In recent years, quantum mechanical (QM) treatment of entire proteins has emerged as a powerful approach to study biomolecular systems. Herein, we apply a semi-empirical divide and conquer (DC) methodology coupled with a dielectric continuum model for the solvent, to explore the contribution of electrostatics, polarization and charge transfer to the interaction energy between barnase and barstar in their complex form. Molecular dynamic (MD) simulation was performed to account for the dynamic behavior of the complex. The results show that electrostatics, charge transfer and polarization favor the formation of the complex. Our study shows that electrostatics dominates the interaction between barnase and barstar ( approximately 73%), while charge transfer and polarization are approximately 21% and approximately 6%, respectively. Close inspection of the polarization and charge-transfer effects on the charge distribution of the complex reveals the existence of two, well localized, regions in barstar. The first region includes the residues between P27 and Y47 and the second region is between N65 and D83. Since no such regions could be detected in barnase clearly suggests that barstar is well optimized for efficiently binding barnase. Furthermore, using our interaction energy decomposition scheme, we were able to identify all residues that have been experimentally determined to be important for the complex formation and to suggest other residues never have been investigated. This suggests that our approach will be useful as an aid in further understanding protein-protein contacts for the ultimate goal to produce successful inhibitors for protein complexes.

Bacterial Proteins↗

PubMed2006

Haptic applications for molecular structure manipulation.

We describe the application of haptic technology to enhance the information available in chemical systems, specifically related to computational drug design. These methods are designed to build upon the visual information presented by molecular viewers and add the sensation of touch, or force feedback. The addition of sensory input can aid in the analysis of molecular structures and the understanding of intermolecular interactions by delivering chemically relevant forces to the end user.

Computer Graphics↗

PubMed2006

Catalyzed decomposition of urea. Molecular dynamics simulations of the binding of urea to urease.

We present the results of molecular dynamics simulations on the urea/urease system. The starting structure was prepared from the 2.0 A crystal structure of Benini et al. [(1999) Struct. Folding Des. 7, 205-216] of DAP-inhibited urease (PDB code ), and the trimeric structure (2479 residues) resulted in 180K atoms after solvation by water. The force field parameters were derived using the bonded model approach described by Hoops et al. [(1991) J. Am. Chem. Soc. 113, 8262-8270]. Three different systems were analyzed, each one modeling a different protonation pattern for the His320 and His219 residues. In each case, the three monomers of urease have been analyzed separately. The time-averaged structures observed in the three monomers suggest that urease could follow two different competitive mechanisms. A "protein-assisted proton transfer" mechanism points to Asp221 as crucial for catalysis. An "Asp-mediated proton transfer" involves the transfer of a proton from the bridging OH to an NH2 moiety of urea, assisted by Asp360 in the active site. The impact of the simulation results on our understanding of urease catalysis is discussed in detail.

Bacillus↗

PubMed2006

X-ray diffraction "fingerprinting" of DNA structure in solution for quantitative evaluation of molecular dynamics simulation.

Solution state x-ray diffraction fingerprinting is demonstrated as a method for experimentally assessing the accuracy of molecular dynamics (MD) simulations. Fourier transforms of coordinate data from MD simulations are used to produce reciprocal space "fingerprints" of atomic pair distance correlations that are characteristic of the ensemble and are the direct numerical analogues of experimental solution x-ray diffraction (SXD). SXD experiments and MD simulations were carried out to test the ability of experiment and simulation to resolve sequence-dependent modifications in helix conformation for B-form DNA. SXD experiments demonstrated that solution-state poly(AT) and poly(A)-poly(T) duplex DNA sequences exist in ensembles close to canonical B-form and B'-form structures, respectively. In contrast, MD simulations analyzed in terms of SXD fingerprints are shown to deviate from experiment, most significantly for poly(A)-poly(T) duplex DNA. Compared with experiment, MD simulation shortcomings were found to include both mismatches in simulated conformer structures and number population within the ensembles. This work demonstrates an experimental approach for quantitatively evaluating MD simulations and other coordinate models to simulate biopolymer structure in solution and suggests opportunities to use solution diffraction data as experimental benchmarks for developing supramolecular force fields optimized for a range of in situ applications.

