Conformationally variable Rab protein surface regions mapped by limited proteolysis and homology modelling.
Conformationally variable Rab protein surface regions mapped by limited proteolysis and homology modelling.
复制标题
通过有限的蛋白水解和同源建模绘制构象可变的 Rab 蛋白表面区域。
DOI：
10.1042/bj3360461
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Hoffenberg,S
中科院分区：
文献类型：
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作者：
Nikolova,L;Soman,K;Nichols,JC;Daniel,DS;Dickey,BF;Hoffenberg,S
小GTP酶Rab 4和Rab 5的胰蛋白酶蛋白水解是一个多步骤的核苷酸依赖性过程。使用N-末端肽测序，基质辅助激光解吸电离飞行时间质谱和分子建模，我们确定了三个初始位点的蛋白水解在Rab 5的Arg-4，Arg-81和Arg-197。Arg-4和Arg-81位于先前涉及Rab 5内吞功能的区域内，Arg-197位于涉及膜靶向的区域内。从拓扑学上讲，Arg-81位于构象可变的开关II区域内，该区域对其他GTP酶的蛋白质-蛋白质相互作用很重要。Rab 5的同源建模研究表明，Arg-81侧链被掩埋在Rab 5 GTP构象中，但在GDP构象中是溶剂可及的，解释了蛋白水解对核苷酸的依赖性。Rab 4的肽图进行利用开关II内的额外易裂键，以更精确地确定核苷酸依赖性蛋白酶可接近区域的限制。Rab 4切割位点对应于Rab 5的Arg-81和Pro-87，并且结合Rab 5在Arg-91处未切割的发现，该分析定义了位于开关II中心的8个残基表面暴露的构象可变区。Rab蛋白的序列比较显示这八个残基具有松散保守的基序，我们将其相对可变性称为开关II（V）。开关II（v）的C端是高度保守的Rab-specific YYRGA基序，我们将其恒定序列称为开关II（c）。开关II（v）的N-末端是参与核苷酸结合和水解的序列不变的G-结构域。我们认为Rab开关II（v）区域赋予核苷酸依赖性蛋白质-蛋白质相互作用特异性。
Tryptic proteolysis of the small GTPases Rab4 and Rab5 is a multi-step, nucleotide-dependent process. Using N-terminal peptide sequencing, matrix-assisted laser desorption ionization–time-of-flight MS and molecular modelling, we identified the three initial sites of proteolysis in Rab5 as Arg-4, Arg-81 and Arg-197. Arg-4 and Arg-81 lie within regions previously implicated in Rab5 endocytic function, and Arg-197 lies in a region involved in membrane targeting. Topologically, Arg-81 lies within the conformationally variable Switch II region shown to be important for protein–protein interactions of other GTPases. Homology modelling studies on Rab5 indicate that the Arg-81 side chain is buried in the Rab5 GTP conformation, but is solvent-accessible in the GDP conformation, explaining the dependence of proteolysis on nucleotides. Peptide mapping of Rab4 was performed to take advantage of additional scissile bonds within Switch II to determine more precisely the limits of the nucleotide-dependent protease-accessible region. The Rab4 cleavage sites corresponded to Arg-81 and Pro-87 of Rab5, and taken together with the finding that Rab5 was not cleaved at Arg-91 this analysis defines an eight-residue surface-exposed conformationally variable region lying in the centre of Switch II. A sequence comparison of Rab proteins shows these eight residues to have a loosely conserved motif that we term Switch II(v) for its relative variability. C-terminal to Switch II(v) is a highly conserved Rab-specific YYRGA motif that we term Switch II(c) for its constant sequence. N-terminal to Switch II(v) is a sequence-invariant G-domain involved in nucleotide binding and hydrolysis. We propose that the Rab Switch II(v) region imparts specificity to nucleotide-dependent protein–protein interactions.