Structural self-organization of C60 and cisplatin in physiological solution.

The specific features of structural self-organization of C60 fullerene and antitumor drug cisplatin (Cis) in physiological solution (0.9% NaCl) have been investigated by means of small-angle neutron scattering, scanning electron and atomic force microscopies, as well as isothermal titration calorimetry, dynamic light scattering and UV-Vis spectroscopy. The formation of C60 + Cis complexes, has been reported, unveiling the mechanism of medico-biological synergy observed during administration of the mixture of these drugs.

Physics-based protein-structure prediction using a hierarchical protocol based on the UNRES force field: assessment in two blind tests.

Recent improvements in the protein-structure prediction method developed in our laboratory, based on the thermodynamic hypothesis, are described. The conformational space is searched extensively at the united-residue level by using our physics-based UNRES energy function and the conformational space annealing method of global optimization. The lowest-energy coarse-grained structures are then converted to an all-atom representation and energy-minimized with the ECEPP/3 force field. The procedure was assessed in two recent blind tests of protein-structure prediction. During the first blind test, we predicted large fragments of alpha and alpha+beta proteins [60-70 residues with C(alpha) rms deviation (rmsd) <6 A]. However, for alpha+beta proteins, significant topological errors occurred despite low rmsd values. In the second exercise, we predicted whole structures of five proteins (two alpha and three alpha+beta, with sizes of 53-235 residues) with remarkably good accuracy. In particular, for the genomic target TM0487 (a 102-residue alpha+beta protein from Thermotoga maritima), we predicted the complete, topologically correct structure with 7.3-A C(alpha) rmsd. So far this protein is the largest alpha+beta protein predicted based solely on the amino acid sequence and a physics-based potential-energy function and search procedure. For target T0198, a phosphate transport system regulator PhoU from T. maritima (a 235-residue mainly alpha-helical protein), we predicted the topology of the whole six-helix bundle correctly within 8 A rmsd, except the 32 C-terminal residues, most of which form a beta-hairpin. These and other examples described in this work demonstrate significant progress in physics-based protein-structure prediction.

The protein folding problem: global optimization of the force fields.

The evolutionary development of a theoretical approach to the protein folding problem, in our laboratory, is traced. The theoretical foundations and the development of a suitable empirical all-atom potential energy function and a global optimization search are examined. Whereas the all-atom approach has thus far succeeded for relatively small molecules and for alpha-helical proteins containing up to 46 residues, it has been necessary to develop a hierarchical approach to treat larger proteins. In the hierarchical approach to single- and multiple-chain proteins, global optimization is carried out for a simplified united residue (UNRES) description of a polypeptide chain to locate the region in which the global minimum lies. Conversion of the UNRES structures in this region to all-atom structures is followed by a local search in this region. The performance of this approach in successive CASP blind tests for predicting protein structure by an ab initio physics-based method is described. Finally, a recent attempt to compute a folding pathway is discussed.

Docking ligands to vasopressin and oxytocin receptors via genetic algorithm.

The aim of the study was to computer-dock selected ligands to neurophyseal receptors in order to identify amino acid residues responsible for ligand-receptor interactions. To this aim, reliable oxytocin receptor (OTR) and arginine-vasopressin receptor (V1aR/V2R) models were built. The OTR-selective agonist [Thr4,Gly7]OT, the OTR-selective cyclohexapeptide antagonist L-366,948 and OT itself were docked via genetic algorithm to OTR, V1aR, and V2R and relaxed using a constrained simulated annealing protocol. For the analysis of receptor/ligand interactions a subset of initial conformations was chosen using energetic and steric criteria. All three ligands seem to prefer similar modes of binding to the receptors, manifested by repetitive residues of the receptors which directly interact with the ligands. Taking into account that many aspects of mechanisms of G protein-coupled receptor (GPCR) action are still unsolved, the results obtained with the docking simulations may propose future experimental research, especially in site-directed mutagenesis analysis and searching for key amino acid residues responsible for drug activities.

Molecular docking-based test for affinities of two ligands toward vasopressin and oxytocin receptors.

Molecular docking simulations are now fast developing area of research. In this work we describe an effective procedure of preparation of the receptor-ligand complexes. The amino-acid residues involved in ligand binding were identified and described.

Molecular modelling study of the role of cholesterol in the stimulation of the oxytocin receptor.

