Hierarchy of simulation models in predicting molecular recognition mechanisms from the binding energy landscapes: structural analysis of the peptide complexes with SH2 domains.

Computer simulations using the simplified energy function and simulated tempering dynamics have accurately determined the native structure of the pYVPML, SVLpYTAVQPNE, and SPGEpYVNIEF peptides in the complexes with SH2 domains. Structural and equilibrium aspects of the peptide binding with SH2 domains have been studied by generating temperature-dependent binding free energy landscapes. Once some native peptide-SH2 domain contacts are constrained, the underlying binding free energy profile has the funnel-like shape that leads to a rapid and consistent acquisition of the native structure. The dominant native topology of the peptide-SH2 domain complexes represents an extended peptide conformation with strong specific interactions in the phosphotyrosine pocket and hydrophobic interactions of the peptide residues C-terminal to the pTyr group. The topological features of the peptide-protein interface are primarily determined by the thermodynamically stable phosphotyrosyl group. A diversity of structurally different binding orientations has been observed for the amino-terminal residues to the phosphotyrosine. The dominant native topology for the peptide residues carboxy-terminal to the phosphotyrosine is tolerant to flexibility in this region of the peptide-SH2 domain interface observed in equilibrium simulations. The energy landscape analysis has revealed a broad, entropically favorable topology of the native binding mode for the bound peptides, which is robust to structural perturbations. This could provide an additional positive mechanism underlying tolerance of the SH2 domains to hydrophobic conservative substitutions in the peptide specificity region.

Deciphering common failures in molecular docking of ligand-protein complexes.

Common failures in predicting crystal structures of ligand-protein complexes are investigated for three ligand-protein systems by a combined thermodynamic and kinetic analysis of the binding energy landscapes. Misdocked predictions in ligand-protein docking are classified as 'soft' and 'hard' failures. While a soft failure arises when the search algorithm is unable to find the global energy minimum corresponding to the crystal structure, a hard failure results from a flaw of the energy function to qualify the crystal structure as the predicted lowest energy conformation in docking simulations. We find that neither the determination of a single structure with the lowest energy nor finding the most common binding mode is sufficient to predict crystal structures of the complexes, which belong to the category of hard failures. In a proposed hierarchical approach, structural similarity clustering of the conformations, generated from equilibrium simulations with the simplified energy function, is followed by energy refinement with the AMBER force field. This protocol, that involves a hierarchy of energy functions, resolves some common failures in ligand-protein docking and detects crystallographic binding modes that were not found during docking simulations.

Towards understanding the mechanisms of molecular recognition by computer simulations of ligand-protein interactions.

The thermodynamic and kinetic aspects of molecular recognition for the methotrexate (MTX)-dihydrofolate reductase (DHFR) ligand-protein system are investigated by the binding energy landscape approach. The impact of 'hot' and 'cold' errors in ligand mutations on the thermodynamic stability of the native MTX-DHFR complex is analyzed, and relationships between the molecular recognition mechanism and the degree of ligand optimization are discussed. The nature and relative stability of intermediates and thermodynamic phases on the ligand-protein association pathway are studied, providing new insights into connections between protein folding and molecular recognition mechanisms, and cooperativity of ligand-protein binding. The results of kinetic docking simulations are rationalized based on the thermodynamic properties determined from equilibrium simulations and the shape of the underlying binding energy landscape. We show how evolutionary ligand selection for a receptor active site can produce well-optimized ligand-protein systems such as MTX-DHFR complex with the thermodynamically stable native structure and a direct transition mechanism of binding from unbound conformations to the unique native structure.

Thermodynamics and kinetics of ligand-protein binding studied with the weighted histogram analysis method and simulated annealing.

The thermodynamics of ligand-protein molecular recognition is investigated by the energy landscape approach for two systems: methotrexate(MTX)--dihydrofolate reductase(DHFR) and biotin-streptavidin. The temperature-dependent binding free energy profile is determined using the weighted histogram analysis method. Two different force fields are employed in this study: a simplified model of ligand-protein interactions and the AMBER force field with a soft core smoothing component, used to soften the repulsive part of the potential. The results of multiple docking simulations are rationalized from the shape of the binding free energy profile that characterizes the thermodynamics of the binding process.

Exploring the energy landscapes of molecular recognition by a genetic algorithm: analysis of the requirements for robust docking of HIV-1 protease and FKBP-12 complexes.

