SOLUTION STRUCTURE AND DYNAMICS OF LINKED CELL ATTACHMENT MODULES OF MOUSE FIBRONECTIN CONTAINING THE RGD AND SYNERGY REGIONS - COMPARISON WITH THE HUMAN FIBRONECTIN CRYSTAL-STRUCTURE

We report the three-dimensional solution structure of the mouse fibron ectin cell attachment domain consisting of the linked ninth and tenth type III modules, mFnFn3(9,10). Because the tenth module contains the RGD cell attachment sequence while the ninth contains the synergy regi on, mFnFn3(9,10) has the cell attachment activity of intact fibronecti n. Essentially complete signal assignments and approximately 1800 dist ance and angle restraints were derived from multidimensional heteronuc lear NMR spectra. These restraints were used with a hybrid distance ge ometry/simulated annealing protocol to generate an ensemble of 20 NMR structures having no distance or angle violations greater than 0.3 Ang strom or 3 degrees. Although the beta-sheet core domains of the indivi dual modules are well-ordered structures, having backbone atom rmsd va lues from the mean structure of 0.51(+/-0.12) and 0.40(+/-0.07) Angstr om, respectively, the rmsd of the core atom coordinates increases to 3 .63(+/-1.41) A when the core domains of both modules are used to align the coordinates. The latter result is a consequence of the fact that the relative orientation of the two modules is not highly constrained by the NMR restraints. Hence, while structures of the beta-sheet core domains of the NMR structures are very similar to the core domains of the crystal structure of hFnFn3(9,10), the ensemble of NMR structures suggests that the two modules form a less extended and more flexible s tructure than the fully extended rod-like crystal structure. The radiu s of gyration, R-g, of mFnFn3(9,10) derived from small-angle neutron s cattering measurements, 20.5(+/-0.5) Angstrom, agrees with the average R-g calculated for the NMR structures, 20.4 Angstrom, and is ca 1 Ang strom less than the value of R-g calculated for the X-ray structure. T he values of the rotational anisotropy, D-parallel to/D-perpendicular to, derived from an analysis of N-15 relaxation data, range from 1.7 t o 2.1, and are significantly less than the anisotropy of 2.67 predicte d by hydrodynamic modeling of the crystal coordinates. In contrast, hy drodynamic modeling of the NMR coordinates yields anisotropies in the range of 1.9 to 2.7 (average 2.4(+/-0.2)), with NMR structures bent by more than 20 degrees relative the crystal structure having calculated anisotropies in best agreement with experiment. In addition, the rela xation parameters indicate that several loops in mFnFn3(9,10), includi ng the RGD loop, are flexible on the nanosecond to picosecond time-sca le. Taken together, our results suggest that, in solution, the Limited set of interactions between the mFnFn3(9,10) modules position the RGD and synergy regions to interact specifically with tell surface integr ins, and at the same time permit sufficient flexibility that allows mF nFn3(9,10) to adjust for some variation in integrin structure or envir onment. (C) 1998 Academic Press Limited.