Combining multi-scale modelling methods to decipher molecular motions of a branching sucrase from glycoside-hydrolase family 70

Among α-transglucosylases from Glycoside-Hydrolase family 70, the ΔN123-GB-CD2 enzyme derived from the bifunctional DSR-E from L. citreum NRRL B-1299 is particularly interesting as it was the first described engineered Branching Sucrase, not able to elongate glucan polymers from sucrose substrate. The previously reported overall structural organization of this multi-domain enzyme is an intricate U-shape fold conserved among GH70 enzymes which showed a certain conformational variability of the so-called domain V, assumed to play a role in the control of product structures, in available X-ray structures. Understanding the role of functional dynamics on enzyme reaction and substrate recognition is of utmost interest although it remains a challenge for biophysical methods. By combining long molecular dynamics simulation (1μs) and multiple analyses (NMA, PCA, Morelet Continuous Wavelet Transform and Cross Correlations Dynamics), we investigated here the dynamics of ΔN123-GB-CD2 alone and in interaction with sucrose substrate. Overall, our results provide the detailed picture at atomic level of the hierarchy of motions occurring along different timescales and how they are correlated, in agreement with experimental structural data. In particular, detailed analysis of the different structural domains revealed cooperative dynamic behaviors such as twisting, bending and wobbling through anti- and correlated motions, and also two structural hinge regions, of which one was unreported. Several highly flexible loops surrounding the catalytic pocket were also highlighted, suggesting a potential role in the acceptor promiscuity of ΔN123-GBD-CD2. Normal modes and essential dynamics underlined an interesting two-fold dynamic of the catalytic domain A, pivoting about an axis splitting the catalytic gorge in two parts. The comparison of the conformational free energy landscapes using principal component analysis of the enzyme in absence or in presence of sucrose, also revealed a more harmonic basin when sucrose is bound with a shift population of the bending mode, consistent with the substrate binding event.

在糖苷水解酶家族70（Glycoside-Hydrolase family 70）的α-转葡萄糖苷酶中，源自柠檬明串珠菌NRRL B-1299（L. citreum NRRL B-1299）双功能酶DSR-E的ΔN123-GB-CD2酶尤为引人关注：它是首款被报道的工程化支链蔗糖酶，无法以蔗糖为底物催化葡聚糖聚合物的延伸反应。此前已报道该多结构域酶的整体结构组织为GH70酶家族保守的复杂U型折叠构象，在已解析的X射线结构中，其所谓的结构域V存在一定构象变异性，该结构域被认为在产物结构的调控过程中发挥功能。尽管解析功能动态性在酶促反应与底物识别中的作用仍是生物物理研究方法面临的挑战，但该问题的解析具有至关重要的科学意义。本研究结合时长1微秒的长时分子动力学模拟（molecular dynamics simulation），以及正态模式分析（Normal Mode Analysis, NMA）、主成分分析（Principal Component Analysis, PCA）、莫雷尔连续小波变换（Morelet Continuous Wavelet Transform）与动态互相关分析（Cross Correlations Dynamics）等多种手段，对游离态以及与蔗糖底物结合的ΔN123-GB-CD2酶的动态特性展开了探究。总体而言，本研究结果在原子层面上详细描绘了不同时间尺度下运动层级的构成及其关联机制，与已有的实验结构数据相符。具体而言，对不同结构域的细致分析揭示了多种协同动态行为：包括借助反相关与相关运动实现的扭转、弯曲与摆动等，同时还鉴定出两个结构铰链区域，其中一个为此前未被报道的新位点。研究还发现了催化口袋周围的多个高度柔性环，提示其可能在ΔN123-GBD-CD2的受体底物混杂性中发挥潜在功能。正态模式分析与本质动力学分析结果显示，催化结构域A存在一种有趣的双模式动态特性，即围绕将催化峡谷一分为二的轴线进行枢转运动。通过主成分分析对比游离态与结合蔗糖的酶的构象自由能景观，研究还发现，当酶与蔗糖结合时，其自由能盆地更为谐调规整，且弯曲模式的种群分布发生偏移，这与底物结合事件的特征相符。