Determining the roles of different chain fragments in recognition of immunoglobulin fold.

We examine sequence-to-structure specificity of beta-structural fragments of immunoglobulin domains. The structure specificity of separate chain fragments is estimated by computing the Z-score values in recognition of the native structure in gapless threading tests. To improve the accuracy of our calculations we use energy averaging over diverse homologs of immunoglobulin domains. We show that the interactions between residues of beta-structure are more determinant in recognition of the native structure than the interactions within the whole chain molecule. This result distinguishes immunoglobulins from more typical proteins where the interactions between residues of the whole chain normally recognize the native fold more accurately than interactions between the residues of the secondary structure residues alone [Reva,B. and Topiol,S. (2000) BIOCOMPUTING: Proceedings of the Pacific Symposium. World Scientific Publishing Co., pp. 168-178]. We also find that the predominant contributions of the secondary structure are produced by the four central beta-strands that form the core of the molecule. The results of this study allow us through quantitative means to understand the architecture of immunoglobulin molecules. Comparing the fold recognition data for different chain fragments one can say that beta-strands form a rigid frame for immunoglobulin molecules, whereas loops, with no structural role, can develop a broad variety of binding specificities. It is well known that protein function is determined by specific portions of a protein chain. This study suggests that the whole protein structure can be predominantly determined by a few fragments of chain which form the structural framework of the molecule. This idea may help in better understanding the mechanisms of protein evolution: strengthening a protein structure in the key framework-forming regions allows mutations and flexibility in other chain regions.

Computer Aided Analysis of Split and Mix Combinatorial Libraries.

Combinatorial chemistry using split and pool synthesis involves making and testing mixtures of compounds in pools which are subsets of the larger compound collection. These subsets are created during the synthesis of the collection through a resin splitting and mixing method. Tests are conducted on each of the final pools of mixtures and the individual compounds within a mixture of interest are then identified through some deconvolution scheme, originally involving selective re-synthesis. It is possible that different schemes for splitting and mixing will have different consequences on the overall effort necessary to deconvolute interesting mixtures. The evaluation of different protocols of splitting and mixing involves consideration of more possibilities than can be exhaustively or optimally determined manually in a realistic time frame for most compound collections. We present herein a computational scheme to aid in this analysis. The approach exhaustively examines possible splitting and mixing strategies for the interrelated values of total library size, number of combinatorial steps, number of reaction vessels, and number of compounds per final pool. Weighting factors may be introduced into the various steps. The resulting complete list of splitting and mixing options is scored based on a variable weighting strategy for the total effort of synthesis and deconvolution. The results indicate the splitting/mixing strategy used has an impact on overall efficiency and should be considered in the design of compound libraries.

Recognition of protein structure: determining the relative energetic contributions of beta-strands, alpha-helices and loops.

We examine the role of residues of secondary structure in recognition of the native structure of proteins. The accuracy of recognition was estimated by computing the Z-score values for fragments of protein chains in threading tests. By testing different combinations of secondary structure fragments of 240 non-homologous proteins we show that the overwhelming majority of proteins can be successfully recognized by the energies of interaction between residues of secondary structure. We also found that beta-structures contribute more significantly to fold recognition than alpha-helices or loops. To validate the Z-score calculations in measuring the accuracy of recognition we evaluated the deviation of the energy distribution from the normal law. The normal law satisfactory approximates the shape of the energy distribution for the majority of proteins and chain fragments; however, deviations are often observed for short fragments and for fragments with relatively high Z-score values. The results of the study justify recognition of remote homologs by threading methods based on a backbone of secondary structure rather than of a whole chain because loops of homologs differ more significantly than strands and helices, and the contribution of loops in structure recognition is relatively small.

Structural and conformational requirements for high-affinity binding to the SH2 domain of Grb2(1).

Following earlier work on cystine-bridged peptides, cyclic phosphopeptides containing nonreducible mimics of cystine were synthesized that show high affinity and specificity toward the Src homology (SH2) domain of the growth factor receptor-binding protein (Grb2). Replacement of the cystine in the cyclic heptapeptide cyclo(CYVNVPC) by D-alpha-acetylthialysine or D-alpha-lysine gave cyclo(YVNVP(D-alpha-acetyl-thiaK)) (22) and cyclo(YVNVP(D-alpha-acetyl-K)) (30), which showed improved binding 10-fold relative to that of the control peptide KPFYVNVEF (1). NMR spectroscopy and molecular modeling experiments indicate that a beta-turn conformation centered around YVNV is essential for high-affinity binding. X-ray structure analyses show that the linear peptide 1 and the cyclic compound 21 adopt a similar binding mode with a beta-turn conformation. Our data confirm the unique structural requirements of the ligand binding site of the SH2 domain of Grb2. Moreover, the potency of our cyclic lactams can be explained by the stabilization of the beta-turn conformation by three intramolecular hydrogen bonds (one mediated by an H2O molecule). These stable and easily accessible cyclic peptides can serve as templates for the evaluation of phosphotyrosine surrogates and further chemical elaboration.

