School of Chemistry, University of Southampton, Southampton, United Kingdom.
Department of Biology and the York Biomedical Research Institute, University of York, York, United Kingdom.
Elife. 2023 Jan 11;12:e81547. doi: 10.7554/eLife.81547.
Acylation of diverse carbohydrates occurs across all domains of life and can be catalysed by proteins with a membrane bound acyltransferase-3 (AT3) domain (PF01757). In bacteria, these proteins are essential in processes including symbiosis, resistance to viruses and antimicrobials, and biosynthesis of antibiotics, yet their structure and mechanism are largely unknown. In this study, evolutionary co-variance analysis was used to build a computational model of the structure of a bacterial O-antigen modifying acetyltransferase, OafB. The resulting structure exhibited a novel fold for the AT3 domain, which molecular dynamics simulations demonstrated is stable in the membrane. The AT3 domain contains 10 transmembrane helices arranged to form a large cytoplasmic cavity lined by residues known to be essential for function. Further molecular dynamics simulations support a model where the acyl-coA donor spans the membrane through accessing a pore created by movement of an important loop capping the inner cavity, enabling OafB to present the acetyl group close to the likely catalytic resides on the extracytoplasmic surface. Limited but important interactions with the fused SGNH domain in OafB are identified, and modelling suggests this domain is mobile and can both accept acyl-groups from the AT3 and then reach beyond the membrane to reach acceptor substrates. Together this new general model of AT3 function provides a framework for the development of inhibitors that could abrogate critical functions of bacterial pathogens.
酰化作用发生在所有生命领域，可由具有膜结合酰基转移酶-3（AT3）结构域（PF01757）的蛋白质催化。在细菌中，这些蛋白质在包括共生、抗病毒和抗微生物以及抗生素生物合成在内的过程中是必不可少的，但它们的结构和机制在很大程度上尚不清楚。在这项研究中，进化协方差分析被用于构建细菌 O-抗原修饰乙酰基转移酶 OafB 的结构计算模型。得到的结构表现出 AT3 结构域的新颖折叠，分子动力学模拟表明其在膜中稳定。AT3 结构域包含 10 个跨膜螺旋，排列成一个由已知对功能至关重要的残基组成的大细胞质腔。进一步的分子动力学模拟支持了一种模型，即酰基辅酶 A 供体通过移动重要的环来穿过膜，从而创建一个孔，该环覆盖内腔，从而使 OafB 能够将乙酰基基团靠近细胞外表面上可能的催化残基。在 OafB 中与融合的 SGNH 结构域之间存在有限但重要的相互作用，建模表明该结构域是可移动的，既能从 AT3 接受酰基，又能超越膜到达受体底物。总之，这种新的 AT3 功能一般模型为开发抑制剂提供了一个框架，该抑制剂可能会破坏细菌病原体的关键功能。