Cholesterol, an integral component of membranes in Eucaryota, is a modifier of membrane properties. In vivo studies have demonstrated that cholesterol can also modulate activities of some G protein-coupled receptors (GPCRs), which are integral membrane proteins. This can result either from an effect of cholesterol on the membrane fluidity or from specific interactions of the membrane cholesterol with the receptor, as recently demonstrated for the cholecystokinin type beta (CCKRbeta) or the oxytocin receptor (OTR). Using molecular modelling, we studied conformational preferences of cholesterol and several of its analogues. Subsequently, we simulated the distributions of their preferred conformations around the surface of OTR, CCKRbeta and a chimeric oxytocin/cholecystokinin receptor. Consequently, we suggest residues on the surface of OTR which are potentially significant in the OTR/cholesterol interaction.

Recent improvements in prediction of protein structure by global optimization of a potential energy function.

Recent improvements of a hierarchical ab initio or de novo approach for predicting both alpha and beta structures of proteins are described. The united-residue energy function used in this procedure includes multibody interactions from a cumulant expansion of the free energy of polypeptide chains, with their relative weights determined by Z-score optimization. The critical initial stage of the hierarchical procedure involves a search of conformational space by the conformational space annealing (CSA) method, followed by optimization of an all-atom model. The procedure was assessed in a recent blind test of protein structure prediction (CASP4). The resulting lowest-energy structures of the target proteins (ranging in size from 70 to 244 residues) agreed with the experimental structures in many respects. The entire experimental structure of a cyclic alpha-helical protein of 70 residues was predicted to within 4.3 A alpha-carbon (C(alpha)) rms deviation (rmsd) whereas, for other alpha-helical proteins, fragments of roughly 60 residues were predicted to within 6.0 A C(alpha) rmsd. Whereas beta structures can now be predicted with the new procedure, the success rate for alpha/beta- and beta-proteins is lower than that for alpha-proteins at present. For the beta portions of alpha/beta structures, the C(alpha) rmsd's are less than 6.0 A for contiguous fragments of 30-40 residues; for one target, three fragments (of length 10, 23, and 28 residues, respectively) formed a compact part of the tertiary structure with a C(alpha) rmsd less than 6.0 A. Overall, these results constitute an important step toward the ab initio prediction of protein structure solely from the amino acid sequence.

Molecular modeling of interactions of the non-peptide antagonist YM087 with the human vasopressin V1a, V2 receptors and with oxytocin receptors.

The nonapeptide hormones arginine vasopressin (CYFQNCPRG-NH2, AVP) and oxytocin (CYIQNCPLG-NH2, OT), control many essential functions in mammals. Their main activities include the urine concentration (via stimulation of AVP V2 receptors, V2R, in the kidneys), blood pressure regulation (via stimulation of vascular V1a AVP receptors, V1aR), ACTH control (via stimulation of V1b receptors, V1bR, in the pituitary) and labor and lactation control (via stimulation of OT receptors, OTR, in the uterus and nipples, respectively). All four receptor subtypes belong to the GTP-binding (G) protein-coupled receptor (GPCR) family. This work consists of docking of YM087, a potent non-peptide V1aR and V2R - but not OTR - antagonist, into the receptor models based on relatively new theoretical templates of rhodopsin (RD) and opiate receptors, proposed by Mosberg et al. (Univ. of Michigan, Ann Arbor, USA). It is simultaneously demonstrated that this RD template satisfactorily compares with the first historical GPCR structure of bovine rhodopsin (Palczewski et al., 2000) and that homology-modeling of V2R, V1aR and OTR using opiate receptors as templates is rational, based on relatively high (20-60%) sequence homology among the set of 4 neurophyseal and 4 opiate receptors. YM087 was computer-docked to V1aR, V2R and OTR using the AutoDock (Olson et al., Scripps Research Institute, La Jolla, USA) and subsequently relaxed using restrained simulated annealing and molecular dynamics, as implemented in AMBER program (Kollman et al., University of California, San Francisco, USA). From about 80 diverse configurations, sampled for each of the three ligand/receptor systems, 3 best energy-relaxed complexes were selected for mutual comparisons. Similar docking modes were found for the YM087/V1aR and YM087/V2R complexes, diverse from those of the YM087/OTR complexes, in agreement with the molecular affinity data.

Signal transmission via G protein-coupled receptors in the light of rhodopsin structure determination.

G protein-coupled receptors (GPCRs) transducing diverse external signals to cells via activation of heterotrimeric GTP-binding (G) proteins, estimated to mediate actions of 60% of drugs, had been resistant to structure determination until summer 2000. The first atomic-resolution experimental structure of a GPCR, that of dark (inactive) rhodopsin, thus provides a trustworthy 3D prototype for antagonist-bound forms of this huge family of proteins. In this work, our former theoretical GPCR models are evaluated against the new experimental template. Subsequently, a working hypothesis regarding the signal transduction mechanism by GPCRs is presented.

