A force field (MSXX) for molecular dynamics simulations of silicon nitride is derived using the Hessian biased technique from ab initio calculations on N(SiH3)3 and Si(NH2)4 clusters. This is used to model the nitrogen and silicon centers of the α and β forms of crystalline Si3N4 for prediction of crystal structures, lattice expansion parameters, elastic constants, phonon states, and thermodynamic properties. Experimental measurements on many of these important physical constants are lacking, so that these calculations provide the first reliable data on such fundamental properties of silicon nitride. This MSXX force field is expected to be useful for molecular dynamics simulations of dislocations and grain boundaries and for studying the reconstruction and energetics of clean, reduced, and oxidized surfaces.

A system for addressing in the construction of macromolecular assemblies can be based on the biospecificity of DNA (cytosine-5) methyltransferases and the capacity of these enzymes to form abortive covalent complexes at targeted 5-f luorocytosine residues in DNA. Using this system, macromolecular assemblies have been created using two representative methyltransferases: M⅐HhaI and M⅐MspI. When 5-f luorocytosine (F) is placed at the targeted cytosine in each recognition sequence in a synthetic oligodeoxynucleotide (GFGC for M⅐HhaI or FCGG for M⅐MspI), we show that the first recognition sequence becomes an address for M⅐HhaI, while the second sequence becomes an address for M⅐MspI. A chimeric enzyme containing a dodecapeptide antigen linked to the C terminus of M⅐HhaI retained its recognition specificity. That specificity served to address the linked peptide to the GFGC recognition site in DNA. With this assembly system components can be placed in a preselected order on the DNA helix. Axial spacing for adjacent addresses can be guided by the observed kinetic footprint of each methyltransferase. Axial rotation of the addressable protein can be guided by the screw axis of the DNA helix. The system has significant potential in the general construction of macromolecular assemblies. We anticipate that these assemblies will be useful in the construction of regular protein arrays for structural analysis, in the construction of protein-DNA systems as models of chromatin and the synaptonemal complex, and in the construction of macromolecular devices.Macromolecular assembly is easily approached with DNA. Branching through the formation of Watson-Crick paired duplexes in the shape of a Y or an X is now well known (1-4), and the feasibility of assembling 2-dimensional quadrilaterals and 3-dimensional cubes on which more extended structures can be based has been demonstrated (5, 6). However, the stable, site-directed attachment of labile enzymes and proteins to a DNA scaffold presents a formidable challenge in macromolecular fabrication. Candidate procedures in which the Watson-Crick base-pairing homology or triple-helix basepairing homology of an oligodeoxynucleotide is used to direct a tethered moiety to a preselected site in DNA (3, 4, 7-9) involve extremes of pH or temperature that can destroy the native structure of these proteins. Attachment systems based on antibodies directed against DNA are likely to lack specificity. On the other hand, antibodies to a hapten could be used to decorate a matrix depending on the pattern of haptens laid down during synthesis. The disadvantage here is that all hapten moieties are equivalent, and thus selective addressing would not be possible unless a series of haptens and antibodies directed to them could be developed. While a system of distinct haptens and antibodies is possible (3), it would be necessary to develop a set of hapten-phosphoramidites and the corresponding series of bifunctional antibodies to utilize this approach. Moreover, the use of noncovalent linkages sacrifice...

To help improve the accuracy of protein-ligand docking as a useful tool for drug discovery, we developed MPSim-Dock, which ensures a comprehensive sampling of diverse families of ligand conformations in the binding region followed by an enrichment of the good energy scoring families so that the energy scores of the sampled conformations can be reliably used to select the best conformation of the ligand. This combines elements of DOCK4.0 with molecular dynamics (MD) methods available in the software, MPSim. We test here the efficacy of MPSim-Dock to predict the 64 protein-ligand combinations formed by starting with eight trypsin cocrystals, and crossdocking the other seven ligands to each protein conformation. We consider this as a model for how well the method would work for one given target protein structure. Using as a criterion that the structures within 2 kcal/mol of the top scoring include a conformation within a coordinate root mean square (CRMS) of 1 A of the crystal structure, we find that 100% of the 64 cases are predicted correctly. This indicates that MPSim-Dock can be used reliably to identify strongly binding ligands, making it useful for virtual ligand screening.

We report the 3D structure predicted for the mouse MrgC11 (mMrgC11) receptor by using the MembStruk computational protocol, and the predicted binding site for the F-M-R-F-NH(2) neuropeptide together with four singly chirally modified ligands. We predicted that the R-F-NH(2) part of the tetrapeptide sticks down into the protein between the transmembrane (TM) domains 3, 4, 5, and 6. The Phe (F-NH(2)) interacted favorably with Tyr110 (TM3), while the Arg makes salt bridges to Asp161 (TM4) and Asp179 (TM5). We predicted that the Met extends from the binding site, but the terminal Phe residue sticks back into an aromatic/hydrophobic site flanked by Tyr237, Leu238, Leu240, and Tyr256 (TM6), and Trp162 (TM4). We carried out subsequent mutagenesis experiments followed by intracellular calcium-release assays that demonstrated the dramatic decrease in activity for the Tyr110Ala, Asp161Ala, and Asp179Ala substitutions, which was predicted by our model. These experiments provide strong evidence that our predicted G protein-coupled receptor (GPCR) structure is sufficiently accurate to identify binding sites for selective ligands. Similar studies were made with the mMrgA1 receptor, which did not bind the R-F-NH(2) dipeptide; we explain this to be due to the increased hydrophobic character of the binding pocket in mMrgA1.

