Mahalingam B, Louis J M, Reed C C, Adomat J M, Krouse J, Wang Y F, Harrison R W, Weber I T
Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, USA.
Eur J Biochem. 1999 Jul;263(1):238-45. doi: 10.1046/j.1432-1327.1999.00514.x.
Mutants of HIV-1 protease that are commonly selected on exposure to different drugs, V82S, G48V, N88D and L90M, showed reduced catalytic activity compared to the wild-type protease on cleavage site peptides, CA-p2, p6pol-PR and PR-RT, critical for viral maturation. Mutant V82S is the least active (2-20% of wild-type protease), mutants N88D, R8Q, and L90M exhibit activities ranging from 20 to 40% and G48V from 50 to 80% of the wild-type activity. In contrast, D30N is variable in its activity on different substrates (10-110% of wild-type), with the PR-RT site being the most affected. Mutants K45I and M46L, usually selected in combination with other mutations, showed activities that are similar to (60-110%) or greater than (110-530%) wild-type, respectively. No direct relationship was observed between catalytic activity, inhibition, and structural stability. The mutants D30N and V82S were similar to wild-type protease in their stability toward urea denaturation, while R8Q, G48V, and L90M showed 1.5 to 2.7-fold decreased stability, and N88D and K45I showed 1.6 to 1.7-fold increased stability. The crystal structures of R8Q, K45I and L90M mutants complexed with a CA-p2 analog inhibitor were determined at 2.0, 1.55 and 1.88 A resolution, respectively, and compared to the wild-type structure. The intersubunit hydrophobic contacts observed in the crystal structures are in good agreement with the relative structural stability of the mutant proteases. All these results suggest that viral resistance does not arise by a single mechanism.
在接触不同药物时通常会被选择出来的HIV-1蛋白酶突变体，即V82S、G48V、N88D和L90M，与野生型蛋白酶相比，在对病毒成熟至关重要的切割位点肽（CA-p2、p6pol-PR和PR-RT）上表现出催化活性降低。突变体V82S活性最低（为野生型蛋白酶的2%-20%），突变体N88D、R8Q和L90M的活性范围为野生型活性的20%-40%，G48V为50%-80%。相比之下，D30N对不同底物的活性变化较大（为野生型的10%-110%），其中PR-RT位点受影响最大。通常与其他突变一起被选择出来的突变体K45I和M46L，其活性分别与野生型相似（60%-110%）或高于野生型（110%-530%）。在催化活性、抑制作用和结构稳定性之间未观察到直接关系。突变体D30N和V82S对尿素变性的稳定性与野生型蛋白酶相似，而R8Q、G48V和L90M的稳定性降低了1.5至2.7倍，N88D和K45I的稳定性增加了1.6至1.7倍。分别以2.0、1.55和1.88 Å的分辨率测定了与CA-p2类似物抑制剂复合的R8Q、K45I和L90M突变体的晶体结构，并与野生型结构进行了比较。在晶体结构中观察到的亚基间疏水接触与突变体蛋白酶的相对结构稳定性高度一致。所有这些结果表明，病毒耐药性并非由单一机制产生。