Heterologously expressed inner lipoyl domain of dihydrolipoyl acetyltransferase inhibits ATP-dependent inactivation of pyruvate dehydrogenase complex. Identification of important amino acid residues.

The activity of the pyruvate dehydrogenase multienzyme complex (PDC), which catalyses the oxidation of pyruvate to acetyl-CoA within the mitochondrion, is diminished in animal models of diabetes. Studies with purified PDC components have suggested that the kinases responsible for inactivating the decarboxylase catalytic subunits of the complex are most efficient in their regulatory role when they are bound to dihydrolipoyl acetyltransferase (E2) subunits, which form the structural core of the complex. We report that the addition of an exogenous E2 subdomain (inner lipoyl domain) to an intact PDC inhibits ATP-dependent inactivation of the complex. By combining molecular modelling, site-directed mutagenesis and biophysical characterizations, we have also identified two amino acid residues in this subdomain (Ile229 and Phe231) that largely determine the magnitude of this effect.

A computational study of a host-guest complex.

The geometry and energetics of a complex involving pyrazine and an acridine diacid cleft-like host designed by Rebek were investigated at several levels of theory. Molecular mechanics (using the Tripos and CHARMm force fields), semiempirical quantum chemical approaches (with the AM1 and PM3 methods), and an ab initio quantum chemical method (RHF/STO-3G) were used in the complete relaxation of the complex. The geometry of the complex optimized by the RHF/STO-3G method is in excellent agreement with a published X-ray structure; upon superposition, the rms deviation between the corresponding cleft heavy atoms is only 0.17 A and the pyrazine molecules are superimposable. In addition, ab initio quantum chemical techniques were used to study the complex when the cleft is modeled by a pair of acetic acid molecules. All the calculations presented herein support a two-point interaction mechanism. The similarities found in the results for the full complex and the truncated model are consistent with a purely structural role for the acridine linker of the host.

The computational design of test compounds with potentially specific biological activity: histamine-H2 agonists derived from 5-HT/H2 antagonists.

The previously proposed models for the recognition and activation of 5-HT and histamine-H2 receptors, which were employed to explain the antagonist activity of LSD at both of these receptors, as well as the selective antagonism for H2 receptors by SKF-10856 and 9,10-dihydro-LSD, are used herein to design a compound to test the H2-receptor model. The design strategy attempts to construct a compound with potentially selective H2 agonism. The design scheme maintains features which were previously used to explain selective recognition of SKF-10856 and 9,10-dihydro-LSD as well as reintroduces the chemical features proposed to be responsible for H2 activation. The existence of the H2 recognition and activation features in the proposed compound is verified, in a previously proposed model, by computational studies of the molecular electrostatic potentials and shifts in the tautomeric preference.

Computational studies of ligand/receptor interactions.

An analysis of common features of many proteins provided the basis for a general model for the origin of receptors (Topiol, 1987). In this model, receptors are derived from fully operational parent systems. The parent system is rendered inactive by "deletion" of some critical component, thereby converting it to a receptor for the deleted entity. Such a model could explain the use of common molecular machinery by different biological systems (e.g., receptors and enzymes), the origin of receptor subtypes, the use of common effector systems by different receptors, the sequence homologies between varied proteins, the relationship between endogenous ligands and biological "building blocks", and the selectivity and compatibility between natural receptors and endogenous ligands. Some examples of the use of this model to analyze a number of different biochemical systems and processes will be given. Preliminary insights from these studies as a guide to developing molecular models for the action of cyclic nucleotide second messengers will be discussed.

A general criterion for molecular recognition: implications for chiral interactions.

A general criterion is formulated for molecular recognition. The criterion for recognition is the inequality of the distance matrices of complexes of different compounds with a resolving agent under ambient experimental conditions. It is shown how this criterion provides for an objective, well-defined, and simple explanation for recognition of chiral compounds. This approach may be used to explain models (e.g., three-point of attachment) and relationships for chiral recognition. It is also shown how one-, two-, or three-point mechanisms are equivalent in this formalism and could result in chiral recognition. Examples are used to illustrate how the so called one- or two-point mechanisms may be operative in many experimental findings. Symmetry requirements of resolving agents may also be derived from considerations of distance matrices. Finally, the reciprocal relationship of chiral resolving agents is easily derived from the present method of analysis.

A molecular model for activation of a 5-hydroxytryptamine receptor.

The extension of a model proposed previously for molecular recognition at a serotonin (5-hydroxytryptamine (5-HT) ) receptor makes possible the formulation of a molecular mechanism of receptor activation. The activation mechanism proposed here is based on the changes induced in the drug and in a model receptor by the interaction mimicking the formation of a drug-receptor complex. This mechanism was simulated by quantum mechanical calculations of molecular interactions between 5-HT and a model for a receptor represented by an imidazolium-ammonia complex that serves as a proton transfer model (PTM). The movement of the proton in the PTM is promoted by the interaction with 5-HT, suggesting a process by which 5-HT can trigger the activation of the receptor. The elements of the activation mechanism revealed by the results of the simulation are: (a) the electrostatic alignment between the PTM and 5-HT, which guides the recognition of 5-HT by the PTM; (b) the contraction of the distance between the hydrogen bonded components of the PTM, induced by the interaction of 5-HT with the PTM, which leads to a decrease in the barrier to proton transfer in the PTM; (c) an additional decrease of the barrier to proton transfer produced by the negative electrostatic potential of 5-HT, which stabilizes the transition state; and (d) the increased preference for product over reactant in the interaction complex between 5-HT and the PTM, which constitutes a driving force for the proton transfer process. According to this model, compounds that activate the 5-HT receptor should bind in a mode that induces the changes described above in the PTM and thus triggers the proton transfer.

