Wu J, Adomat J M, Ridky T W, Louis J M, Leis J, Harrison R W, Weber I T
Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107,
Biochemistry. 1998 Mar 31;37(13):4518-26. doi: 10.1021/bi972183g.
The Rous sarcoma virus (RSV) protease S9 variant has been engineered to exhibit high affinity for HIV-1 protease substrates and inhibitors in order to verify the residues deduced to be critical for the specificity differences. The variant has 9 substitutions (S38T, I42D, I44V, M73V, A100L, V104T, R105P, G106V, and S107N) of structurally equivalent residues from HIV-1 protease. Unlike the wild-type enzyme, RSV S9 protease hydrolyzes peptides representing the HIV-1 protease polyprotein cleavage sites. The crystal structure of RSV S9 protease with the inhibitor, Arg-Val-Leu-r-Phe-Glu-Ala-Nle-NH2, a reduced peptide analogue of the HIV-1 CA-p2 cleavage site, has been refined to an R factor of 0.175 at 2.4-A resolution. The structure shows flap residues that were not visible in the previous crystal structure of unliganded wild-type enzyme. Flap residues 64-76 are structurally similar to residues 47-59 of HIV-1 protease. However, residues 61-63 form unique loops at the base of the flaps. Mutational analysis indicates that these loop residues are essential for catalytic activity. Side chains of flap residues His 65 and Gln 63' make hydrogen bond interactions with the inhibitor P3 amide and P4' carbonyl oxygen, respectively. Other interactions of RSV S9 protease with the CA-p2 analogue are very similar to those observed in the crystal structure of HIV-1 protease with the same inhibitor. This is the first crystal structure of an avian retroviral protease in complex with an inhibitor, and it verifies our knowledge of the molecular basis for specificity differences between RSV and HIV-1 proteases.
劳氏肉瘤病毒（RSV）蛋白酶S9变体经过改造，对HIV-1蛋白酶底物和抑制剂表现出高亲和力，以验证推断对特异性差异至关重要的残基。该变体具有来自HIV-1蛋白酶的9个结构等效残基的取代（S38T、I42D、I44V、M73V、A100L、V104T、R105P、G106V和S107N）。与野生型酶不同，RSV S9蛋白酶可水解代表HIV-1蛋白酶多蛋白切割位点的肽段。RSV S9蛋白酶与抑制剂Arg-Val-Leu-r-Phe-Glu-Ala-Nle-NH2（一种HIV-1 CA-p2切割位点的还原肽类似物）的晶体结构已在2.4埃分辨率下精修至R因子为0.175。该结构显示了在未结合配体的野生型酶的先前晶体结构中不可见的侧翼残基。侧翼残基64 - 76在结构上与HIV-1蛋白酶的残基47 - 59相似。然而，残基61 - 63在侧翼基部形成独特的环。突变分析表明，这些环残基对催化活性至关重要。侧翼残基His 65和Gln 63'的侧链分别与抑制剂的P3酰胺和P4'羰基氧形成氢键相互作用。RSV S9蛋白酶与CA-p2类似物的其他相互作用与在具有相同抑制剂的HIV-1蛋白酶晶体结构中观察到的相互作用非常相似。这是禽逆转录病毒蛋白酶与抑制剂复合物的首个晶体结构，它验证了我们对RSV和HIV-1蛋白酶之间特异性差异的分子基础的认识。
