The NH2-terminal fibrin-binding site of fibronectin is formed by interacting fourth and fifth finger domains. Studies with recombinant finger fragments expressed in Escherichia coli.
The NH2-terminal 29-kDa Fib-1 fragment consisting of the first five finger modules of fibronectin (F1-5) binds reversibly to fibrin and facilitates cross-linking by Factor XIII. To narrow down the fibrin-binding site within this region, we have used recombinant technology to express a number of individual fingers, rF1, rF2, rF3, rF4, and rF5, and their pairs, rF1-2 rF2-3, and rF4-5, as fusion proteins in Escherichia coli. These recombinant fragments were separated from the carrier maltose-binding protein by digestion with human factor Xa or other proteases, and their structural integrity was confirmed by spectroscopic and calorimetric methods. The recombinant F1 and F4-5 exhibited fluorescence-detected melting transitions of the same magnitude and with the same midpoint (Tm) as their natural analogues prepared from Fib-1 by proteolysis. Differential scanning calorimetry experiments further demonstrated that these fragments are properly folded and have compact structures identical to the natural ones. Isolated rF4 melts at a much lower temperature than rF5 or the bimodular fragment rF4-5, indicating the loss of a stabilizing interaction between fingers 4 and 5. Comparison 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 rF2-3, suggesting a stabilizing interaction between the second and third fingers as well. When tested on fibrin-Sepharose, only the bimodular fragment rF4-5 was able to bind. All other fragments, including individual fingers 4 and 5, failed to bind. Thus, fibrin binding is not a common property of all fingers. The results indicate that a recognition site for fibrin is located within fingers 4 and 5. The interaction between these neighboring domains may play an important role in proper orientation of the residues forming this site.
Matsuka
Y V
YV
J. Holland Laboratory, American Red Cross, Rockville, Maryland 20855.
Medved
L V
LV
Brew
S A
SA
Ingham
K C
KC
D000595:Amino Acid Sequence; D001483:Base Sequence; D002152:Calorimetry, Differential Scanning; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D017931:DNA Primers; D004926:Escherichia coli; D005176:Factor XIII / Q000378:metabolism; D005337:Fibrin / Q000378:metabolism*; D005353:Fibronectins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008961:Models, Structural; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013816:Thermodynamics
D017931:DNA Primers; D005353:Fibronectins; D011994:Recombinant Proteins; D005337:Fibrin; D005176:Factor XIII