Type IV pili and pseudopili: structure, dynamics, assembly and molecular function – FiberSpace

Type 2 secretion pseudopili and type 4 pili (T4P) are bacterial fibers stabilized by non covalent weak and flexible bonds. They are anchored in the membrane and dynamic, in a permanent state of assembly or (in the case of T4P) disassembly. To determine their structure and understand the molecular basis of their dynamics and function, we have adopted an integrative and multidisciplinary approach. Owing to the complementary expertise of four partners, we are performing in parallel and in synergy the in vivo, in vitro and in silico analyses. The Partner 1 team (Francetic) is studying the capacity of bacteria and their mutants to assemble fibers, by using biochemical fractionation methods or immunofluorescence microscopy. This partners is studying the membrane assembly complex by co-purification, cross linking and bacterial two-hybrid assay to define inter-protomer interfaces. Partner 2 (Nilges team) has developed methods to integrate distance information (experimentally determined by NMR, cross-linking, site-directed mutagenesis, coevolution and other methods) to generate structural models of proteins or their polymers. The expertise of Parter 3 team (Izadi-Pruneyre) is essential for structural analysis and determination of protein components of fibers and assembly complex by solution NMR. This method allows us to study conformational changes and interactions between biomolecules in real time. Finally, the expertise of Partner 4 (Egelman lab) in high-resolution cryo-electron microscopy provides us with information on fiber structure and is essential for the success of this project.

Important advances of the program include structure determination of the major pseudopilin PulG by NMR and characterization of its calcium-dependent folding and stability in vivo and in vitro. CryoEM reconstruction of pseudopilus fibers at <5 Å resolution was combined with a novel modeling strategy to fit NMR and cryoEM data, providing us with the atomic structure of the PulG pseudopilus. This strutcure revealed unfolding of central region of the PulG stem, suggesting a novel conformational change coupled to fibre assembly. The role of calcium in fibre stability suggests a mechanism of pseudopilus length control.
Studies of PulG interaction with assembly factors PulM and PulF have revealed conserved pseudopilin residues that determine interaction with PulM as well as PulG binding to the membrane. A novel role for the N-methylation of fibre subunits has been proposed. Soluble domains of three minor pseudopilins and PulM have been purified for NMR, interaction studies and X-ray crystallography.
The T4P assembly system has been reconstituted in Escherichia coli and allowed us to identify the effect of assembly system on the structure of assembled fibres. The struture of PpdD T4P subunit has been determined by NMR and residues involved in inter-molecular interactions have been identified by BAC2H and functional studies. Ongoing cryoEM analysis of PpdD pili assembled by two different nanomachines will provide us with high-resolution structures of fibres with two distinct conformations. The discovery of the major structural role of calcium in PulG but also in PpdD folding opens up perspectives for further studies. Calcium role in the stabilization of the pseudopilin complex will be analysed by biophysical methods (P1 and P3).
The functional reconstitution of the E. coli T4P assembly system opens the way to more extensive comparative analysis of pili assembled by the two machineries - T4P and T2SS, and also to the analysis of T4P function in motility, DNA uptake, etc.

The results obtained so far open numerous research directions, in particular concerning the role of calcium in the biogenesis and function of these systems. The reconstitution of the T4P assembly machinery is an important achievement, which allowed us to study a system from a class 3 pathogen in the safe E. coli K-12 background. It also allowed us to observe differences between pili assembled by a heterologous T2SS and the native reconstituted T4P system. These differences so far characterized by fluorescence microscopy are awaiting detailed cryoEM analyses, structural modeling and biological validation.

