Unravelling the molecular basis of subunit specificity in bacterial pilus assembly mechanisms

半个多世纪以来，人们已经知道一些致病菌（如大肠杆菌和肠炎沙门氏菌）在其表面产生毛发状结构，促进血液凝集。这些“菌毛”或“菌毛”的尖端含有一种特殊的蛋白质分子，称为黏附素，它允许细菌附着在宿主细胞表面，从而启动感染过程。这些毛发状结构是由数百个具有相似结构的蛋白质亚基拷贝组装成一个长长的聚合物链，通过每个亚基之间的非共价相互作用连接而成。一种复杂的折叠和组装机制，称为伴侣-引导通路，负责这些菌毛的组装，以尿路致病性大肠杆菌的P菌毛为典型。该途径的关键成分包括(i)一个特定的伴侣蛋白，它需要将毛囊亚基折叠成一个能够组装的构象，并防止亚基在质周空间过早组装；（ii）毛菌亚基本身，在毛杆菌的情况下，涉及六种不同的亚基类型；（iii）外膜嵌入的引导蛋白，它作为组装平台，将伴侣蛋白：菌毛亚基复合物带到生长的菌毛的基端，随后并入生长的纤维中。以一种目前尚不清楚的方式，并且完全独立于ATP， usher催化菌毛组装，在生长的菌毛的基部添加一个明确的，特别选择的亚基，并将其挤压到细菌的外表面。引子也起到锚的作用，将菌毛固定在细菌表面，武装细菌进行攻击。近年来，一些毛蕊伴侣的x射线结构已经被阐明，以及许多毛蕊亚基（称为毛蕊）。令人着迷的是，这些研究表明，pilins具有基于免疫球蛋白（Ig）折叠的共同结构。然而，虽然标准的Ig折叠包含七个？而柱状结构只有6股，因此结构不完整且不稳定。监护人的一个角色是捐赠一枚？-链转移到菌毛亚基，暂时完成-链的内容。在皮勒斯集会期间，监护人？然后-stand被进入的pilin亚基从其结合位点移位，形成新的？从其最初无序的n端区域（称为n端延伸（Nte））中分离出-链，从而形成非常稳定的分子间Ig分子链。细菌毛的有序组装是结构生物学和分子识别领域的一个重要问题，具有深远的影响。首先，它提出了关于分子自组装机制的重要基本问题，并询问这些问题在多大程度上由氨基酸链的生物物理特性（其序列，或相互作用的动力学或热力学）决定，以及这些特性如何在体内被控制、调节和/或协调。其次，同样重要的是，阐明菌毛形成的分子机制对开发新的抗菌剂具有重要意义，可以对抗由菌毛介导的细菌感染。在这个提案中，我们描述了一系列的实验涉及三个申请人与互补的专业知识，旨在揭示蛋白质如何组装成毛前所未有的细节。具体来说，我们的目的是确定n端延伸（Nte），伴侣：亚基复合物和膜结合的可溶性n端结构域在定义和控制亚基组装顺序中的作用。最后，利用我们纯化完整功能usher蛋白的能力，我们的目标是开发一种能够首次深入了解体外膜表面菌毛组装的检测方法。

It has been known for more than half a century that some pathogenic bacteria (e.g. Escherichia coli and Salmonella enterica) produce hair-like structures on their surfaces that promote haemagglutination. These 'pili' or 'fimbriae' contain at their tip a special protein molecule, called an adhesin, which allows the bacteria to attach to the host cell surface, thereby initiating the process of infection. These hair-like structures are formed from the assembly of hundreds of copies of protein subunits with similar structure into a long polymer chain linked by non-covalent interactions between each subunit. A complex folding and assembly machinery known as the chaperone-usher pathway is responsible for the assembly of these pili, typified by the P pili of uropathogenic E. coli. Key components of this pathway include (i) a specific chaperone which is needed to fold the pilus subunit into an assembly-competent conformation and to prevent premature subunit assembly in the periplasmic space; (ii) the pilus subunits themselves which, in the case of P pili, involves six different subunit types; and (iii) an outer membrane-embedded usher protein, which acts as the assembly platform where chaperone:pilus subunit complexes are brought to the basal end of the growing pilus for subsequent incorporation into the growing fibre. In a manner that is currently not understood, and is entirely independent of ATP, the usher catalyzes pilus assembly, adds a defined, specially chosen subunit to the base of the growing pilus, and extrudes it to the outer surface of the bacterium. The usher also serves as an anchor, tethering the pilus to the bacterial surface, arming the bacterium for attack. In recent years the X-ray structures of several pilus chaperones have been elucidated, along with many pilus subunits (known as pilins). Fascinatingly, these studies have shown that pilins have a common structure, based on an immunoglobulin (Ig) fold. However, whilst the canonical Ig fold contains seven ?-strands, the pilins have only six strands and their structure is thus incomplete and unstable. One role of the chaperone is to donate a ?-strand to the pilus subunit, temporarily completing its content of ?-strands. During pilus assembly the chaperone's ?-stand is then displaced from its binding site by the incoming pilin subunit, which forms a new ?-strand from its initially disordered N-terminal region (known as the N-terminal extension (Nte)), resulting in a very stable, intermolecular chain of Ig molecules. The ordered assembly of bacterial pili provides a fascinating problem in structural biology and molecular recognition that has far-reaching impact. First, it poses important fundamental questions about molecular self-assembly mechanisms and asks to what extent these are dictated by the biophysical properties of the amino acid chain (its sequence, or the kinetics or thermodynamics of the interactions) and how these are controlled, modulated and/or coordinated in vivo. Secondly, and equally importantly, elucidation of the molecular mechanism of pilus formation has immense importance for the possible development of new anti-microbial agents against bacterial infection mediated by pili. In this proposal we describe a series of experiments involving three applicants with complementary expertise that aim to reveal how proteins assemble into pili in unprecedented detail. Specifically, our aims are to determine the role of the N-terminal extension (Nte), the chaperone:subunit complex, and the soluble N-terminal domain of the membrane-bound usher in defining and controlling the order of subunit-assembly. Finally using our ability to purify intact functional usher protein we aim to develop an assay capable of providing the first insights into pilus assembly at a membrane surface in vitro.