ESR13 – An integrative strategy for structure determination of bacterial type IV pili

Institut Pasteur, University of Paris (France)
To interact with their environment, bacteria have acquired a number of proteinaceous appendages that decorate their surface. These appendages, such as pili, secretion systems or flagella, are large macromolecular assemblies that participate in adhesion, cell-cell interactions, auto-aggregation, DNA exchange, motility and virulence factor transport. Type IV pili (T4P) are a class of fimbrial adhesins present in numerous pathogenic bacteria and are crucial for host colonization and virulence of many gram-negative bacteria. T4P assembly system is very closely related to type II protein secretion systems (T2S) that are involved in the transport of folded proteins from the periplasm across the outer membrane. The common architecture of T4P and T2S is based on the assembly of helical fibers in the plasma membrane with membrane protein subunits called pilins. Other proteins called minor pilins are crucial for the initiation of this assembly. However, their molecular complexity, their dynamics, and membrane localization seriously hamper structural characterization of these fibers at atomic resolution with traditional methods (X-ray crystallography, NMR, and EM). It has become clear that a combination of structural information from many sources is the key to success. In this project, we will implement an integrative strategy to determine the structure of a T4P, improving upon our work on T2S (see Campos et al., PNAS 107, 13081-13086 (2010); Campos et al., J Struct Biol. 173, 436-444 (2011); Campos et al., Structure 22, 685-696 (2014). First, the high-resolution structure of the soluble domain of the major pilin will be determined by NMR. In a second stage, helical parameters of pili will be obtained by EM and this data will be then combined with the pilin structure and co-evolutionary data to construct a model of the assembled pilus using a flexible molecular modeling approach. Finally, to better understand the fiber assembly mechanism, molecular interactions between major and minor pilins will be characterized by NMR and other biophysical techniques (Microcalorimetry, SPR and fluorescence). The project involves secondments with other network partners for labeled protein production (Tubingen University), training in cryo-EM data analysis (University of Helsinki) and at the Centre for Nanotechnology and smart materials (CeNTI) in Portugal to design antiadhesive surfaces.