The overall goals of this research program over a more than a 30-year period are to define structure function relationships of vitamin K-dependent coagulation proteins, both in vitro and in vivo, with specific attention paid to interactions of the gamma-carboxyglutamic acid (Gla) domains (GD) with metal ions, membranes, and receptors. Over this period of time, we have addressed these issues by a variety of biophysical techniques, and by protein chemistry, molecular/cell biology, and gene targeting strategies. In the current renewal application, efforts will be concentrated on the structures, dynamics, and functions of these Gla-containing peptides with specific attention on the Protein C (PC)/Endothelial Cell Protein C Receptor (EPCR) system, and on Gla-containing conantokins, which are neuroactive models of GDs of coagulation proteins. Three interrelated specific aims are proposed: (1) To chemically synthesize specific Gla-containing conantokins, viz., conG, conT, conR (1-17), and conL, as well as strategic variants of these peptides, and: (a) to employ electrophysiology and [3H]MK801 binding to quantitate their inhibitory potency toward recombinant NMDA receptors (NMDAR) comprised of known combinations of NR1 and NR2 subunits and to delineate the specific residues involved in their NMDAR subunit specificity; (b) to clone and express a recombinant construct encoding the noncontiguous Glu-binding domains of the NR2B subunit of the NMDAR and to more fully assess the nature of the inhibition of the conantokins with the coagonist, Glu, on this receptor segment; (c) to identify conR residues involved in its binding to the NR2B subunit of the NMDAR and to study the solution structure of conR (1-17) when bound to its NMDAR receptor segment using multidimensional hetero- and homonuclear NMR on isotopically-enriched conR and receptor; and (d) to study the interactions that stabilize the unique metal ion-induced GD dimeric superstructure that is adopted by conG in the presence of Ca 2+. 2) to express murine soluble (s) recombinant (r) perdeuterated [2H/13C/15N]-EPCR and to determine backbone molecular dynamics of lipidated sr-EPCR + GDPC (1-47). 3) To chemically synthesize the GD of murine PC (GDPC1-47), along with analogs containing individual amino acid replacements, and: (a) to examine the interactions of these domains with Ca 2+, Mg 2+, and Zn 2+, and with murine sr-EPCR, using biophysical methods; and (b) to determine the solution and crystal structures and backbone dynamics of Ca 2+ - and Mg 2+ -complexed murine GDPC (1-47) using NMR and X-ray crystallography. Accomplishment of these goals will assist in our understanding of the critical structural features of these GDs that allow the specific functional binding of proteins containing these regions to their receptors.
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