The Solution Structure of Melanoma Growth Stimulating Activity

Abstract

The solution structure of melanoma growth stimulating activity (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, 60 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, Cα, C)atomic r.m.s. distribution about the mean coordinates for residues 8 to 69 being 0·44(±0·10) Å for the dimer and 0·34(±0·07) Å for the individual monomers. 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 β-sheet packed against two C-terminal antiparallel α-helices. A best fit superposition of the NMR structure of MGSA on the X-ray and NMR structures of IL-8 yields backbone atomic r.m.s. differences of 0·99 and 1·28 Å, respectively for individual monomers, and 1·08 and 1·82 Å, 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 β-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 Å in the IL-8 NMR structure and 11·7 (±0·4) Å in the MGSA structure).