DNA↗

PubMed2006

Quantum mechanical and molecular dynamics simulations of ureases and Zn beta-lactamases.

Herein we briefly review theoretical contributions that have increased our understanding of the structure and function of metallo-beta-lactamases and ureases. Both are bimetallic metalloenzymes, with the former containing two zinc ions and the latter containing two nickel ions. We describe the use of several different methodologies, including quantum chemical calculations, molecular dynamic simulations, as well as mixed QM/MM approaches and how they have impacted our understanding of the structure and function of metallo-beta-lactamases and ureases.

Binding Sites↗

PubMed2006

Critical assessment of quantum mechanics based energy restraints in protein crystal structure refinement.

A critical evaluation of the performance of X-ray refinement protocols using various energy functions is presented using the bovine pancreatic trypsin inhibitor (BPTI) protein. The four potential energy functions we explored include: (1) fully quantum mechanical calculations; (2) one based on an incomplete molecular mechanics (MM) energy function employed in the Crystallography and NMR System (CNS) with empirical parameters developed by Engh and Huber (EH), which lacks electrostatic and attractive van der Waals terms; (3) one based on a complete MM energy function (AMBER ff99 parameter set); and (4) the same as 3, with the addition of a Generalized Born (GB) implicit solvation term. The R, R (free), real space R values of the refined structures and deviations from the original experimental structure were used to assess the relative performance. It was found that at 1 Angstrom resolution the physically based energy functions 1, 3, and 4 performed better than energy function 2, which we attribute to the better representation of key interactions, particularly electrostatics. The observed departures from the experimental structure were similar for the refinements with physically based energy functions and were smaller than the structure refined with EH. A test refinement was also performed with the reflections truncated at a high-resolution cutoff of 2.5 Angstrom and with random perturbations introduced into the initial coordinates, which showed that low-resolution refinements with physically based energy functions held the structure closer to the experimental structure solved at 1 Angstrom resolution than the EH-based refinements.

Animals↗

PubMed2006

Quantum mechanics in structure-based drug design.

In principle, quantum mechanics provides a more accurate representation of molecular systems than other modeling approaches. While this notion is not a matter of dispute, it has not yet been definitively demonstrated within the realm of structure-based drug design that the use of quantum mechanical methods over the use of classical modeling approaches is justified in consideration of the increase in expense associated with quantum mechanical methods. Demonstrating that quantum mechanics-based methods can be superior to simpler models, and resolving problems relating to estimating the effects of conformational entropy, will provide key areas of interest in the coming years for in silico structure-based drug design. Recent applications using quantum mechanical methods in structure-based drug design are reviewed herein, and applications ranging from scoring receptor-ligand interactions using quantum mechanics to the generation of quantitative structure-activity relationships using quantum mechanics-derived descriptors are discussed.

Drug Design↗

PubMed2005

Computational studies of the farnesyltransferase ternary complex part I: substrate binding.

Farnesyltransferase (FTase) catalyzes the transfer of farnesyl from farnesyl diphosphate (FPP) to cysteine residues at or near the C-terminus of protein acceptors with a CaaX motif (a, aliphatic; X, Met). Farnesylation is a critical modification to many switch proteins involved in the extracellular signal transduction pathway, which facilitates their fixation on the cell membrane where the extracellular signal is processed. The malfunction caused by mutations in these proteins often causes uncontrolled cell reproduction and leads to tumor formation. FTase inhibitors have been extensively studied as potential anticancer agents in recent years, several of which have advanced to different phases of clinical trials. However, the mechanism of this biologically important enzyme has not been firmly established. Understanding how FTase recruits the FPP substrate is the first and foremost step toward further mechanistic investigations and the design of effective FTase inhibitors. Molecular dynamic simulations were carried out on the ternary structure of FTase complexed with the FPP substrate and an acetyl-capped tetrapeptide (acetyl-CVIM), which revealed that the FPP substrate maintains an inactive conformation and the binding of the diphosphate group can be largely attributed to residues R291beta, K164alpha, K294beta, and H248beta. The FPP substrate assumes an extended conformation in the binding site with restricted rotation of the backbone dihedral angles; however, it does not have a well-defined conformation when unbound in solution. This is evident from multinanosecond MD simulations of the FPP substrate in a vacuum and solution. Our conclusion is further supported by theoretical J coupling calculations. Our results on the FPP binding are in good agreement with previous experimental kinetic studies on FTase mutants. The hypothetical conformational activation of the FPP substrate is currently under investigation.