Mrg receptors are orphan G protein-coupled receptors (GPCRs) located mainly at the specific set of sensory neurons in the dorsal root ganglia, suggesting a role in nociception. We report here the 3-D structure of rat MrgA (rMrgA) receptor [obtained from homology modeling to the recently validated predicted structures of mouse MrgA1 and MrgC11] and the structure of adenine (a known agonist, K i =18nM) bound to rMrgA. This predicted binding site is located within transmembrane helical domains (TMs) 3, 4, 5 and 6, with Asn residues in TM3 and TM4 identified as the key residues for adenine binding. Here the side chain of Asn88 (TM3) forms two pairs of hydrogen bonds with N3 and N9 of adenine while Asn146 (TM4) makes two pairs of hydrogen bonds with N1 and N6 of adenine. These interactions lock adenine tightly in the binding pocket. We also predict the binding site of guanine (not an agonist) and seven other derivatives. Guanine cannot make the hydrogen bond to Asn146 (TM4), leading to binding too weak to be observed experimentally. The predicted binding affinity for other adenine derivatives correlates with the availability of the hydrogen bonds to these two Asn residues. These results validate the predicted structure for rat MrgA and suggest mutation experiments that could further validate the structure. Moreover the predicted structure and binding site should be useful for seeking other small molecule agonists and antagonists.

Bacterial DNA methyltransferases offer an approach to addressable protein targeting in macromolecular assembly that permits the construction of ordered arrays of functional proteins or peptides. This approach uses the natural recognition specificity of the bacterial DNA cytosine methyltransferase to target fusion proteins to preselected sites on a DNA scaffold through the formation of a stable covalent attachment to a mechanism-based methyltransferase inhibitor. Addressable targets on DNA molecules can readily be created with 5FdC§ at the enzyme recognition site. This is because the substitution of the targeted pyrimidine ring with fluorine at C5 generates a kinetic barrier to the methyltransfer step in the reaction and a thermodynamic barrier to the beta-elimination step that stall the enzyme after it forms a covalent bond with DNA. In this report we have studied the capacity of dU to substitute for 5FdC in addressable targeting. When dU is used in place of 5FdC as the base targeted by the enzyme for nucleophilic attack, it forms a mispair with dG. Like 5FdC, dU creates a kinetic barrier to completion of the reaction causing the enzyme to stall. Unlike 5FdC, dU does not create a thermodynamic barrier to beta-elimination and release after methyltransfer, however, the mispaired state of the dU in the dG:dU basepair appears to create a transition-state analog that cannot be easily released by the enzyme. The data show that dU can be used to substitute for 5FdC in the production of ordered nucleoprotein-based macromolecular assemblies.

Dynamic modelling was carried out on the binding in water of several substances to the conserved membrane-adjacent heptapeptide of the 15-residue C-terminal cytoplasmic fragment (C-tail) of mammalian dopamine D2 receptors, PheAsnIleGluPheArgLys. Particularly important in the establishment of binding pockets for ligands were the carboxyl, phenyl, guanidino, and epsilon-amino groups of the last 4 residues. A broad array of chemical structures was found to be potentially capable of binding to this site, among which were dopamine, dopamine D2 receptor agonists and antagonists, GABA, muscimol, GABA(B) receptor agonists and antagonists, homocamosine, and carnosine. Since the C-tail is critical for G protein binding, it is suggested that many naturally-occurring and synthetic substances may be modulators of activation of G proteins by G-coupled receptors.

The cover picture shows the predicted structure of the mouse MrgC11 G protein-coupled receptor (GPCR) with the predicted binding site for a signaling tetrapeptide (Phe-(d)Met-Arg-Phe-NH 2 , a member of the FMRFa family of signal peptides). MrgC11 belongs to the MAS-related gene family and is thought to be involved in pain sensation or modulation. These predictions led to the identification of several residues critical for ligand binding (see inset), while mutagenesis and intracellular-calcium-release experiments confirm the dramatic decrease in activity predicted for the Y110A, D161A, and D179A mutants. This validates the accuracy of these predicted structures, which can now be used for structurebased drug design to find small nonpeptide agonists and antagonists for mMrgC11 and perhaps to identify the endogenous ligand. This also validates the MembStruk methodology used in these predictions, which is equally applicable to all GPCR proteins. Details of the modeling, docking, and experimental verification are in the article by W. A. Goddart III et al. on p. 1527 ff.