Babé L M, Rosé J, Craik C S
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0446.
Protein Sci. 1992 Oct;1(10):1244-53. doi: 10.1002/pro.5560011003.
Retroviral proteases are obligate homodimers and play an essential role in the viral life cycle. Dissociation of dimers or prevention of their assembly may inactivate these enzymes and prevent viral maturation. A salient structural feature of these enzymes is an extended interface composed of interdigitating N- and C-terminal residues of both monomers, which form a four-stranded beta-sheet. Peptides mimicking one beta-strand (residues 95-99), or two beta-strands (residues 1-5 plus 95-99 or 95-99 plus 95-99) from the human immunodeficiency virus 1 (HIV1) interface were shown to inhibit the HIV1 and 2 proteases (PRs) with IC50's in the low micromolar range. These interface peptides show cognate enzyme preference and do not inhibit pepsin, renin, or the Rous sarcoma virus PR, indicating a degree of specificity for the HIV PRs. A tethered HIV1 PR dimer was not inhibited to the same extent as the wild-type enzymes by any of the interface peptides, suggesting that these peptides can only interact effectively with the interface of the two-subunit HIV PR. Measurements of relative dissociation constants by limit dilution of the enzyme show that the one-strand peptide causes a shift in the observed Kd for the HIV1 PR. Both one- and two-strand peptides alter the monomer/dimer equilibrium of both HIV1 and HIV2 PRs. This was shown by the reduced cross-linking of the HIV2 PR by disuccinimidyl suberate in the presence of the interface peptides. Refolding of the HIV1 and HIV2 PRs with the interface peptides shows that only the two-strand peptides prevent the assembly of active PR dimers. Although both one- and two-strand peptides seem to affect dimer dissociation, only the two-strand peptides appear to block assembly. The latter may prove to be more effective backbones for the design of inhibitors directed toward retroviral PR dimerization in vivo.
逆转录病毒蛋白酶是专性同源二聚体，在病毒生命周期中起关键作用。二聚体的解离或其组装的阻止可能使这些酶失活并防止病毒成熟。这些酶的一个显著结构特征是由两个单体的相互交错的N端和C端残基组成的延伸界面，其形成一个四链β-折叠。模拟来自人类免疫缺陷病毒1（HIV1）界面的一条β-链（残基95 - 99）或两条β-链（残基1 - 5加95 - 99或95 - 99加95 - 99）的肽被证明能抑制HIV1和2蛋白酶（PRs），其IC50在低微摩尔范围内。这些界面肽显示出同源酶偏好，并且不抑制胃蛋白酶、肾素或劳氏肉瘤病毒PR，表明对HIV PRs有一定程度的特异性。与野生型酶相比，任何界面肽对拴系的HIV1 PR二聚体的抑制程度都不同，这表明这些肽只能与双亚基HIV PR的界面有效相互作用。通过酶的极限稀释测量相对解离常数表明，单链肽导致HIV1 PR观察到的Kd发生偏移。单链和双链肽都改变了HIV1和HIV2 PRs的单体/二聚体平衡。这通过在界面肽存在下辛二酸二琥珀酰亚胺酯对HIV2 PR交联的减少得以证明。用界面肽对HIV1和HIV2 PRs进行重折叠表明，只有双链肽能阻止活性PR二聚体的组装。虽然单链和双链肽似乎都影响二聚体解离，但只有双链肽似乎能阻止组装。后者可能被证明是在体内设计针对逆转录病毒PR二聚化的抑制剂的更有效骨架。