Farnesyltranstransferase↗

PubMed2005

Quantum mechanical description of the interactions between DNA and water.

In recent years, a lot of attention has been focused on the electronic properties of DNA. With recent advances in linear scaling quantum mechanics there are now new tools available to enhance our understanding of the electronic properties of DNA among other biomolecules. Using both explicit solvent models and implicit (continuum) solvent models, the electronic characteristics of a dodecamer duplex DNA have been fully studied using both divide and conquer (D&C), semi-empirical quantum mechanics and non-D&C semi-empirical quantum mechanics. According to the AM1 Hamiltonian, approximately 3.5 electrons (approximately 0.3 electron/base pair) are transferred from the duplex to the solvent. According to the density of state (DOS) analysis, in vacuo DNA has a band gap of approximately 1 eV showing that in the absence of solvent, the DNA may exhibit similar properties to those of a semiconductor. Upon increasing solvation (2.5-5.5 A), the band gap ranges from approximately 3 eV to approximately 6 eV. For the implicit solvent model, the band gap continues this widening trend to approximately 7 eV. Therefore, upon solvation and in the absence of dopants, the DNA should begin to loose its conductive properties. Finally, when one considers the energy and localization of the frontier orbitals (HOMO and LUMO), solvent has a stabilizing effect on the DNA system. The energy of the HOMO drops from approximately 15 eV in vacuo to approximately 2 eV for 5.5 A of water to approximately -8 eV for the implicit solvent model. Similarly, the LUMO drops from approximately 16 eV for in vacuo to approximately 9 eV for 5.5 A of water to approximately -1 eV for the implicit model. Beyond the importance of the computed results on the materials properties of DNA, the present work also shows that the behavior of intercalators will be affected by the electronic properties of DNA. This could have an impact on our understanding of how DNA based drugs interact with DNA and on the design of new DNA based small molecule drugs.

Computer Simulation↗

PubMed2005

Effects of fluorine substitution on the edge-to-face interaction of the benzene dimer.

To gain some insight into the effects of fluorination on the aromatic-aromatic interactions found in protein-ligand complexes, like those observed in the set of N-(4-sulfamylbenzoyl)benzylamine (SBB) inhibitors bound to Human Carbonic Anhydrase II (HCAII), we have produced potential energy curves for the edge-to-face interactions of a set of fluorinated benzene dimer compounds. All calculations were carried out at the MP2/aug-cc-pVDZ level of theory using the counterpoise method of Boys and Bernardi (Boys, S. F.; Bernardi, F. Mol. Phys. 1970, 19, 553) to account for the basis set superposition error. Fluorine substitutions are made onto the face molecule of the edge-to-face benzene dimer. As one might expect, the substitution of additional fluorines into this system generally resulted in a decrease of the binding energy. It was also found that the positioning of the fluorine substituents on isosubstituted compounds has a large effect on the total binding energy of these types of systems. More specifically, complexes with fluorines that are substituted closer to the hydrogen atoms of the edge benzene will tend to be stabilized by an electrostatic interaction between the partially negative fluorine atoms and the partially positive hydrogen atoms. However, our findings do not explain the recent crystallographic findings for the SBB-HCAII protein-ligand complex, where increased fluorination resulted in closer edge-to-face contacts, which suggests that there are factors, other than edge-to-face aromatic interactions, influencing this system's behavior.

