THE SOLUTION STRUCTURE OF MELANOMA GROWTH-STIMULATING ACTIVITY

The solution structure of melanoma growth stimulating activity (M MGSA), a dimeric chemokine consisting of 73 residues per monomer, has been determined using two-dimensional homonuclear and three-dimensional heteronuclear NMR spectroscopy. Structure calculations were carried out using a hybrid distance geometry-simulated annealing approach with the programs DGII and X-PLOR. The structure is based on a total of 2362 experimental restraints, comprising 2150 NOE-derived distance restraints (2076 unambiguous intrasubunit restraints, 6O unambiguous intersubunit restraints, and 14 ambiguous restraints with potential contributions from both intra and intersubunit NOEs), 84 distance restraints for 42 backbone hydrogen bonds, and 128 torsion angle restraints. The ambiguous distance restraints were treated using a target function which accounts for both intra- and intermolecular contributions to the NOE intensity. A total of 25 structures were calculated, with the backbone (N, C-chi, C) atomic r.m.s. distribution about the mean coordinates for residues 8 to 89 being 0.44(+/- 0.10)Angstrom for the dimer and 0.34(+/- 0.07)Angstrom for the individual monorners. The N- and C-terminal residues (1 to 7 and 70 to 73, respectively) are disordered. The overall structure of the MGSA dimer is similar to that reported previously for the NMR and X-ray structures of interleukin-8 (IL-8), and consists of a six-stranded antiparallel P-sheet packed against two C-terminal antiparallel cr-helices. A best fit superposition of the NMR structure of NGSA on the X-ray and NMR structures of IL-8 yields backbone atomic r.m.s. differences of 0.99 and 1.28 Angstrom, respectively for individual monomers, and 1.O8 and 1.82 Angstrom, respectively for the dimers (using MGSA residues 8 to 14 and 19 to 69). In general, the MGSA structure resembles the IL-8 X-ray structure more than it does the IL-8 NMR structure. At the tertiary (monomer) level the two main differences between the MGSA solution structure and IL-8 NMR structure involve the loops between residues 14 to 19 and between residues 30 to 38. At the quaternary (dimer) level the difference results from differing angles between the P-strands which form the dimer interface, and is manifest as a different interhelical separation (distance of closest approach between the two helices is 15.3 Angstrom in the IL-8 NMR structure and 11. 7 (+/- 04)Angstrom in the MGXA structure).