Theoretical conformational analysis of six arginine vasopressin analogs with the L-naphthylalanine in position 3.

Introduction of the naphthylalanine residue into either position 3 of arginine vasopressin (AVP), or its analogs results in peptides with interesting pharmacological properties. The single substituted analog of AVP with L-2-Nal in position 3 causes moderate antiduretic activity, whereas [Mpa1, (L-1-Nal)3, (D-Arg)8] VP and [Mpa1, (L-2-Nal)3, (D-Arg)8] VP are potent and selective V2 agonists. Moreover [(L-2-Nal)3, (D-Arg)8] VP is among the most potent and selective antagonists of V1a receptors. In this study we carried out conformational calculations on [(L-1-Nal)3] AVP, [(L-2-Nal)3] AVP, [(L-1-Nal)3, (D-Arg)8] VP, [(L-2-Nal)3, (D-Arg)8] VP, [Mpa1, (L-1-Nal)3, (D-Arg)8] VP, [Mpa1, (L-2-Nal)3, (D-Arg)8] VP, using the ECEPP/3 force field with and without including hydration to simulate aqueous and nonpolar environments. It was found that in all six compound studied, the low-energy conformations have common geometry and relative energies. Therefore, the modifications of the Phe in position 3 influence the binding to the receptor by changing the size of the third residue, rather than by changing the conformational space. The lowest-energy conformations in the presence and absence of water had beta-turns at residues Phe3-Gln4 and Gln4-Asn5 and Gln4-Asn5, respectively. The conformation at the Gln4-Asn5 turn was most similar to the crystal structure of the pressinoic acid (the cyclic moiety of vasopressin).

Essential dynamics/factor analysis for the interpretation of molecular dynamics trajectories.

Subject of this work is the analysis of molecular dynamics (MD) trajectories of neurophysins I (NPI) and II (NPII) and their complexes with the neurophyseal nonapeptide hormones oxytocin (OT) and vasopresssin (VP), respectively, simulated in water. NPs serve in the neurosecretory granules as carrier proteins for the hormones before their release to the blood. The starting data consisted of two pairs of different trajectories for each of the (NPII/VP)2 and (NPI/OT)2 heterotetramers and two more trajectories for the NPII2 and NPI2 homodimers (six trajectories in total). Using essential dynamics which, to our judgement, is equivalent to factor analysis, we found that only about 10 degrees of freedom per trajectory are necessary and sufficient to describe in full the motions relevant for the function of the protein. This is consistent with these motions to explain about 90% of the total variance of the system. These principal degrees of freedom represent slow anharmonic motional modes, clearly pointing at distinguished mobility of the atoms involved in the protein's functionality.

Molecular modeling of the human vasopressin V2 receptor/agonist complex.

The V2 vasopressin renal receptor (V2R), which controls antidiuresis in mammals, is a member of the large family of heptahelical transmembrane (7TM) G protein-coupled receptors (GPCRs). Using the automated GPCR modeling facility available via Internet (http:/(/)expasy.hcuge.ch/swissmod/SWISS-MODEL.+ ++html) for construction of the 7TM domain in accord with the bovine rhodopsin (RD) footprint, and the SYBYL software for addition of the intra- and extracellular domains, the human V2R was modeled. The structure was further refined and its conformational variability tested by the use of a version of the Constrained Simulated Annealing (CSA) protocol developed in this laboratory. An inspection of the resulting structure reveals that the V2R (likewise any GPCR modeled this way) is much thicker and accordingly forms a more spacious TM cavity than most of the hitherto modeled GPCR constructs do, typically based on the structure of bacteriorhodopsin (BRD). Moreover, in this model the 7TM helices are arranged differently than they are in any BRD-based model. Thus, the topology and geometry of the TM cavity, potentially capable of receiving ligands, is in this model quite different than it is in the earlier models. In the subsequent step, two ligands, the native [arginine8]vasopressin (AVP) and the selective agonist [D-arginine8]vasopressin (DAVP) were inserted, each in two topologically non-equivalent ways, into the TM cavity and the resulting structures were equilibrated and their conformational variabilities tested using CSA as above. The best docking was selected and justified upon consideration of ligand-receptor interactions and structure-activity data. Finally, the amino acid residues were indicated, mainly in TM helices 3-7, as potentially important in both AVP and DAVP docking. Among those Cys112, Val115-Lys116, Gln119, Met123 in helix 3; Glu174 in helix 4; Val206, Ala210, Val213-Phe214 in helix 5; Trp284, Phe287-Phe288, Gln291 in helix 6; and Phe307, Leu310, Ala314 and Asn317 in helix 7 appeared to be the most important ones. Many of these residues are invariant for either the GPCR superfamily or the neurophyseal (vasopressin V2R, V1aR and V1bR and oxytocin OR) subfamily of receptors. Moreover, some of the equivalent residues in V1aR have already been found critical for the ligand affinity.

