Role of residues in the tryptophan repeat motif for HIV-1 reverse transcriptase dimerization
Role of residues in the tryptophan repeat motif for HIV-1 reverse transcriptase dimerization
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DOI：
10.1016/s0022-2836(02)01433-x
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发表时间：
2003-02-14
影响因子：
5.6
通讯作者：
Goff, SP
中科院分区：
文献类型：
--
作者：
Tachedjian, G;Aronson, HEG;Goff, SP
人类免疫缺陷病毒1型（HIV-1）逆转录酶（RT）的色氨酸重复基序由密码子398、401、402、406、410和414上的6个色氨酸残基组成，在灵长类慢病毒RT中高度保守。为了确定这些残基对HIV-1 RT二聚化的贡献，我们在克隆DNA中引入了变化，并测试了突变亚基介导异源二聚化的能力。在酵母双杂交系统中。与表达野生型p66饵料和p51的酵母相比，报告基因产生的p -半乳糖苷酶活性显著降低，残基401变为亮氨酸或丙氨酸（但不包括苯丙氨酸），残基414变为亮氨酸。融合的猎物。亚基选择性突变表明这些突变的作用主要是通过p66亚基介导的。将色氨酸突变体引入细菌表达载体pRT6H/NB-PROT后，发现含有W401A或W401L（但不含W401F）和W414L的RTs在体外二聚化时存在缺陷。与二聚化缺陷一致，W401A、W401L和W414L突变体缺乏RT活性。利用酵母双杂交系统，我们在p66中发现了几个第二位点抑制子，这些抑制子恢复了p66W401A诱饵与p5lW401A猎物的相互作用。抑制因子（T4091、D110G、V372A和1393M）也恢复了细菌表达的W401A亚基的异源二聚化。当引入W401A突变体时，T4091能够将RT活性恢复到野生型水平的50%。RT结构检查显示p51中存在K331。与W401和W414跨出的p66环中的残基形成多个氢键接触。与这一观察结果一致，K331A RT突变体存在二聚化缺陷。我们得出结论，p66中密码子401和414的突变通过改变与p51有重要接触的这些残基之间结构元件的正确定位而损害了二聚化。2003爱思唯尔科学有限公司版权所有。
The tryptophan repeat motif of the human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) is comprised of a cluster of six tryptophan residues at codons 398, 401, 402, 406, 410 and 414 that are highly conserved amongst primate lentiviral RTs. To determine the contributions of each of these residues for HIV-1 RT dimerization, we introduced changes into cloned DNA and tested the mutant subunits for their capacity to mediate heterodimerization. in the yeast two-hybrid system. Changes of residue 401 to either leucine or alanine (but not phenylalanine) and residue 414 to leucine resulted in major reductions in P-galactosidase activity produced from the reporter gene as compared to yeast expressing wild-type p66 bait and p51. prey fusions. Subunit selective mutagenesis revealed that the effect of these mutations was mediated mainly through the p66 subunit. Introduction of tryptophan mutants into the bacterial expression vector pRT6H/NB-PROT showed that RTs containing W401A or W401L substitutions (but not W401F) and W414L were defective for dimerization in vitro. Consistent with their dimerization defect, the W401A, W401L and W414L mutants were devoid of RT activity. Using the yeast two-hybrid system, we identified several second-site suppressors in p66 that restored interaction of the p66W401A bait to the p5lW401A prey. The suppressors (T4091, D110G, V372A and 1393M) also restored heterodimerization of bacterially expressed W401A subunits. When introduced into the W401A mutant, T4091 was able to restore RT activity to 50% of the wild-type level. Examination of the RT structures revealed that K331 in p51. makes multiple hydrogen bond contacts with residues in the p66 loop spanned by W401 and W414. Consistent with this observation, the K331A RT mutant was dimerization-defective. We conclude that mutations at codons 401 and 414 in p66 impair dimerization by altering the proper positioning of structural elements in between these residues that make important contacts with p51. (C) 2003 Elsevier Science Ltd. All rights reserved.