Yi J, Arthur J W, Dunbrack R L, Skalka A M
Fox Chase Cancer Center, Institute for Cancer Research, Philadelphia, Pennsylvania 19111, USA.
J Biol Chem. 2000 Dec 8;275(49):38739-48. doi: 10.1074/jbc.M005499200.
With the increase in our understanding of its structure and enzymatic mechanism, HIV-1 integrase (IN) has become a promising target for designing drugs to treat patients with AIDS. To investigate the structure and function of IN, a panel of monoclonal antibodies (mAbs) directed against HIV-1 IN was raised and characterized previously in this laboratory. Among them, mAbs17, -4, and -33 were found to inhibit IN activity in vitro. In this study, we investigated the interaction of N-terminal-specific mAb17 and its isolated Fab fragment with full-length HIV-1 IN(1-288) and its isolated N-terminal, Zn(2+)-binding domain IN(1-49). Our results show that binding of Zn(2+) to IN(1-49) stabilizes the mAb17-IN complex and that dimer dissociation is not required for binding of the Fab. To identify the epitope recognized by mAb17, we developed a protein footprinting technique based on controlled proteolysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Binding was mapped to a region within amino acids Asp(25)-Glu(35). This peptide corresponds to the end of a helix-turn-helix motif in the IN(1-55) NMR structure and contributes to the dimerization of the N-terminal domain. Antibody binding also appears to destabilize the N-terminal helix in this domain. A molecular model of the IN(1-49).(Fab)(1) complex shows Fab binding across the dimer protein and suggests a potential target for drug design. These data also suggest that mAb17 inhibits integrase activity by blocking critical protein-protein interactions and/or by distorting the orientation of the N-terminal alpha-helix. The relevance of our results to an understanding of IN function is discussed.
随着我们对HIV-1整合酶（IN）结构和酶促机制认识的增加，HIV-1整合酶已成为设计治疗艾滋病患者药物的一个有前景的靶点。为了研究IN的结构和功能，本实验室先前制备并鉴定了一组针对HIV-1 IN的单克隆抗体（mAb）。其中，发现mAb17、-4和-33在体外可抑制IN活性。在本研究中，我们研究了N端特异性mAb17及其分离的Fab片段与全长HIV-1 IN(1-288)及其分离的N端锌结合结构域IN(1-49)之间的相互作用。我们的结果表明，锌离子（Zn(2+)）与IN(1-49)的结合稳定了mAb17-IN复合物，并且Fab的结合不需要二聚体解离。为了确定mAb17识别的表位，我们基于可控蛋白酶解和基质辅助激光解吸电离飞行时间质谱开发了一种蛋白质足迹技术。结合被定位到氨基酸Asp(25)-Glu(35)内的一个区域。该肽对应于IN(1-55)核磁共振结构中一个螺旋-转角-螺旋基序的末端，并有助于N端结构域的二聚化。抗体结合似乎也使该结构域中的N端螺旋不稳定。IN(1-49).(Fab)(1)复合物的分子模型显示Fab跨二聚体蛋白结合，并提示了一个潜在的药物设计靶点。这些数据还表明，mAb17通过阻断关键的蛋白质-蛋白质相互作用和/或通过扭曲N端α-螺旋的方向来抑制整合酶活性。讨论了我们的结果与理解IN功能的相关性。
