Matsuka Y V, Medved L V, Brew S A, Ingham K C
J. Holland Laboratory, American Red Cross, Rockville, Maryland 20855.
J Biol Chem. 1994 Apr 1;269(13):9539-46.
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.
由纤连蛋白的前五个指状结构域组成的NH2末端29 kDa的Fib-1片段（F1-5）与纤维蛋白可逆结合，并促进因子XIII介导的交联。为了缩小该区域内的纤维蛋白结合位点，我们利用重组技术在大肠杆菌中表达了多个单个指状结构域，即rF1、rF2、rF3、rF4和rF5，以及它们的配对组合，rF1-2、rF2-3和rF4-5，作为融合蛋白。这些重组片段通过人因子Xa或其他蛋白酶消化与载体麦芽糖结合蛋白分离，并用光谱和量热法确认其结构完整性。重组的F1和F4-5表现出与通过蛋白水解从Fib-1制备的天然类似物相同幅度和相同中点（Tm）的荧光检测熔解转变。差示扫描量热法实验进一步证明这些片段折叠正确，具有与天然片段相同的紧密结构。分离的rF4在比rF5或双模块片段rF4-5低得多的温度下熔解，表明指状结构域4和5之间稳定相互作用的丧失。单个rF4和rF5的荧光光谱与rF4-5的荧光光谱比较也与影响色氨酸残基环境的相互作用一致。rF2在比rF3或rF2-3低的温度下熔解，表明第二和第三个指状结构域之间也存在稳定相互作用。当在纤维蛋白-琼脂糖上进行测试时，只有双模块片段rF4-5能够结合。所有其他片段，包括单个指状结构域4和5，均未能结合。因此，纤维蛋白结合并非所有指状结构域的共同特性。结果表明，纤维蛋白的识别位点位于指状结构域4和5内。这些相邻结构域之间的相互作用可能在形成该位点的残基的正确取向中起重要作用。
