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首页> 外文期刊>Journal of Molecular Biology >Metal-dependent conformational changes in a recombinant vWF-A domain from human factor B: a solution study by circular dichroism, fourier transform infrared and (1)H NMR spectroscopy.
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Metal-dependent conformational changes in a recombinant vWF-A domain from human factor B: a solution study by circular dichroism, fourier transform infrared and (1)H NMR spectroscopy.

机译：重组vWF-A域中人源因子B的金属依赖性构象变化：通过圆二色性，傅立叶变换红外光谱和（1）H NMR光谱研究溶液。

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摘要

Factor B is a key component of the alternative pathway of complement and is cleaved by factor D into the Ba and Bb fragments when complexed with the activated form of C3, namely C3b. The Bb fragment contains a von Willebrand factor type A (vWF-A) domain, which is composed of an open twisted almost-parallel beta-sheet flanked on both sides by seven alpha-helices A1 to A7, with a metal coordination site at its active-site cleft. Homology modelling of this vWF-A domain shows that the metal-binding site was present. Two recombinant vWF-A domains (Gly229-Ile444 and Gly229-Gln448) were examined by circular dichroism and Fourier transform infrared spectroscopy and indicated a significant conformational transition in the presence and absence of Mg(2+). Two upfield-shifted signals in the (1)H NMR spectrum were used as sensitive probes of the vWF-A protein structure, one of which was assigned to a methyl group and demonstrated metal- and pH-dependent properties between two distinct conformations. Temperature denaturation studies followed by spectroscopy showed that metal-binding caused the vWF-A structure to become significantly more stable. Ring current calculations based on a homology model for the vWF-A structure correlated one upfield-shifted signal with a methyl group on the alpha-helices in the vWF-A structure and the other one with individual single protons. An allosteric property of the vWF-A domain has thus been identified, and its implications for factor B activation were examined. Since the vWF-A domain after alpha-helix A7 is connected by a short link to the catalytic serine protease domain in the Bb fragment, the identification of a metal-free and a more stable metal-bound conformation for the vWF-A domain implies that the vWF-A interaction with C3b may alter its Mg(2+)-bound coordination in such a way as to induce conformational changes that may regulate the proteolytic activity of factor B. Copyright 2000 Academic Press.
机译：因子B是补体替代途径的关键组成部分，当与激活形式的C3（即C3b）复合时，因子D会被D切割成Ba和Bb片段。该Bb片段包含一个von Willebrand因子A型（vWF-A）结构域，该结构域由两侧均开有七个α-螺旋A1至A7的开放扭曲，几乎平行的β-折叠构成，其金属配位点活动部位裂口。该vWF-A结构域的同源性建模表明存在金属结合位点。通过圆二色性和傅立叶变换红外光谱法检查了两个重组vWF-A域（Gly229-Ile444和Gly229-Gln448），并表明在存在和不存在Mg（2+）的情况下，显着的构象转变。 （1）H NMR光谱中的两个高场移位信号被用作vWF-A蛋白结构的敏感探针，其中一个被分配给甲基，并证明了两个不同构象之间的金属和pH依赖性。温度变性研究以及光谱分析表明，金属结合导致vWF-A结构变得更加稳定。基于针对vWF-A结构的同源性模型的环电流计算，使一个高场移位信号与vWF-A结构中的α螺旋上的甲基相关，而另一个与单个质子相关。因此，已经确定了vWF-A结构域的变构性质，并研究了其对因子B激活的影响。由于α-螺旋A7之后的vWF-A结构域通过短链连接到Bb片段中的催化丝氨酸蛋白酶结构域，因此鉴定vWF-A结构域的无金属且更稳定的金属结合构象意味着vWF-A与C3b的相互作用可能会改变其Mg（2+）结合的配位，从而诱导可能调节B因子蛋白水解活性的构象变化。版权所有2000，学术出版社。

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