Mutational Analysis of a Conserved Glutamic Acid Required for Self-Catalyzed Cross-Linking of Bacteriophage HK97 Capsids
Mutational Analysis of a Conserved Glutamic Acid Required for Self-Catalyzed Cross-Linking of Bacteriophage HK97 Capsids
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DOI：
10.1128/jvi.02000-08
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发表时间：
2009-03-01
影响因子：
5.4
通讯作者：
Duda, Robert L.
中科院分区：
文献类型：
--
作者：
Dierkes, Lindsay E.;Peebles, Craig L.;Duda, Robert L.
噬菌体HK97的衣壳是由类似于400个亚基之间的共价交联键稳定的，这些交联键不需要任何外部酶或辅助因子的作用就可以形成。只有在大规模的结构变化将每个交联点的三个亚基的侧链聚集在一起后，才会在完全组装的颗粒中发生交联。天冬酰胺和赖氨酸侧链在两个亚基上形成异肽交联物。第三个亚基的谷氨酸363(E363)的羧酸类似于含有交联键的部分疏水口袋中的异肽键的2.4埃。此前有报道称，在没有支持数据的情况下，将E363改为丙氨酸会消除交联键，这表明E363在交联键中起到了作用。对这个丙氨酸突变体和E363的另外六个替代进行了充分的鉴定，并测试了突变体产生的蛋白质在各种条件下的交联性。天冬氨酸和组氨酸取代在很大程度上支持交联，而丙氨酸、天冬酰胺、谷氨酰胺和酪氨酸不支持，这表明残基363在交联过程中起到质子受体的作用。这些结果支持一种尚未完全测试的化学机制，该机制结合了这一观点以及交联点的结构特征。最近在细菌菌毛中发现的化学上相同的等肽键在其交联点上具有惊人的相似的化学几何结构，这表明与噬菌体蛋白具有共同的化学机制，但两种蛋白完全不同的结构和折叠表明，噬菌体衣壳和细菌菌毛蛋白通过聚合进化实现了共同的交联化学。
The capsid of bacteriophage HK97 is stabilized by similar to 400 covalent cross-links between subunits which form without any action by external enzymes or cofactors. Cross-linking only occurs in fully assembled particles after large-scale structural changes bring together side chains from three subunits at each cross-linking site. Isopeptide cross-links form between asparagine and lysine side chains on two subunits. The carboxylate of glutamic acid 363 (E363) from a third subunit is found similar to 2.4 angstrom from the isopeptide bond in the partly hydrophobic pocket that contains the cross-link. It was previously reported without supporting data that changing E363 to alanine abolishes cross-linking, suggesting that E363 plays a role in cross-linking. This alanine mutant and six additional substitutions for E363 were fully characterized and the proheads produced by the mutants were tested for their ability to cross-link under a variety of conditions. Aspartic acid and histidine substitutions supported cross-linking to a significant extent, while alanine, asparagine, glutamine, and tyrosine did not, suggesting that residue 363 acts as a proton acceptor during cross-linking. These results support a chemical mechanism, not yet fully tested, that incorporates this suggestion, as well as features of the structure at the cross-link site. The chemically identical isopeptide bonds recently documented in bacterial pili have a strikingly similar chemical geometry at their cross-linking sites, suggesting a common chemical mechanism with the phage protein, but the completely different structures and folds of the two proteins argues that the phage capsid and bacterial pilus proteins have achieved shared cross-linking chemistry by convergent evolution.