Interactions of HIV-1 Inhibitory Peptide T20 with the gp41 N-HR Coiled Coil
Interactions of HIV-1 Inhibitory Peptide T20 with the gp41 N-HR Coiled Coil
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DOI：
10.1074/jbc.m809269200
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发表时间：
2009-02-06
影响因子：
4.8
通讯作者：
Root, Michael J.
中科院分区：
文献类型：
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作者：
Champagne, Kelly;Shishido, Akira;Root, Michael J.
人类免疫缺陷病毒1型（HIV-1）进入细胞涉及病毒和细胞膜的融合，并由病毒糖蛋白gp 41的结构转变介导。抗病毒C肽T20靶向gp 41 N-末端七肽重复区（N-HR），阻断进入过程所必需的gp 41构象变化。为了探测T20结构-活性关系，我们使用5-Hisp设计策略设计了整个gp 41 N-HR卷曲螺旋的分子模拟物。T20以纳摩尔亲和力（K-D = 30 nM）结合该人工蛋白（表示为5 H-ex），接近其IC 50浓度（类似于3 nM），但远弱于相关抑制性C肽C37的亲和力（K-D = 0.0007 nM）。T20/C37竞争性结合测定证实，T20与N-HR卷曲螺旋表面上的疏水沟相互作用，该疏水沟位于对C37结合至关重要的深口袋区域之外。我们使用5 H-ex来研究T20 N和C末端如何有助于抑制剂结合活性。在T20 C末端突变三个芳香族残基（WNWF -> ANAA）对亲和力没有影响，表明这些氨基酸不参与T20与gp 41 N-HR的结合。G.，Gallo，S.一、布卢门塔尔，R.，Shai，Y.（2003）J.Biol.Chem.278，21012-21017; Liu，S.，Jing，W.，Cheung，B.，吕，H.，孙，J.，Yan，X.，Niu，J.，Farmar，J.，吴，S.，和Jiang，S.（2007）J.Biol.Chem.282，9612-9620）。相比之下，T20 N末端附近的突变显著影响抑制剂结合强度。当Ile取代第二个T20位置的Thr时，测得结合亲和力增加40倍（K-D = 0.75 nM）。这种亲和力增强对T20抑制效力的影响在不同的病毒株中不同。原始T20和更高亲和力的T20变体对野生型HIV-1具有相似的效力。然而，更高亲和力的T20变体对T20抗性病毒的效力显著更强。这些发现表明，除了结合亲和力之外，其他因素也在限制T20效力方面发挥作用。作为完整gp 41 N-HR卷曲螺旋区的模拟物，5 H-ex将是进一步阐明C肽抑制剂作用机制的有用工具。
Cellular entry of human immunodeficiency virus type 1 (HIV-1) involves fusion of viral and cellular membranes and is mediated by structural transitions in viral glycoprotein gp41. The antiviral C-peptide T20 targets the gp41 N-terminal heptad repeat region (N-HR), blocking gp41 conformational changes essential for the entry process. To probe the T20 structure-activity relationship, we engineered a molecular mimic of the entire gp41 N-HR coiled coil using the 5-Helix design strategy. T20 bound this artificial protein (denoted 5H-ex) with nanomolar affinity (K-D = 30 nM), close to its IC50 concentration (similar to 3 nM) but much weaker than the affinity of a related inhibitory C-peptide C37 (K-D = 0.0007 nM). T20/C37 competitive binding assays confirmed that T20 interacts with the hydrophobic groove on the surface of the N-HR coiled coil outside of a deep pocket region crucial for C37 binding. We used 5H-ex to investigate how the T20 N and C termini contributed to the inhibitor binding activity. Mutating three aromatic residues at the T20 C terminus (WNWF -> ANAA) had no effect on affinity, suggesting that these amino acids do not participate in T20 binding to the gp41 N-HR. The results support recent evidence pointing to a different role for these residues in T20 inhibition (Peisajovich, S. G., Gallo, S. A., Blumenthal, R., and Shai, Y. (2003) J. Biol. Chem. 278, 21012-21017; Liu, S., Jing, W., Cheung, B., Lu, H., Sun, J., Yan, X., Niu, J., Farmar, J., Wu, S., and Jiang, S. (2007) J. Biol. Chem. 282, 9612-9620). By contrast, mutations near the T20 N terminus substantially influenced inhibitor binding strength. When Ile was substituted for Thr in the second T20 position, a 40-fold increase in binding affinity was measured (K-D = 0.75 nM). The effect of this affinity enhancement on T20 inhibitory potency varied among different viral strains. The original T20 and the higher affinity T20 variant had similar potency against wild type HIV-1. However, the higher affinity T20 variant was significantly more potent against T20-resistant virus. The findings suggest that other factors in addition to binding affinity play a role in limiting T20 potency. As a mimetic of the complete gp41 N-HR coiled coil region, 5H-ex will be a useful tool to further elucidate mechanistic profiles of C-peptide inhibitors.