Molecular determinants for the agonist activity of 2-methylhistamine and 4-methylhistamine at H2-receptors.

A model for drug action at the histamine H2-receptor has been evaluated computationally for the agonists 2- and 4-methylhistamine. Based on molecular properties calculated for molecular structures optimized with ab initio quantum mechanical methods, the activities of these compounds and their potencies relative to histamine are found to be explained by the previously proposed model. Recognized in the N3-H tautomeric form of their monocations, both compounds exhibit a change in ring tautomeric preference when the cationic side chain is neutralized. This change makes possible their participation in a proposed proton relay event that was postulated to initiate the receptor response of H2-agonists. The relative concentrations of the mono- and dication forms of the molecules in equimolar concentrations of histamine and the two derivatives are calculated from the values of the molecular electrostatic potentials at the ring protonation sites. Because the monocation is the species recognized at the H2-receptor, the reduced potency of 2-methylhistamine relative to histamine and to the 4-methyl derivative is explained by the finding that 2-methylhistamine will have the lowest concentration of the recognized species. The rank order of potencies obtained from the ratio of monocationic species of the molecules is in agreement with experimental results.

Theoretical studies on the activation mechanism of the histamine H2-receptor: the proton transfer between histamine and a receptor model.

A proposed molecular mechanism for the activation of the H2-receptor of histamine was simulated with ab initio calculations including geometry optimization with several basis sets. The system is modeled by a proton-relay chain produced by the binding of a histamine molecule to a receptor model consisting of an anionic anchoring site, and proton donor and acceptor sites. The anchoring of histamine cation at a negative receptor site is simulated by the interaction with a hydroxyl anion or by calculations on neutral histamine; the proton donor and acceptor sites are modeled by ammonium and ammonia groups, respectively. Results of the calculations reveal that a significant decrease in the barrier for the movement of the proton from the donor site to the N1 nitrogen in the imidazole portion of histamine occurs as a consequence of the neutralization of the side chain and the simultaneous interaction of the N3 nitrogen with the proton acceptor. An increase of the driving force for the proton transfer process is produced by these interactions, as observed from the relative energies of the initial and final steps of the charge relay. The barrier and the driving force depend on the nature of the proton acceptor site. This simulation of the receptor activation mechanism provides the basis for exploration of the partial receptor activation by molecules characterized as partial agonists and the lack of activation by molecules that act as antagonists on this receptor.

A molecular theory of recognition and activation at a 5-HT receptor based on a quantum chemical approach to structure activity relationships.

The study of structure activity relationships (SAR) is based on the delineation of the causal relationships between the properties of molecules and the observed responses evoked by the interaction of these molecules with biological systems. The methods of theoretical and quantum chemistry describe accurately the molecular properties that are determined by molecular structure and provide a rigorous link between structure and activity. We study the molecular events in the pharmacological mechanism of drugs interacting with the receptor of 5-hydroxytryptamine (5-HT, serotonin) by defining the elements of recognition and by analyzing the changes induced in a molecular model for the receptor. These steps define the relationship between the properties of the drugs and their ability to be recognized and cause the activation of the receptors. Consequently, our quantum chemical studies of drug-receptor interactions explain the selectivity of receptors and the molecular determinants for agonism and antagonism on the 5-HT receptor.

A theoretical investigation of histamine tautomerism.

Geometry optimizations of the structures of histamine (neutral and monocation) in the N(3)-H and N(1)-H tautomeric forms were performed at the ab initio Hartree-Fock level with the STO-3G basis set. Values of the structural parameters and their changes upon protonation and/or tautomerization are in good agreement with data from X-ray crystal-structure analysis of histamine and several analogues. Earlier predictions of the tautomeric preference from calculations using frozen geometries based on crystal-structure data are confirmed by calculations of energies of histamine in the fully optimized geometries with both the STO-3G and LP-3G basis sets and by comparisons of the minima in the molecular electrostatic potentials of the two tautomers. These results support a previously proposed model for the activation of the histamine H2 receptor.

The two-modes-of-binding model for partial agonism and the design of partial agonists.

A model of drug action is developed for a drug which can bind in two modes to a given receptor: an agonist mode which elicits a response and an antagonist mode which does not. It is shown that this combination of agonist and antagonist binding, which is tantamount to self-antagonism, can lead to agonist, partial agonist or antagonist behavior. The proposed two-modes-of-binding model has applications to the design of partial agonists. Finally, partial agonism arising from two modes of binding is indistinguishable from partial agonism occurring by two-state model mechanisms using experimental equilibrium studies.