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首页> 外文期刊>Acta crystallographica. Section D, Structural biology. >Structure determination of the CAMP factor of Streptococcus agalactiae with the aid of an MBP tag and insights into membrane-surface attachment
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Structure determination of the CAMP factor of Streptococcus agalactiae with the aid of an MBP tag and insights into membrane-surface attachment

机译：结构测定的因素链球菌agalactiae MBP的援助标签和见解膜表面附件

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摘要

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 A 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 chaper-ones 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.
机译：营的因素是一个独特的α螺旋形细菌毒素co-hemolytic活动而闻名结合葡萄球菌鞘磷脂酶。这是首次发现在人类病原体链球菌agalactiae(也称为B组链球菌),但同源基因发现在许多其它的革兰氏阳性病原体。这项研究中,导致的努力确定第一个结构CAMP-family毒素报告。本机的可能产生晶体蛋白衍射至接近2.45决议。是在路上遇到的障碍结构的决心。五年,许多方法,包括硒代蛋氨酸标签,突变,结晶chaper-ones和重原子浸泡,未遂,但这些尝试导致进展有限。最后解决了使用碘的组合浸泡和分子替换使用结晶女伴maltose-binding蛋白质(MBP)作为搜索模型。MBP-tagged CAMP-factor结构确定了保存在c端交互界面域(CTD)。被绑定到酸性配体的关键。此外,突变的交互CTD完全废除其接口co-hemolytic活动。新颖的见解的机制membrane-permeabilizing营活动的因素。

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