Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, VT 05405, USA.
J Struct Biol. 2012 Feb;177(2):439-46. doi: 10.1016/j.jsb.2011.11.024. Epub 2011 Dec 2.
Adhesion to collagen is an important virulence determinant for the periodontal pathogen Aggregatibacter actinomycetemcomitans. Binding to collagen is mediated by the extracellular-matrix protein adhesin-A (EmaA). EmaA is a homotrimeric autotransporter protein that forms flexible antenna-like appendages on the bacterium surface. An ellipsoidal structure at the distal end of the appendage, composed of three subdomains, contains the functional domain of the molecule. A correlation between amino-acid sequence and subdomain structure (SI and SII) was proposed based on an analysis of the volume/molecular weight ratio. EmaA from three mutant strains (deletions of amino-acids 70-206 and 70-386 and a substitution mutation G162S) has been studied by electron microscopy to test this hypothesis. 3D structures were analyzed using single-axis tilt tomography of negatively stained preparations of bacteria combined with subvolume averaging. Additionally, a large number of 2D images of the apical domain of the adhesins from the mutants were extracted from micrographs of the bacterial surface, aligned and classified. The combined data showed that amino-acids 70-206 localize to subdomain SI and 70-386 comprise subdomains SI and SII. Moreover, we showed that the substitution mutation G162S, which abolishes collagen binding activity, does not affect the overall structural integrity of the functional domain. However, the structure of subdomain SI in this mutant is slightly altered with respect to the wild-type strain. These data also have allowed us to interpret the architectural features of each subdomain of EmaA in more detail and to correlate the 3D structure of the functional domain of EmaA with the amino-acid sequence.
黏附在胶原蛋白上是牙周病原体伴放线放线杆菌的一个重要毒力决定因素。结合胶原蛋白是由细胞外基质蛋白黏附素-A (EmaA)介导的。EmaA 是一种同三聚体自转运蛋白，在细菌表面形成灵活的天线状附属物。附属物末端的一个椭圆形结构，由三个亚结构域组成，包含分子的功能域。基于体积/分子量比的分析，提出了氨基酸序列和亚结构域结构（SI 和 SII）之间的相关性。通过电子显微镜研究了来自三个突变株（氨基酸 70-206 和 70-386 的缺失以及 G162S 取代突变）的 EmaA，以检验这一假设。通过对细菌负染色制备物进行单轴倾斜断层摄影术和子体积平均化分析，研究了 3D 结构。此外，从细菌表面的显微照片中提取了大量突变体黏附素顶端结构域的 2D 图像，对其进行了对齐和分类。综合数据表明，氨基酸 70-206 定位于亚结构域 SI，70-386 包含亚结构域 SI 和 SII。此外，我们还表明，导致胶原结合活性丧失的取代突变 G162S 不会影响功能域的整体结构完整性。然而，该突变体中 SI 亚结构域的结构与野生型菌株略有改变。这些数据还使我们能够更详细地解释 EmaA 每个亚结构域的结构特征，并将 EmaA 功能域的 3D 结构与氨基酸序列相关联。
