Kazmirski S L, Howard M J, Isaacson R L, Fersht A R
Medical Research Council Centre for Protein Engineering and the Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom.
Proc Natl Acad Sci U S A. 2000 Sep 26;97(20):10706-11. doi: 10.1073/pnas.180310097.
Familial amyloidosis-Finnish type (FAF) results from a single mutation at residue 187 (D187N or D187Y) within domain 2 of the actin-regulating protein gelsolin. The mutation somehow allows a masked cleavage site to be exposed, leading to the first step in the formation of an amyloidogenic fragment. We have performed NMR experiments investigating structural and dynamic changes between wild-type (WT) and D187N gelsolin domain 2 (D2). On mutation, no significant structural or dynamic changes occur at or near the cleavage site. Areas in conformational exchange are observed between beta-strand 4 and alpha-helix 1 and within the loop region following beta-strand 5. Chemical shift differences are noted along the face of alpha-helix 1 that packs onto the beta-sheet, suggesting an altered conformation. Conformational changes within these areas can have an effect on actin binding and may explain why D187N gelsolin is inactive. [(1)H-(15)N] nuclear Overhauser effect and chemical shift data suggest that the C-terminal tail of D187N gelsolin D2 is less structured than WT by up to six residues. In the crystal structure of equine gelsolin, the C-terminal tail of D2 lies across a large cleft between domains 1 and 2 where the masked cleavage site sits. We propose that the D187N mutation destabilizes the C-terminal tail of D2 resulting in a more exposed cleavage site leading to the first proteolysis step in the formation of the amyloidogenic fragment.
家族性淀粉样变性芬兰型（FAF）是由肌动蛋白调节蛋白凝溶胶蛋白第2结构域中第187位残基（D187N或D187Y）的单个突变引起的。该突变以某种方式使一个隐蔽的切割位点暴露出来，从而导致了淀粉样生成片段形成的第一步。我们进行了核磁共振实验，研究野生型（WT）和D187N凝溶胶蛋白第2结构域（D2）之间的结构和动态变化。发生突变时，切割位点或其附近没有发生明显的结构或动态变化。在β链4和α螺旋1之间以及β链5之后的环区域内观察到构象交换区域。沿着堆积在β折叠上的α螺旋1的表面发现了化学位移差异，表明构象发生了改变。这些区域内的构象变化可能会影响肌动蛋白结合，这也许可以解释为什么D187N凝溶胶蛋白没有活性。[（1）H-（15）N]核Overhauser效应和化学位移数据表明，D187N凝溶胶蛋白D2的C末端尾巴的结构比野生型少了多达六个残基。在马凝溶胶蛋白的晶体结构中，D2的C末端尾巴横跨结构域1和2之间的一个大裂缝，隐蔽的切割位点就位于此处。我们提出，D187N突变使D2的C末端尾巴不稳定，导致切割位点更加暴露，从而引发了淀粉样生成片段形成过程中的第一步蛋白水解反应。