Molecular modelling of the vasopressin V2 receptor/antagonist interactions.

We predict some essential interactions between the V2 vasopressin renal receptor (V2R) and its selective peptide antagonist desGly9-[Mca1,D-Ile2,Ile4]AVP, and compare these predictions with the earlier ones for the non-peptide OPC-36120 antagonist- and the [Arg8]vasopressin (AVP) agonist-V2 receptor interactions. V2R controls antidiuresis in mammals and belongs to the superfamily of the heptahelical transmembrane (7TM) G protein-coupled receptors (GPCR)s. V2R was built, the ligands docked and the structures relaxed using advanced molecular modeling techniques. Both the agonist and the antagonists (no matter whether of peptide- or non-peptide type) appear to prefer a common V2R compartment for docking. The receptor amino-acid residues, potentially important in ligand binding, are mainly in the TM3-TM7 helices. A few of these residues are invariant for the whole GPCR superfamily while most of them are conserved in the subfamily of neurohypophyseal receptors, to which V2R belongs. Some of the equivalent residues in a related V1a receptor have been earlier reported as critical for the ligand affinity.

Molecular modeling of the neurophysin I/oxytocin complex.

Neurophysins I and II (NPI and NPII) act in the neurosecretory granules as carrier proteins for the neurophyseal hormones oxytocin (OT) and vasopressin (VP), respectively. The NPI/OT functional unit, believed to be an (NPI/OT)2 heterotetramer, was modeled using low-resolution structure information, viz. the C alpha carbon atom coordinates of the homologous NPII/dipeptide complex (file 1BN2 in the Brookhaven Protein Databank) as a template. Its all-atom representation was obtained using standard modeling tools available within the INSIGHT/Biopolymer modules supplied by Biosym Technologies Inc. A conformation of the NPI-bound OT, similar to that recently proposed in a transfer NOE experiment, was docked into the ligand-binding site by a superposition of its Cys1-Tyr2 fragment onto the equivalent portion of the dipeptide in the template. The starting complex for the initial refinements was prepared by two alternative strategies, termed Model I and Model II, each ending with a approximately 100 ps molecular dynamics (MD) simulation in water using the AMBER 4.1 force field. The free homodimer NPI2 was obtained by removal of the two OT subunits from their sites, followed by a similar structure refinement. The use of Model I, consisting of a constrained simulated annealing, resulted in a structure remarkably similar to both the NPII/dipeptide complex and a recently published solid-state structure of the NPII/OT complex. Thus, Model I is recommended as the method of choice for the preparation of the starting all-atom data for MD. The MD simulations indicate that, both in the homodimer and in the heterotetramer, the 3(10)-helices demonstrate an increased mobility relative to the remaining body of the protein. Also, the C-terminal domains in the NPI2 homodimer are more mobile than the N-terminal ones. Finally, a distinct intermonomer interaction is identified, concentrated around its most prominent, although not unique, contribution provided by an H-bond from Ser25 O gamma in one NPI unit to Glu81 O epsilon in the other unit. This interaction is present in the heterotetramer (NPI/OT)2 and absent or weak in the NPI2 homodimer. We speculate that this interaction, along with the increased mobility of the 3(10)-helices and the carboxy domains, may contribute to the allosteric communication between ligand binding and NPI dimerization.

Elucidation of neurophysin/bioligand interactions from molecular modeling.

