CRYSTALLOGRAPHIC STUDIES OF REGULATED MYOSINS: MUSCLE CONTRACTION

Cornell University Ithaca, Ithaca NY

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Abstract

In the past year, Doolittle and colleagues have determined the structures of the terminal human fragment D domain (M.W. 86 kDa) and the crosslinked D-D fragment obtained from fibrin (Spraggon et al., 1997). Using molecular replacement techniques (with X-ray data collected both at the Brookhaven and at CHESS), we have succeeded in positioning the fragment D domains in the unit cells of the flash-frozen (as well as 4 C) crystals of the intact bovine fibrinogen molecule. We find that the end-to-end contacts made between symmetry-related molecules in our crystals are the same as those found by Doolittle and colleagues in the human D-D dimer. Using electron density difference maps, we clearly see coiled-coil regions connecting the terminal D and central E regions of the molecule, and can now locate the bends in these coiled coils. These bends appear to be the primary points of flexibility of the molecule. Using a variety of techniques, including improved data processing programs, refinement, and density modification, we are now extending the resolution of the electron density map to 3.5[unreadable] (the present limit of the flash-frozen data sets). We are now tracing the chains in the Fragment E and connecting coiled coil regions, and are on the threshold of the first near-atomic resolution picture of virtually the whole fibrinogen molecule (Brown et al., in preparation). Using protein supplied to us by L. Medved, we have also crystallized the central Fragment E of bovine fibrinogen (which consists of a disulphide bridge-stabilized globular domain connecting two short coiled coils). Data to 2.8[unreadable] resolution have been collected at CHESS from a native and 7 heavy metal-soaked Fragment E crystals. In attempting to solve this crystal structure, we are also using models of Fragment E derived from our results on the whole molecule. This information is vital for understanding the assembly of fibrinogen into the blood clot and for establishing a comprehensive rational drug design approach for treatment of clotting disorders.