Janowski Robert, Abrahamson Magnus, Grubb Anders, Jaskolski Mariusz
Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Grunwaldzka 6, 60-780 Poznan, Poland.
J Mol Biol. 2004 Jul 30;341(1):151-60. doi: 10.1016/j.jmb.2004.06.013.
Human cystatin C (HCC) inhibits papain-like cysteine proteases by a binding epitope composed of two beta-hairpin loops and the N-terminal segment. HCC is found in all body fluids and is present at a particularly high level in the cerebrospinal fluid. Oligomerization of HCC leads to amyloid deposits in brain arteries at advanced age but this pathological process is greatly accelerated with a naturally occurring Leu68Gln variant, resulting in fatal amyloidosis in early adult life. When proteins are extracted from human cystatin C amyloid deposits, an N-terminally truncated cystatin C (THCC) is found, lacking the first ten amino acid residues of the native sequence. It has been shown that the cerebrospinal fluid may cause this N-terminal truncation, possibly because of disintegration of the leucocytes normally present in this fluid, and the release of leucocyte proteolytic enzymes. HCC is the first disease-causing amyloidogenic protein for which oligomerization via 3D domain swapping has been observed. The aggregates arise in the crystallization buffer and have the form of 2-fold symmetric dimers in which a long alpha-helix of one molecule, flanked by two adjacent beta-strands, has replaced an identical domain of the other molecule, and vice versa. Consistent with a conformational change at one of the beta-hairpin loops of the binding epitope, the dimers (and also any other oligomers, including amyloid aggregates) are inactive as papain inhibitors. Here, we report the structure of N-truncated HCC, the dominant form of cystatin C in amyloid deposits. Although the protein crystallized under conditions that are drastically different from those for the full-length protein, the structure reveals dimerization by the same act of domain swapping. However, the new crystal structure is composed of four independent HCC dimers, none of which has the exact 2-fold symmetry of the full-length dimer. While the four dimers have the same overall topology, the exact relation between the individual domains shows a variability that reflects the flexibility at the dimer-specific open interface, which in the case of 3D domain-swapped HCC consists of beta-interactions between the open hinge loops and results in an unusually long intermolecular beta-sheet. The dimers are engaged in further quaternary interactions resulting in spherical, closed octameric assemblies that are identical to that present in the crystal of the full-length protein. The octamers interact via hydrophobic patches formed on the surface of the domain-swapped dimers as well as by extending the dimer beta-sheet through intermolecular contacts.
人胱抑素C（HCC）通过由两个β-发夹环和N端片段组成的结合表位抑制木瓜蛋白酶样半胱氨酸蛋白酶。HCC存在于所有体液中，在脑脊液中的含量特别高。HCC的寡聚化会在老年时导致脑动脉中的淀粉样沉积物，但这种病理过程会因一种天然存在的Leu68Gln变体而大大加速，导致成年早期出现致命的淀粉样变性。当从人胱抑素C淀粉样沉积物中提取蛋白质时，会发现一种N端截短的胱抑素C（THCC），它缺少天然序列的前十个氨基酸残基。研究表明，脑脊液可能导致这种N端截短，可能是因为该液体中正常存在的白细胞解体，以及白细胞蛋白水解酶的释放。HCC是第一种通过三维结构域交换观察到寡聚化的致病淀粉样蛋白。聚集体在结晶缓冲液中形成，呈2倍对称二聚体形式，其中一个分子的长α-螺旋两侧有两个相邻的β-链，取代了另一个分子的相同结构域，反之亦然。与结合表位的一个β-发夹环处的构象变化一致，二聚体（以及任何其他寡聚体，包括淀粉样聚集体）作为木瓜蛋白酶抑制剂无活性。在此，我们报道了N端截短的HCC的结构，它是淀粉样沉积物中胱抑素C的主要形式。尽管该蛋白质在与全长蛋白质截然不同的条件下结晶，但结构显示通过相同的结构域交换行为形成二聚体。然而，新的晶体结构由四个独立的HCC二聚体组成，其中没有一个具有全长二聚体的确切2倍对称性。虽然这四个二聚体具有相同的总体拓扑结构，但各个结构域之间的确切关系显示出变异性，这反映了二聚体特异性开放界面处的灵活性，在三维结构域交换的HCC情况下，该界面由开放铰链环之间的β-相互作用组成，并导致异常长的分子间β-折叠。二聚体参与进一步的四级相互作用，形成球形、封闭的八聚体组装体，与全长蛋白质晶体中的组装体相同。八聚体通过在结构域交换二聚体表面形成的疏水区域相互作用，以及通过分子间接触扩展二聚体β-折叠相互作用。
