Cryo-EM Structure of the Type IV Pilus Extension ATPase from Enteropathogenic Escherichia coli.
Cryo-EM Structure of the Type IV Pilus Extension ATPase from Enteropathogenic Escherichia coli.
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4型皮利（T4 P）是在许多细菌和古细菌上发现的可伸缩表面附属物，在各种微生物功能（包括病原体的宿主定殖）中发挥重要作用。ATP酶是T4 P延伸所必需的，但是将化学能转换为机械能以用于菌毛延伸的机制尚未阐明。在这里，我们报告的冷冻电镜（cryo-EM）结构的BfpD ATP酶从肠致病性大肠杆菌（EPEC）的存在下，无论是ADP或ADP和AMP-PNP的混合物。这两种结构，在3 μ m分辨率下解析，揭示了AAA+ ATP酶的典型环形形状和明确的6重对称性。这种6重对称性与先前报道的其他T4 P延伸ATP酶结构的2重对称性形成对比，所有这些结构都来自嗜热菌并通过晶体学解决。在核苷酸混合物的存在下，BfpD仅结合AMP-PNP，并且与ADP存在下的结构相比，这种结合导致适度的向外扩张，表明水解的协调模型。从头分子模型揭示了所有亚基的部分开放构型，其中核苷酸结合位点可能不是催化的最佳位置。ATP酶功能研究显示，适度的活动类似于其他延伸ATP酶，而计算表明，这种活动是不足以权力菌毛延伸。我们的研究结果表明，尽管在一级序列和三级结构的相似性，T4 P延伸ATP酶表现出不同的四级结构。我们的数据提出了新的可能性的机制，T4 P延伸ATP酶的权力菌毛的形成。
Type 4 pili (T4P) are retractable surface appendages found on numerous bacteria and archaea that play essential roles in various microbial functions, including host colonization by pathogens. An ATPase is required for T4P extension, but the mechanism by which chemical energy is transduced to mechanical energy for pilus extension has not been elucidated. Here, we report the cryo-electron microscopy (cryo-EM) structure of the BfpD ATPase from enteropathogenic Escherichia coli (EPEC) in the presence of either ADP or a mixture of ADP and AMP-PNP. Both structures, solved at 3 Å resolution, reveal the typical toroid shape of AAA+ ATPases and unambiguous 6-fold symmetry. This 6-fold symmetry contrasts with the 2-fold symmetry previously reported for other T4P extension ATPase structures, all of which were from thermophiles and solved by crystallography. In the presence of the nucleotide mixture, BfpD bound exclusively AMP-PNP, and this binding resulted in a modest outward expansion in comparison to the structure in the presence of ADP, suggesting a concerted model for hydrolysis. De novo molecular models reveal a partially open configuration of all subunits where the nucleotide binding site may not be optimally positioned for catalysis. ATPase functional studies reveal modest activity similar to that of other extension ATPases, while calculations indicate that this activity is insufficient to power pilus extension. Our results reveal that, despite similarities in primary sequence and tertiary structure, T4P extension ATPases exhibit divergent quaternary configurations. Our data raise new possibilities regarding the mechanism by which T4P extension ATPases power pilus formation.
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