Zhou Hao, Li Shangyang, Badger John, Nalivaika Ellen, Cai Yufeng, Foulkes-Murzycki Jennifer, Schiffer Celia, Makowski Lee
Department of Electrical and Computer Engineering, Northeastern University, Boston, Massachusetts.
DeltaG Technologies, San Diego, California.
Proteins. 2015 Nov;83(11):1929-39. doi: 10.1002/prot.24737. Epub 2015 Sep 29.
The flexibility of HIV protease (HIVp) plays a critical role in enabling enzymatic activity and is required for substrate access to the active site. While the importance of flexibility in the flaps that cover the active site is well known, flexibility in other parts of the enzyme is also critical for function. One key region is a loop containing Thr 80, which forms the walls of the active site. Although not situated within the active site, amino acid Thr80 is absolutely conserved. The mutation T80N preserves the structure of the enzyme but catalytic activity is completely lost. To investigate the potential influence of the T80N mutation on HIVp flexibility, wide-angle X-ray scattering (WAXS) data was measured for a series of HIVp variants. Starting with a calculated WAXS pattern from a rigid atomic model, the modulations in the intensity distribution caused by structural fluctuations in the protein were predicted by simple analytic methods and compared with the experimental data. An analysis of T80N WAXS data shows that this variant is significantly more rigid than the WT across all length scales. The effects of this single point mutation extend throughout the protein, to alter the mobility of amino acids in the enzymatic core. These results support the contentions that significant protein flexibility extends throughout HIVp and is critical to catalytic function.
HIV蛋白酶（HIVp）的灵活性在实现酶活性方面起着关键作用，并且是底物进入活性位点所必需的。虽然覆盖活性位点的瓣区灵活性的重要性已为人所知，但酶的其他部分的灵活性对其功能也至关重要。一个关键区域是包含苏氨酸80的环，它构成了活性位点的壁。尽管苏氨酸80并不位于活性位点内，但它是绝对保守的。T80N突变保留了酶的结构，但催化活性完全丧失。为了研究T80N突变对HIVp灵活性的潜在影响，我们测量了一系列HIVp变体的广角X射线散射（WAXS）数据。从刚性原子模型计算出的WAXS模式开始，通过简单的分析方法预测了蛋白质结构波动引起的强度分布调制，并与实验数据进行了比较。对T80N WAXS数据的分析表明，在所有长度尺度上，该变体都比野生型明显更刚性。这个单点突变的影响延伸到整个蛋白质，改变了酶核心中氨基酸的流动性。这些结果支持了这样的观点，即显著的蛋白质灵活性贯穿HIVp始终，并且对催化功能至关重要。
