Dynamics of Scabin toxin. A proposal for the binding mode of the DNA substrate

Scabin is a mono-ADP-ribosyltransferase enzyme and is a putative virulence factor produced by the plant pathogen, Streptomyces scabies. Previously, crystal structures of Scabin were solved in the presence and absence of substrate analogues and inhibitors. Herein, experimental (hydrogen-deuterium exchange), simulated (molecular dynamics), and theoretical (Gaussian Network Modeling) approaches were systematically applied to study the dynamics of apo-Scabin in the context of a Scabin·NAD+·DNA model. MD simulations revealed that the apo-Scabin solution conformation correlates well with the X-ray crystal structure, beyond the conformation of the exposed, mobile regions. In turn, the MD fluctuations correspond with the crystallographic B-factors, with the fluctuations derived from a Gaussian network model, and with the experimental H/D exchange rates. An Essential Dynamics Analysis identified the dynamic aspects of the toxin as a crab-claw-like mechanism of two topological domains, along with coupled deformations of exposed motifs. The “crab-claw” movement resembles the motion of C3-like toxins and emerges as a property of the central β scaffold of catalytic single domain toxins. The exposure and high mobility of the cis side motifs in the Scabin β-core suggest involvement in DNA substrate binding. A ternary Scabin·NAD+·DNA model was produced via an independent docking methodology, where the intermolecular interactions correspond to the region of high mobility identified by dynamics analyses and agree with binding and kinetic data reported for wild-type and Scabin variants. Based on data for the Pierisin-like toxin group, the sequence motif Rβ1–RLa–NLc–STTβ2–WPN–WARTT–(QxE)ARTT emerges as a catalytic signature involved in the enzymatic activity of these DNA-acting toxins. However, these results also show that Scabin possesses a unique DNA-binding motif within the Pierisin-like toxin group.Scabin是一种单ADP核糖基转移酶（mono-ADP-ribosyltransferase），同时是植物病原菌Streptomyces scabies产生的假定毒力因子。此前，研究人员已解析了Scabin在存在底物类似物与抑制剂、以及不存在这些物质时的晶体结构。本文系统采用实验（氢氘交换法）、模拟（分子动力学）与理论（高斯网络模型，Gaussian Network Modeling）手段，针对Scabin·烟酰胺腺嘌呤二核苷酸（NAD+）·DNA模型中的脱辅基Scabin（apo-Scabin）的动态特性展开研究。分子动力学模拟结果显示，脱辅基Scabin的溶液构象与X射线晶体结构具有较高的相关性，这一相关性在暴露的柔性区域之外同样成立。进一步而言，分子动力学波动与晶体学B因子、高斯网络模型推导的波动值以及实验测得的氢氘交换速率均具有较好的一致性。本征动力学分析（Essential Dynamics Analysis）识别出该毒素的动态特性表现为两个拓扑结构域的“蟹钳样”运动机制，同时伴随暴露基序的耦合形变。这种“蟹钳”运动模式与C3样毒素的运动特征相似，且可作为催化单结构域毒素中央β折叠支架的固有特性。Scabin的β折叠核心中顺式侧基序的暴露与高流动性，提示其参与DNA底物的结合过程。研究人员通过独立的对接方法构建了Scabin·NAD+·DNA三元复合物模型，该模型的分子间相互作用区域与动力学分析鉴定的高流动性区域相匹配，同时与已报道的野生型及Scabin变体的结合及动力学数据一致。基于Pierisin样毒素家族（Pierisin-like toxin group）的相关研究数据，序列基序Rβ1–RLa–NLc–STTβ2–WPN–WARTT–(QxE)ARTT可作为一类催化特征基序，参与这类作用于DNA的毒素的酶促活性。但本研究结果同时表明，在Pierisin样毒素家族中，Scabin拥有独特的DNA结合基序。