Binding kinetics and substrate selectivity in HIV-1 protease-Gag interactions probed at atomic resolution by chemical exchange NMR
Binding kinetics and substrate selectivity in HIV-1 protease-Gag interactions probed at atomic resolution by chemical exchange NMR
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DOI：
10.1073/pnas.1716098114
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发表时间：
2017-11-14
影响因子：
11.1
通讯作者：
Clore, G. Marius
中科院分区：
文献类型：
--
作者：
Deshmukh, Lalit;Tugarinov, Vitali;Clore, G. Marius
未成熟的非感染性HIV-1颗粒向感染性病毒粒子的转化依赖于HIV-1蛋白酶对前体群特异性抗原（Gag）多蛋白的顺序切割。蛋白酶识别不同Gag切割位点的确切机制尚不清楚，这些位点位于连接Gag球形结构域的内在无序连接体中。在这里，我们利用carr - purcell - meiboomm - gill弛豫分散和化学交换饱和转移核磁共振实验探讨了Gag大片段与各种蛋白酶变体（包括耐药结构）相互作用的动力学。我们发现，在催化裂口上形成盖子的HIV-1蛋白酶瓣内的构象动力学在底物特异性和有序Gag加工中起着重要作用。封闭和开放的蛋白酶瓣构象之间的快速相互转换促进了蛋白酶和Gag底物之间短暂的、稀疏分布的生产复合体的形成。皮瓣闭合在蛋白酶的催化裂口内捕获Gag裂解位点。通过蛋白酶-Gag相互作用调节皮瓣打开微调生产复合体的寿命，因此Gag蛋白水解的可能性。在非同源底物存在的情况下，也可以形成生产性复合体，但由于蛋白酶的活性位点裂缝与底物之间缺乏最佳的互补性，导致酶瓣快速打开和底物释放，从而使蛋白酶能够区分同源底物和非同源底物。
The conversion of immature noninfectious HIV-1 particles to infectious virions is dependent upon the sequential cleavage of the precursor group-specific antigen (Gag) polyprotein by HIV-1 protease. The precise mechanism whereby protease recognizes distinct Gag cleavage sites, located in the intrinsically disordered linkers connecting the globular domains of Gag, remains unclear. Here, we probe the dynamics of the interaction of large fragments of Gag and various variants of protease (including a drug resistant construct) using Carr-Purcell-Meiboom-Gill relaxation dispersion and chemical exchange saturation transfer NMR experiments. We show that the conformational dynamics within the flaps of HIV-1 protease that form the lid over the catalytic cleft play a significant role in substrate specificity and ordered Gag processing. Rapid interconversion between closed and open protease flap conformations facilitates the formation of a transient, sparsely populated productive complex between protease and Gag substrates. Flap closure traps the Gag cleavage sites within the catalytic cleft of protease. Modulation of flap opening through protease-Gag interactions fine-tunes the lifetime of the productive complex and hence the likelihood of Gag proteolysis. A productive complex can also be formed in the presence of a noncognate substrate but is short-lived owing to lack of optimal complementarity between the active site cleft of protease and the substrate, resulting in rapid flap opening and substrate release, thereby allowing protease to differentiate between cognate and noncognate substrates.