Tomin M, Tomić S
Division of Organic Chemistry and Biochemistry, Rudjer Boskovic Institute, Bijenička cesta 54, 10000, Zagreb, Croatia.
Mol Biosyst. 2017 Oct 24;13(11):2407-2417. doi: 10.1039/c7mb00310b.
Dipeptidyl peptidase III (DPP III) from the human gut symbiont Bacteroides thetaiotaomicron (Bt) is the first identified prokaryotic DPP III orthologue. It has low sequence similarity to the thoroughly studied human DPP III, and differently from eukaryotic orthologues it has a cysteine (Cys450) residue in the zinc-binding motif HEXXGH (HECLGH). The recently determined crystal structure of BtDPP III showed that its 3D structure, similar to the structure of the human DPP III, consists of two domains with a wide cleft in between. Although such a striking similarity of the 3D structures of orthologues with low sequence similarity is not surprising, it is no guarantee for similarity of their dynamic properties and the catalytic performance. Here, we report the results of the molecular modelling study of BtDPP III, wild type and its C450S mutant, as well as their complexes with characteristic DPP III substrates Arg-Arg-2-naphthylamide (RRNA) and Lys-Ala-2-naphtylamide (KANA). During several hundred nanoseconds of all-atom MD simulations of the wild type protein, the long range conformational changes, which can be described as protein 'closing', have been traced. We have determined a similar conformational change for the human orthologue as well. However, the amplitude of the change is lower for BtDPP III than for the human DPP III. The MD simulations have been performed using ff03, ff12SB and ff14SB force fields wherein the results of the last two better fit to the experimental results. The hydrogen bond analysis indicates reasons for higher substrate specificity of BtDPP III towards RRNA than KANA as well as for the decrease of the RRNA hydrolysis rate induced by the Cys450 to Ser mutation. The obtained results are in line with the experimental data.
来自人类肠道共生菌嗜热栖热放线菌（Bt）的二肽基肽酶III（DPP III）是首个被鉴定出的原核生物DPP III直系同源物。它与经过深入研究的人类DPP III的序列相似性较低，并且与真核生物直系同源物不同的是，它在锌结合基序HEXXGH（HECLGH）中有一个半胱氨酸（Cys450）残基。最近测定的BtDPP III晶体结构表明，其三维结构与人类DPP III的结构相似，由两个结构域组成，中间有一个宽裂缝。尽管序列相似性低的直系同源物三维结构有如此显著的相似性并不奇怪，但这并不能保证它们的动力学性质和催化性能相似。在此，我们报告了BtDPP III野生型及其C450S突变体的分子建模研究结果，以及它们与DPP III特征底物精氨酸-精氨酸-2-萘酰胺（RRNA）和赖氨酸-丙氨酸-2-萘酰胺（KANA）的复合物。在对野生型蛋白质进行数百纳秒的全原子分子动力学模拟过程中，追踪到了可描述为蛋白质“关闭”的长程构象变化。我们也确定了人类直系同源物有类似的构象变化。然而，BtDPP III的变化幅度比人类DPP III的小。使用ff03、ff12SB和ff14SB力场进行了分子动力学模拟，其中后两者的结果与实验结果拟合得更好。氢键分析表明了BtDPP III对RRNA比对KANA具有更高底物特异性的原因，以及半胱氨酸450突变为丝氨酸导致RRNA水解速率降低的原因。所得结果与实验数据一致。
