THE NH2-TERMINAL FIBRIN-BINDING SITE OF FIBRONECTIN IS FORMED BY INTERACTING 4TH AND 5TH FINGER DOMAINS - STUDIES WITH RECOMBINANT FINGER FRAGMENTS EXPRESSED IN ESCHERICHIA-COLI

The NH2-terminal 29-kDa Fib-1 fragment consisting of the first five fi nger modules of fibronectin (F1-5) binds reversibly to fibrin and faci litates cross-linking by Factor XIII. To narrow down the fibrin-bindin g site within this region, we have used recombinant technology to expr ess a number of individual fingers, rF1, rF2, rF3, rF4, and rF5, and t heir pairs, rF1-2 rF2-3, and rF4-5, as fusion proteins in Escherichia coli. These recombinant fragments were separated from the carrier malt ose-binding protein by digestion with human factor Xa or other proteas es, and their structural integrity was confirmed by spectroscopic and calorimetric methods. The recombinant F1 and F4-5 exhibited fluorescen ce-detected melting transitions of the same magnitude and with the sam e midpoint (T(m)) as their natural analogues prepared from Fib-1 by pr oteolysis. Differential scanning calorimetry experiments further demon strated that these fragments are properly folded and have compact stru ctures identical to the natural ones. Isolated rF4 melts at a much low er temperature than rF5 or the bimodular fragment rF4-5, indicating th e loss of a stabilizing interaction between fingers 4 and 5. Compariso n of fluorescence spectra of individual rF4 and rF5 with that of rF4-5 was also consistent with an interaction that affects the environment of Trp residue(s). rF2 also melts at a lower temperature than rF3 or r F2-3, suggesting a stabilizing interaction between the second and thir d fingers as well. When tested on fibrin-Sepharose, only the bimodular fragment rF4-5 was able to bind. All other fragments, including indiv idual fingers 4 and 5, failed to bind. Thus, fibrin binding is not a c ommon property of all fingers. The results indicate that a recognition site for fibrin is located within fingers 4 and 5. The interaction be tween these neighboring domains may play an important role in proper o rientation of the residues forming this site.