H-1 NMR-BASED DETERMINATION OF THE 3-DIMENSIONAL STRUCTURE OF THE HUMAN PLASMA FIBRONECTIN FRAGMENT CONTAINING INTER-CHAIN DISULFIDE BONDS

Human plasma fibronectin is a plasma glycoprotein that plays an import ant role in many biological processes. It consists of two identical 23 0-250-kDa subunits that are joined by two disulfide bonds near their c arboxyl termini. Each subunit contains various binding domains compose d of three types of homologous repeats. Recent work has determined the three-dimensional structures of various repeat fragments, but little is known about the three-dimensional structure of the carboxyl-termina l region. A recent NMR study of a plasmin-digested carboxyl-terminal i nter-chain disulfide-linked heptapeptide dimer has proposed that the t wo subunits are arranged in an antiparallel fashion (An et al. (1992) Biochemistry 31, 9927-9933). We have now determined the three-dimensio nal structure for a substantial portion of a trypsin-digested inter-ch ain disulfide-linked 52-residue (6 kDa) fragment of the carboxyl-termi nal of human plasma fibronectin (which includes the above-mentioned he ptapeptide dimer) using two-dimensional NMR methods and a new strategy for NMR-based protein structure determination. The NMR data requires that the two chains in the dimer be linked in a symmetric, antiparalle l arrangement. The resulting monomer conformation consists of two twis ted or coiled segments, Thr3-Asn6 and Ile9-Phe12, connected by the Cys 7-Pro8 residues in extended conformations, with the two monomer chains cross-linked at residues Cys7 and Cys11. The conformation of the hept apeptide dimer region differs substantially from the conformations pro posed by An et al.