This is a review of our recent modeling work aimed at: (i) development and assessment of techniques for reliable refinement of low-resolution protein structures and (ii) using these techniques, at solving specific problems pertinent to neurophysin-bioligand interactions. Neurophysins I and II (NPI and NPII) serve in the neurosecretory granules of the posterior pituitary as carrier proteins for the neurophyseal hormones oxytocin (OT) and vasopressin (VP), respectively, until the latter are released into blood. NPs are homologous two-domain, sulphur rich small proteins (93-95 residues, 7 disulphide bridges per monomer), capable of being aggregated. The C2 symmetrical NPI2 and NPII2 homodimers, and the (NPI/OT)2 and (NPII/VP)2 heterotetramers, all believed to be the smallest functional units, were modeled using low-resolution structure information, i.e. the C alpha-carbon coordinates of the homologous NPII/dipeptide complex as a template. The all-atom representations of the models were obtained using the SYBYL suite of programs (by Tripos, Inc.). Subsequently, they were relaxed, using a constrained simulated annealing (CSA) protocol, and submitted to about 100 ps molecular dynamics (MD) in water, using the AMBER 4.1 force field. The (NPI/OT)2 and (NPII/VP)2 structures, averaged after the last 20 ps of MD, were remarkably similar to those recently reported either for NPII/dipeptide or NPII/oxytocin complex in the solid state (Chen et al., 1991, Proc. Natl. Acad. Sci., U.S.A. 88, 4240-4244; Rose et al., 1996, Nature Struct. Biol. 3, 163-169). The results indicate that the 3(10) helices (terminating the amino domains) and the carboxyl domains are more mobile than the remainder of the NP monomers. The hormones become anchored by residues 1-3 and 6 to the host, leaving residues 4-5 and 7-9 exposed on the surface and free to move. A cluster of attractive interactions, extending from the ligand binding site, Tyr-24-Ile-26 of unit 1(2), to the inter-monomer interface Val-36 of unit 1(2), Cys-79 and Ile-72 of unit 2(1), is clearly seen. We suggest that both these interactions as well as the increased mobility of the 3(10) helix and the carboxyl domain may contribute to the allosteric communication between the ligand and the unit1-unit2 interface.

Design of a knowledge-based force field for off-lattice simulations of protein structure.

Prediction of protein structure from amino-acid sequence still continues to be an unsolved problem of theoretical molecular biology. One approach to solve it is to construct an appropriate (free) energy function that recognizes the native structures of some selected proteins (whose native structures are known) as the ones distinctively lowest in (free) energy and then to carry out a search of the lowest-energy structure of a new protein. In order to reduce the complexity of the problem and the cost of energy evaluation, the so-called united-residue representation of the polypeptide chain is often applied, in which each amino-acid residue is represented by only a few interaction sites. Once the global energy minimum of the simplified chain has been found, the all-atom structure can easily and reliably be constructed. The search of the lowest-energy structure is usually carried out by means of Monte Carlo methods, though use of more efficient global-optimization methods, especially those of deformation of original energy surface is potentially promising. Monte Carlo search of the conformational space can be accelerated greatly, if the chain is superposed on a discrete lattice (the on-lattice approach). On the other hand, the on-lattice approach prohibits the use of many efficient global-optimization methods, because they require both energy and its space derivatives. The on-lattice methods in which the chain is embedded in the continuous 3D space are, therefore, also worth developing. In this paper we summarize the work on the design and implementation of an off-lattice united-residue force field that is underway in our group, in cooperation with Professor HA. Scheraga of Cornell University, U.S.A.

Theoretical conformational analysis of three vasopressin antagonists with a modified cyclohexyl ring in the first thioacid residue.

Analogues of arginine vasopressin (AVP) with bulky thioacid residues in position 1 of the amino acid sequence are known to be effective antagonists of the pressor response. Some of the most effective ones are those that have the first cysteine residue replaced with beta,beta-cyclopentamethylene-beta'-mercaptopropionic acid (Cpp) and its derivatives, such as 4-mercapto-4-tetrahydropyraneacetic acid (OCA) and 4-mercapto-4-tetrahydrothiopyraneacetic acid (SCA). The SCA analogues are more potent and the OCA ones slightly less potent antagonists than the Cpp ones. In this study we carried out conformational calculations on [Cpp1]AVP, [OCA1]AVP and [SCA1]AVP, using the ECEPP/3 force field both with and without hydration (to simulate an aqueous and non-polar receptor environment, respectively). It was found that most of the low-energy conformations are common in geometry and relative energy for all three compounds studied. It can therefore be concluded that the modifications of the cyclohexyl ring in position 1 influence the binding to the receptor because of changing the lipophilicity of the first residue, rather than by changing the conformational space. This is further supported by the fact that the lowest-energy conformations in the absence of water have closely located the Phe3 side chain (which is critical for the interaction with vasopressin receptors) and the (modified) cyclohexyl ring. The lowest-energy conformations in the presence and absence of water had beta-turns at residues Phe3-Gln4 and Gln4-Asn5, and Gln4-Asn5, respectively. The conformation with the turn at Gln4-Asn5 was most similar to the crystal structure of the pressinoic acid (the cyclic moiety of vasopressin).