Energy landscapes of molecular recognition are explored by performing "semi-rigid" docking of FK-506 and rapamycin with the Fukisawa binding protein (FKBP-12), and flexible docking simulations of the Ro-31-8959 and AG-1284 inhibitors with HIV-1 protease by a genetic algorithm. The requirements of a molecular recognition model to meet thermodynamic and kinetic criteria of ligand-protein docking simultaneously are investigated using a family of simple molecular recognition energy functions. The critical factor that determines the success rate in predicting the structure of ligand-protein complexes is found to be the roughness of the binding energy landscape, in accordance with a minimal frustration principle. The results suggest that further progress in structure prediction of ligand-protein complexes can be achieved by designing molecular recognition energy functions that generate binding landscapes with reduced frustration.

Empirical free energy calculations of ligand-protein crystallographic complexes. I. Knowledge-based ligand-protein interaction potentials applied to the prediction of human immunodeficiency virus 1 protease binding affinity.

The steadily increasing number of high-resolution human immunodeficiency virus (HIV) 1 protease complexes has been the impetus for the elaboration of knowledge-based mean field ligand-protein interaction potentials. These potentials have been linked with the hydrophobicity and conformational entropy scales developed originally to explain protein folding and stability. Empirical free energy calculations of a diverse set of HIV-1 protease crystallographic complexes have enabled a detailed analysis of binding thermodynamics. The thermodynamic consequences of conformational changes that HIV-1 protease undergoes upon binding to all inhibitors, and a substantial concomitant loss of conformational entropy by the part of HIV-1 protease that forms the ligand-protein interface, have been examined. The quantitative breakdown of the entropy-driven changes occurring during ligand-protein association, such as the hydrophobic contribution, the conformational entropy term and the entropy loss due to a reduction of rotational and translational degrees of freedom, of a system composed to ligand, protein and crystallographic water molecules at the ligand-protein interface has been carried out. The proposed approach provides reasonable estimates of distinctions in binding affinity and gives an insight into the nature of enthalpyentropy compensation factors detected in the binding process.

Molecular recognition of the inhibitor AG-1343 by HIV-1 protease: conformationally flexible docking by evolutionary programming.

BACKGROUND: An important prerequisite for computational structure-based drug design is prediction of the structures of ligand-protein complexes that have not yet been experimentally determined by X-ray crystallography or NMR. For this task, docking of rigid ligands is inadequate because it assumes knowledge of the conformation of the bound ligand. Docking of flexible ligands would be desirable, but requires one to search an enormous conformational space. We set out to develop a strategy for flexible docking by combining a simple model of ligand-protein interactions for molecular recognition with an evolutionary programming search technique. RESULTS: We have developed an intermolecular energy function that incorporates steric and hydrogen-bonding terms. The parameters in this function were obtained by docking in three different protein systems. The effectiveness of this method was demonstrated by conformationally flexible docking of the inhibitor AG-1343, a potential new drug against AIDS, into HIV-1 protease. For this molecule, which has nine rotatable bonds, the crystal structure was reproduced within 1.5 A root-mean-square deviation 34 times in 100 simulations, each requiring eight minutes on a Silicon Graphics R4400 workstation. The energy function correctly evaluates the crystal structure as the global energy minimum. CONCLUSIONS: We believe that a solution of the docking problem may be achieved by matching a simple model of molecular recognition with an efficient search procedure. The necessary ingredients of a molecular recognition model include only steric and hydrogen-bond interaction terms. Although these terms are not necessarily sufficient to predict binding affinity, they describe ligand-protein interactions faithfully enough to enable a docking program to predict the structure of the bound ligand. This docking strategy thus provides an important tool for the interdisciplinary field of rational drug design.

De novo design of enzyme inhibitors by Monte Carlo ligand generation.

A new computational method for the in situ generation of small molecules within the binding site of a protein is described. The method has been evaluated using two well-studied systems, dihydrofolate reductase and thymidylate synthase. The method has also been used to guide improvements to inhibitors of HIV-1 protease. One such improvement resulted in a compound selected for preclinical studies as an antiviral agent against AIDS.

Dihydrofolate reductase from Lactobacillus casei. Stereochemistry of NADPH binding.