Benzene Derivatives↗

PubMed2005

Large-scale validation of a quantum mechanics based scoring function: predicting the binding affinity and the binding mode of a diverse set of protein-ligand complexes.

Computational methods to calculate binding affinity in protein-ligand interaction are of immense interest because of obvious practical applications in structure-based drug design. Scoring functions attempt to calculate the variation in binding affinity of ligands-inhibitors bound to protein targets at various levels of theory. In this study we use semiempirical quantum mechanics to design a scoring function that can calculate the electrostatic interactions and solvation free energy expected during complexation. This physically based approach has the ability to capture binding affinity trends in a diverse range of protein-ligand complexes. We also show the predictive power of this scoring function within protein targets and its ability to score ligand poses docked to a protein target. We also demonstrate the ability of this scoring function to discriminate between native and decoy poses and highlight the crucial role played by electrostatic interactions in molecular recognition. Finally we compare the performance of our scoring function with other available scoring functions in the literature.

HIV Protease↗

PubMed2005

Pairwise decomposition of residue interaction energies using semiempirical quantum mechanical methods in studies of protein-ligand interaction.

Pairwise decomposition of the interaction energy between molecules is shown to be a powerful tool that can increase our understanding of macromolecular recognition processes. Herein we calculate the pairwise decomposition of the interaction energy between the protein human carbonic anhydrase II (HCAII) and the fluorine-substituted ligand N-(4-sulfamylbenzoyl)benzylamine (SBB) using semiempirical quantum mechanics based methods. We dissect the interaction between the ligand and the protein by dividing the ligand and the protein into subsystems to understand the structure-activity relationships as a result of fluorine substitution. In particular, the off-diagonal elements of the Fock matrix that is composed of the interaction between the ionic core and the valence electrons and the exchange energy between the subsystems or atoms of interest is examined in detail. Our analysis reveals that the fluorine-substituted benzylamine group of SBB does not directly affect the binding energy. Rather, we find that the strength of the interaction between Thr199 of HCAII and the sulfamylbenzoyl group of SBB affects the binding affinity between the protein and the ligand. These observations underline the importance of the sulfonamide group in binding affinity as shown by previous experiments (Maren, T. H.; Wiley: C. E. J. Med. Chem. 1968, 11, 228-232). Moreover, our calculations qualitatively agree with the structural aspects of these protein-ligand complexes as determined by X-ray crystallography.

Benzylamines↗

PubMed2005

Validation of the binding site structure of the cellular retinol-binding protein (CRBP) by ligand NMR chemical shift perturbations.

We have calculated proton chemical shift perturbations (CSPs) of retinol in the cellular retinol-binding protein (CRBP) through the use of a recently developed computational approach (Wang et al. J. Chem. Phys. 2004, 120, 11392-11400). Excellent agreement with experimental values was obtained for the X-ray structure, whereas the lack of a key hydrogen bond and the distorted isoprene tail of retinol for some NMR models lead to large CSP RMSDs. Therefore, a comparison of computed CSPs of retinol with experiment offers a convenient way to validate the structure of retinol and its orientation in the binding site for the NMR structures.

Binding Sites↗

PubMed2005

Hybrid QM/MM and DFT investigations of the catalytic mechanism and inhibition of the dinuclear zinc metallo-beta-lactamase CcrA from Bacteroides fragilis.

