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首页> 外文期刊>Biochemistry >Identification of the principal binding site for RGD-containing ligands in the alpha(V)beta(3) integrin: a photoaffinity cross-linking study.
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Identification of the principal binding site for RGD-containing ligands in the alpha(V)beta(3) integrin: a photoaffinity cross-linking study.

机译：鉴定α（v）β（3）细胞蛋白中含RGD配体的主要结合位点：光边和交联研究。

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摘要

By superimposing data obtained by photo-cross-linking RGD-containing ligands to the human alpha(V)beta(3) integrin onto the crystal structure of the ectopic domain of this receptor (Xiong et al. (2001) Science 294, 339-345), we have identified the binding site for the RGD triad within this integrin. We synthesized three novel analogues of the 49-amino acid disintegrin, echistatin: [Bpa(21),Leu(28)]-, [Bpa(23),Leu(28)]-, and [Bpa(28)]echistatin. Each contains a photoreactive p-benzoyl-phenylalanyl (Bpa) residue in close proximity to the RGD motif which spans positions 24-26; together, the photoreactive positions flank the RGD motif. The analogues bind with high affinity to the purified recombinant alpha(V)beta(3) integrin, but very poorly to the closely related human alpha(IIb)beta(3) platelet integrin. While echistatin analogues containing Bpa in either position 23 or 28 cross-link specifically and almost exclusively to the beta(3) subunit of alpha(V)beta(3), [Bpa(21),Leu(28)]echistatin cross-links to both the alpha(V) and the beta(3) subunits, with cross-linking to the former favored. [Bpa(23),Leu(28)]echistatin cross-links 10-30 times more effectively than the other two analogues. We identified beta(3)[109-118] as the domain that encompasses the contact site for [Bpa(28)]echistatin. This domain is included in beta(3)[99-118] (Bitan et al. (2000) Biochemistry 39, 11014-11023), a previously identified contact domain for a cyclic RGD-containing heptapeptide with a benzophenone moiety in a position that is similar to the placement of the benzophenone in [Bpa(28)]echistatin relative to the RGD triad. Recently, we identified beta(3)[209-220] as the contact site for an echistatin analogue with a photoreactive group in position 45, near the C-terminus of echistatin (Scheibler et al. (2001) Biochemistry 40, 15117-14126). Taken together, these results support the hypothesis that the very high binding affinity of echistatin to alpha(V)beta(3) results from two distinct epitopes in the ligand, a site including the RGD triad and an auxiliary epitope at the C-terminus of echistatin. Combining our results from photoaffinity cross-linking studies with data now available from the recently published crystal structure of the ectopic domain of alpha(V)beta(3), we characterize the binding site for the RGD motif in this receptor.
机译：通过将通过光交联的RGD的配体获得的数据叠加到人α（v）β（3）整合蛋白上的该受体的异位结构域的晶体结构上（熊等（2001）科学294,339- 345），我们已经确定了该整合素内的RGD三合会的结合位点。我们合成了49-氨基酸脱胶素的三种新型类似物，Echistatin：[BPA（21），Leu（28）] - ，[BPA（23），LeU（28）] - ，和[BPA（28）] Echistatin。各自含有光反应性P-苯甲酰基 - 苯丙烯基（BPA）残余物，其紧邻跨越24-26的RGD基序。在一起，光反应性位置侧翼侧翼。类似物与纯化的重组α（v）β（3）整联蛋白的高亲和力结合，但对密切相关的人α（IIB）β（3）血小板整联蛋白非常差。虽然EchistatIN在一个位置23或28中的BPA含有BPA，但特别是α（v）β（3），[BPA（21），Leu（28）] Echistatin交叉链接的β（3）亚基α（v）和β（3）亚基，与前者交联。 [BPA（23），Leu（28）]超声链接比其他两个类似物更有效地交叉链接10-30倍。我们鉴定了β（3）[109-118]作为包含[BPA（28）] Echistatin的接触部位的结构域。该域包含在Beta（3）中（3）[99-118]（Bitan等）（2000）生物化学39,11014-11023），以前鉴定了含环RGD的肝肽的接触结构域，其位置类似于二苯酮在[BPA（28）] Echistatin相对于RGD三合会的位置。最近，我们鉴定了β（3）[209-220]作为EchistatiN类似物的接触部位，在Echistatin的C-末端附近与位置45的光反应性基团（Scheibler等人。（2001）生物化学40,15117-14126 ）。总之，这些结果支持Hechistatin对α（v）β（3）的非常高结合亲和力的假设来自配体中的两个不同表位，包括RGD三合会的部位和C-末端的辅助表位Echistatin。将我们的结果由光谱素质交联研究与现在可从最近公开的α（v）β（3）的异位结构域的晶体结构相结合，我们在该受体中表征RGD基序的结合位点。

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