The NADPH molecule binds to dihydrofolate reductase in an extended conformation. Several of the individual dihedral angles, especially in the adenine mononucleotide portion of the coenzyme, differ from their minimum energy conformations. The ribose phosphate portions of the coenzyme are involved in numerous specific hydrogen-bonded and charge-charge interactions. The adenine ring resides in an apparently nonspecific hydrophobic cleft and the nicotinamide ring is bound within an intricately constructed cavity, one wall of which includes the pyrazine ring of bound methotrexate. Two rather extended loops (residues 10 to 24 and 117 to 135) connecting beta A to alpha B and beta F to beta G, respectively, move 2 to 3 A when NADPH binds to dihydrofolate reductase. No overall structural homology is evident between the dinucleotide binding domains of dihydrofolate reductase on the one hand and the four NAD+-dependent dehydrogenases of known structure on the other. However, binding does occur in both cases at the carboxyl edge of a region of parallel beta sheet flanked by a pair of alpha helices.

Dihydrofolate reductase: x-ray structure of the binary complex with methotrexate.

A central eight-stranded beta-pleated sheet is the main feature of the polypeptide backbone folding in dihydrofolate reductase. The innermost four strands and two bridging helices are geometrically similar to but are connected in a different way from those in the dinucleotide binding domains found in nicotinamide-adenine dinucleotide-linked dehydrogenases. Methotrexate is bound in a 15-angstrom-deep cavity with the pteridine ring buried in a primarily hydrophobic pocket, although a strong interaction occurs between the side chain of aspartic acid 27 and N(1), N(8), and the 2-amino group of methotrexate.

A detailed structural comparison between the charge relay system in chymotrypsinogen and in alpha-chymotrypsin.

An improved 2.5-A electron density map of chymotrypsinogen was calculated by incorporating heavy-atom anomalous scattering effects and a new model of the molecule was constructed. Phases from x-ray structure factors (R = 0.43) computed from this model were then used in the calculation of another electron density map against which the model was further refined. The catalytic Ser-195 side chain in the new model is in the "down" or "acyl" orientation and its Ogamma atom is in position to form a normal hydrogen bond with Nepsilon2 of His-57. In contrast, the corresponding hydrogen bond in alpha-chymotrypsin (Birktoft, J.J., and Blow, D.M. (1972), J.Mol. Biol. 68, 187) is severely distorted, probably as a consequence of a 1.5-A shift in the relative positions of the two cylindrical folding domains composing most of the molecule. We suggest that this activiation induced distortion of the charge-relay, hydrogen-bonding system plays an important role in the genesis of enzymic activity, in accord with an earlier proposal by Wang concerning the role of bent hydrogen bonds in enzyme catalysis (Wang, J.J. (1970), Proc. Natl. Acad. Sci. U.S.A. 66, 874).

Polypeptide halomethyl ketones bind to serine proteases as analogs of the tetrahedral intermediate. X-ray crystallographic comparison of lysine- and phenylalanine-polypeptide chloromethyl ketone-inhibited subtilisin.

1. A detailed study of cytochrome C oxidse activity with Keilin-Hartree particles and purified beef heart enzyme, at low ionic strength and low cytochrome C concentrations, showed biphasic kinetics with apparent Km1 = 5 x 10(-8) M, and apparent Km2 = 0.35 to 1.0 x 10(-6) M. Direct binding studies with purified oxidase, phospholipid-containing as well as phospholipid-depleted, demonstrated two sites of interaction of cytochrome c with the enzyme, with KD2 less than or equal to 10(-7) M, and KD2 = 10(-6) M. 2...

X-ray crystallographic study of boronic acid adducts with subtilisin BPN' (Novo). A model for the catalytic transition state.

We have studied the structures of adducts formed between subtilisin BPN' and both benzeneboronic acid and 2-phenylethaneboronic acid by x-ray diffraction techniques. Electron density and difference maps at 2.5 A resolution were computed with phases calculated from a partially refined structure of the native enzyme (R = 0.23 at 2.0 A). Both adducts contain a covalent bond between Ogamma of the catalytic Ser-221 and the inhibitor boron atom. The boron atom is coordinated tetrahedrally, with one of the two additional boronic acid oxygen atoms lying in the "oxyanion hole" and the other at the leaving group site identified in previous studies (ROBERTUS, J.D., Kraut, J. ALDEN, R.A., and BIRKTOFT, J.J. (1972) Biochemistry 11, 4293-4303). Moreover, the previously postulated structure of the tetrahedral intermediate for substrate hydrolysis is isosteric with these boronic acid adducts, which can therefore be considered good models for the transition state complex (KOEHLER, K.K., and LIENHARD, G.E. (1972) Biochemistry 10, 2477-2483). These observations further support the suggestion that an important contribution to stabilization of this transition state complex, relative to both the Michaelis complex and the acyl intermediate, occurs as a consequence of hydrogen bond donation to the substrate carbonyl oxygen atom from the side chain amido group of Asn-155 and from the backbone amido group of Ser-221.