Based on hybrid QM/MM molecular dynamics simulation and density functional theoretical (DFT) calculations, we investigate the mechanistic and energetic features of the catalytic action of dizinc metallo-beta-lactamase CcrA from Bacteroides fragilis. The 200 ps QM/MM simulation of the CcrA enzyme in complex with nitrocefin shows that the substrate beta-lactam moiety is directed toward the active site dizinc center through the interactions of aminocarbonyl and carboxylate groups with the two active site zinc ions and the two conserved residues, Lys167 and Asn176. From the determination of the potential energy profile of a relevant enzymatic reaction model, it is found that the nucleophilic displacement reaction step proceeds with a low-barrier height, leading to the formation of an energetically favored reaction intermediate. The results also show that the high catalytic activity of the CcrA enzyme stems from a simultaneous operation of three catalytic components: activation of the bridging hydroxide nucleophile by zinc-coordinated Asp86; polarization of the substrate aminocarbonyl group by the first zinc ion; stabilization of the negative charge developed on the departing amide nitrogen by the second zinc ion. Consistent with the previous experimental finding that the proton-transfer reaction step is rate-limiting, the activation energy of the second step is found to be 1.6 kcal/mol higher than that of the first step. Finally, through an examination of the structural and energetic features of binding of a thiazolidinecarboxylic acid inhibitor to the active site dizinc center, a two-step inhibition mechanism involving a protonation-induced ligand exchange reaction is proposed for the inhibitory action of a tight-binding inhibitor possessing a thiol group.

Bacterial Proteins↗

PubMed2005

Force field design and molecular dynamics simulations of the carbapenem- and cephamycin-resistant dinuclear zinc metallo-beta-lactamase from Bacteroides fragilis and its complex with a biphenyl tetrazole inhibitor.

On the basis of molecular dynamics simulations, we investigate the dynamic properties of the carbapenem- and cephamycin-resistant dinuclear zinc metallo-beta-lactamase from Bacteroides fragilis and its complex with a biphenyl tetrazole inhibitor, 2-butyl-6-hydroxy-3-[2'-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-quinazolin-4-one 1 (L-159061). The results obtained with the newly developed force field parameters for the coordination environment of the catalytic zinc ions show that the active site gorge comprising major and minor loops gets deeper and narrower upon binding of the inhibitor, which supports the previous experimental implication that the structural flexibility of the loop structures plays a significant role in enzymatic action. In the presence of the inhibitor, the Trp32 side chain at the apex of the major loop covers the entrance of active site channel, thereby contributing to the stabilization of the enzyme-inhibitor complex. In addition to a direct coordination of the inhibitor tetrazole ring to the second zinc ion in the active site, the hydrogen bonding of Lys167 to the inhibitor carbonyl group and hydrophobic interactions between the inhibitor and side chains of loop residues prove to be significant binding forces of the enzyme-inhibitor complex.

Anti-Bacterial Agents↗

PubMed2005

Refinement of protein crystal structures using energy restraints derived from linear-scaling quantum mechanics.

A novel method is proposed in which combined restraints derived from linear-scaling semiempirical quantum-mechanical (QM) calculations and X-ray diffraction data are combined to refine crystal structures of proteins. Its performance has been tested on a small protein molecule, bovine pancreatic trypsin inhibitor (BPTI). The refinement involves minimization of the sum of a geometric energy function and an X-ray target function based on either the least-squares residual or the maximum-likelihood formalism. For comparison, similar refinement runs have also been performed using energy restraints derived from the force field available in the Crystallography & NMR System (CNS) program. The QM refinements were carried out with weights that were varied by several orders of magnitude and the optimal weights were identified by observing the trend in the final free R values, QM heats of formation and coordinate root-mean-square deviations (r.m.s.d.s) from the crystal structure. It is found that the QM weights are typically smaller but generally on the same scale as the molecular-mechanics (MM) weights for the respective X-ray target functions. The crystallographic R, free R, real-space R values and correlation coefficients based on the structures refined with the energy restraints derived from our QM calculations and Engh and Huber parameters are comparable, suggesting that the QM restraints are capable of maintaining reasonable stereochemistry to a similar degree as the force-field parameters. A detailed inspection of the structures refined with the QM and MM energy restraints reveals that one of the common differences between them and the crystal structure is that the strained bond angles in the crystal structure are corrected after energetically restrained refinements. Systematic differences in certain bond lengths between the QM-refined structures and the statistical averages of experimental structures have also been observed and discussed.

Crystallography, X-Ray↗