Tachedjian Gilda, Aronson Hans-Erik G, de los Santos Martha, Seehra Jas, McCoy John M, Goff Stephen P
Department of Biochemistry and Molecular Biophysics, Columbia University College of Physicians and Surgeons, 701 West 168th St, New York, NY 10032, USA.
J Mol Biol. 2003 Feb 14;326(2):381-96. doi: 10.1016/s0022-2836(02)01433-x.
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 beta-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 p51W401A prey. The suppressors (T409I, D110G, V372A and I393M) also restored heterodimerization of bacterially expressed W401A subunits. When introduced into the W401A mutant, T409I 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.
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处的六个色氨酸残基簇组成，这些残基在灵长类慢病毒RT中高度保守。为了确定这些残基中每个残基对HIV-1 RT二聚化的贡献，我们对克隆的DNA进行了改变，并在酵母双杂交系统中测试了突变亚基介导异源二聚化的能力。将残基401突变为亮氨酸或丙氨酸（但不是苯丙氨酸）以及将残基414突变为亮氨酸，与表达野生型p66诱饵和p51猎物融合体的酵母相比，导致报告基因产生的β-半乳糖苷酶活性大幅降低。亚基选择性诱变表明，这些突变的影响主要通过p66亚基介导。将色氨酸突变体引入细菌表达载体pRT6H/NB-PROT表明，含有W401A或W401L替代（但不是W401F）以及W414L的RT在体外二聚化存在缺陷。与其二聚化缺陷一致，W401A、W401L和W414L突变体没有RT活性。利用酵母双杂交系统，我们在p66中鉴定出几个第二位点抑制子，它们恢复了p66W401A诱饵与p51W401A猎物之间的相互作用。这些抑制子（T409I、D110G、V372A和I393M）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，T409I能够将RT活性恢复到野生型水平的50%。对RT结构的研究表明，p51中的K331与由W401和W414跨越的p66环中的残基形成多个氢键。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处的突变通过改变这些残基之间与p51形成重要接触的结构元件的正确定位来损害二聚化。
