Division of Molecular Medicine, Hefei National Laboratory for Physical Sciences at Microscale, CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences, University of Science and Technology of China, Hefei 230027, People's Republic of China.
Shanghai Jiao Tong University Affiliated Sixth People's Hospital South Campus, Shanghai, People's Republic of China.
Acta Crystallogr D Struct Biol. 2019 Aug 1;75(Pt 8):772-781. doi: 10.1107/S205979831901057X. Epub 2019 Jul 31.
CAMP factor is a unique α-helical bacterial toxin that is known for its co-hemolytic activity in combination with staphylococcal sphingomyelinase. It was first discovered in the human pathogen Streptococcus agalactiae (also known as group B streptococcus), but homologous genes have been found in many other Gram-positive pathogens. In this study, the efforts that led to the determination of the first structure of a CAMP-family toxin are reported. Initially, it was possible to produce crystals of the native protein which diffracted to near 2.45 Å resolution. However, a series of technical obstacles were encountered on the way to structure determination. Over a period of more than five years, many methods, including selenomethionine labeling, mutations, crystallization chaperones and heavy-atom soaking, were attempted, but these attempts resulted in limited progress. The structure was finally solved using a combination of iodine soaking and molecular replacement using the crystallization chaperone maltose-binding protein (MBP) as a search model. Analysis of native and MBP-tagged CAMP-factor structures identified a conserved interaction interface in the C-terminal domain (CTD). The positively charged surface may be critical for binding to acidic ligands. Furthermore, mutations on the interaction interface at the CTD completely abolished its co-hemolytic activities. This study provides novel insights into the mechanism of the membrane-permeabilizing activity of CAMP factor.
CAMP 因子是一种独特的α-螺旋细菌毒素，因其与葡萄球菌神经鞘磷脂酶的协同溶血活性而闻名。它最初在人类病原体酿脓链球菌（也称为 B 群链球菌）中被发现，但在许多其他革兰氏阳性病原体中也发现了同源基因。本研究报告了确定第一个 CAMP 家族毒素结构的努力。最初，可以生产出具有近 2.45Å 分辨率的天然蛋白晶体。然而，在结构确定的过程中遇到了一系列技术障碍。在五年多的时间里，尝试了许多方法，包括硒代蛋氨酸标记、突变、结晶伴侣和重金属原子浸泡，但这些尝试仅取得了有限的进展。最终，使用碘浸泡和使用结晶伴侣麦芽糖结合蛋白（MBP）作为搜索模型的分子置换的组合解决了结构问题。对天然和 MBP 标记的 CAMP 因子结构的分析确定了 C 末端结构域（CTD）中的保守相互作用界面。带正电荷的表面可能对于与酸性配体的结合至关重要。此外，CTD 上相互作用界面的突变完全消除了其协同溶血活性。本研究为 CAMP 因子的膜透性活性的机制提供了新的见解。