Original and review articles in peer rieviewed journals:

1. 1H, 15N and 13C resonance assignments and secondary structure of PulG, the major pseudopilin from Klebsiella oxytoca type 2 secretion system. López-Castilla A, Vitorge B, Khoury L, Morellet N, Francetic O, Izadi-Pruneyre N. Biomol NMR Assign. 2017 Mar 3. doi: 10.1007/s12104-017-9738-7. PMID: 28258547
2. Nivaskumar, M., Santos-Moreno, J., Malosse, C., Nadeau, N., Chamot-Rooke, J., Tran Van Nhieu, G. and Francetic, O. (2016) Pseudopilin residue E5 is essential for recruitment by the type 2 secretion system assembly platform. Molecular Microbiology 101(6):924-41.
3. Santos-Moreno J, East A, Guilvout I, Nadeau N, Bond PJ, Tran Van Nhieu G, Francetic O. (2017) Polar N-terminal residues conserved in type 2 secretion pseudopilins determine subunit targeting and membrane extraction steps during fibre assembly. Journal of Molecular Biology, 2017 Jun 2;429(11):1746-1765.
4. Thomassin JL, Santos Moreno J, Guilvout I, Tran Van Nhieu G, Francetic O. (2017) The trans-envelope architecture and function of the type 2 secretion system: new insights raising new questions. Molecular Microbiology May 9. doi: 10.1111/mmi.13704. [Epub ahead of print] Review.
5. Maffei B, Francetic O, Subtil A. (2017) Tracking Proteins Secreted by Bacteria: What's in the Toolbox? Front Cell Infect Microbiol. 2017 May31;7: 221.doi: 10.3389/f cimb.2017. 00221. eCollection 2017.

Microorganisms shape their environment using sophisticated devices that allow them to transport molecules across their cell envelope or assemble surface organelles providing substrate attachment and motility. Some of the most ancient and powerful biological machines are members of the cytoplasmic membrane fiber (CMF) family, found in all domains of life. In bacteria, CMF members - the type II protein secretion (T2SS) and type IV pilus (T4P) assembly systems - play crucial roles in adaptation and virulence. Both systems use ATPase motors to assemble helical fibers from membrane protein subunits called pilins. Fiber dynamics and surface properties determine their specific functions: in T2SS periplasmic pseudopilus fibrils drive transport of folded periplasmic proteins across the outer membrane channels, while T4P are long surface fibers that promote bacterial adherence, aggregation, motility, cell signaling, DNA or protein transport. Highly relevant from medical, environmental and biotechnological viewpoints, these systems are studied in dozens of laboratories worldwide. However, mechanisms underlying their functions have remained elusive due to their complexity, dynamic nature and membrane localization.
Recently, we have developed a powerful integrative methodology that has provided molecular insights into several steps crucial for CMF function. We determined the detailed structure of the T2SS pseudopilus and described the initiation steps of assembly involving the minor fiber subunits. We developed an original neural network-based approach to analyze and compare large sets of structures and study detailed conformational changes. Functional analysis based on these structural predictions allowed us to define distinct pseudopilus assembly steps and their functions, which suggest a rotational mechanism of CMF assembly.
Here we propose to elucidate the fiber assembly mechanism and unravel the molecular basis of fiber dynamics and function, integrating the information obtained by high-resolution electron microscopy, solution NMR, in silico modeling, site-directed mutagenesis, biochemical and functional in vivo analysis.
Using enterobacterial T2SS and T4P as experimental models we will address the following questions: (1) What are the molecular determinants of T4P assembly and stability? What is the detailed structure of the T4P and what are the conformational transitions involved in fiber dynamics? What are the functional determinants of fiber surface and what is the molecular basis of binding specificity? (2) What is the composition and structure of native periplasmic pseudopili? How do minor pseudopilin subunits activate ATP dependent fiber elongation? Do minor pseudopilins remain associated with the fibers and do they play a late functional role? (3) What is the composition and organization of the fiber assembly platform? Which components interact directly with pilin subunits and how is ATP hydrolysis energy transduced to promote fiber assembly? Is the assembly platform a rotary motor? (4) We will develop a set of modeling and structural analysis tools that would be widely applicable to the analysis of flexible and dynamic biological complexes.
This project is built on the top-level complementary expertise of 3 teams in the Institut Pasteur in T2SS biology and functional analysis (Partner 1), structural modeling and molecular dynamics (Partner 2) and NMR structural and interaction analysis (Partner 3) and includes Partner 4, Edward Egelman at the University of Virginia, USA, one of the world-leading experts in electron microscopic analysis of protein fibers. The wealth of preliminary results and the synergy between the teams is the guarantee of success of this study. The results of these studies will greatly improve our fundamental understanding of these widespread and important biological machines and will have important medical and biotechnological implications.