CRYSTALLOGRAPHIC STUDIES OF PROKARYOTIC RNA POLYMERASE

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Fibrinogen is a large glycoprotein found in the blood which becomes transformed into the insoluble polymer called fibrin under the catalytic influence of the enzyme thrombin. The transformation is brought about by the removal of small peptides from the amino-termini of two different chains in fibrinogen, exposing two new amino-terminal sequences. The newly exposed ends dubbed "knobs," were thought to fit into complementary sites ("holes") near the carboxyl-termini of other fibrinogen molecules. Synthetic peptides corresponding to the "knobs" bind to fibrinogen, but only one of the two (A-knob = Gly-Pro-Arg-X) is effective in preventing fibrin formation. The other peptide (B-knob = Gly-His-Arg-X) binds to fibrinogen but does not block polymerization. Fibrinogen and fibrin can be broken down into large mol. wt. core fragments by various proteases. The largest of these fragments is called fragment D, and it is known to bind the synthetic peptide knobs. We recently reported the structure of fragment D, from human fibrinogen, and also that of double-D from fibrin (Spraggon et al, Nature, 389:455-462, 1997). At the time, we had only been able to crystallize the latter in the presence of one of the (synthetic) A-knob. Last year we succeeded in growing crystals in the presence of both synthetic peptides. We came to CHESS and managed a complete data set at 2.3 A resolution. The two peptides were found bound to two different but homologous sites. This was the first direct demonstration that the Gly-His-Arg-knob fit into a hole on the beta-chain corresponding to a hole on the gamma-chain that binds Gly-Pro-Arg. The two holes are aimed in opposite directions, explaining why polymerization is only prevented by the A-knob. The B-knob is apparently involved in secondary (lateral). associations. The structure also revealed a weakly bound calcium ion near the beta-chain hole, explaining a longstanding mystery of why calcium influences the binding of the B-knob. This work is currently in press in Biochemistry, schedule for publication in early July, 1998.