European Commission logo
italiano italiano
CORDIS - Risultati della ricerca dell’UE

3D structures of bacterial supramolecular assemblies by solid-state NMR

Final Report Summary - ASSEMBLYNMR (3D structures of bacterial supramolecular assemblies by solid-state NMR)

In the last decade, solid-state NMR has emerged as a powerful technique in structural biology as it gives access to structural information for systems that are insoluble or do not crystallize easily. Furthermore, the technique allows for the characterization of chemical details (e.g. protonation of side chains), the interaction with water and/or lipid molecules, and functionally important protein dynamics. For solid-state NMR investigations, samples are placed in a strong superconducting magnet (external field up to 20 T, i.e. ~400,000 times stronger than the earth’s magnetic field), spun rapidly (up to 100,000 rotations per second; magic-angle spinning), and probed by radio waves. Important applications concern membrane proteins in the context of native-like liposomes and the 3D atomic structure determination of molecular machines.
In our ERC project we focused on the characterization of protein structure, dynamics and function of three different bacterial systems of interest: The bacteria-specific cytoskeletal element bactofilin (Caulobacter crescentus: subunit name BacA), the type i pilus rod (Uropathogenic E. coli: subunit name FimA), and the type iii secretion system needle (Shigella flexneri: subunit name MxiH). Those systems have in common that the respective subunits polymerize and form supramolecular assemblies that may be part of complex molecular machines. As a consequence of their size and insolubility they pose a significant challenge to the standard techniques for 3D protein structure determination at atomic resolution, which are X-ray crystallography and solution NMR. In contrast, solid-state NMR neither requires solubility nor long-range order and can thus in principle be readily applied to those systems. It was a major goal of this project to demonstrate that structure, dynamics and function of such systems can indeed be characterized by solid-state NMR, possibly in combination with data from cryo-electron microscopy (cryo-EM). Cryo-EM has recently witnessed tremendous progress and it perfectly complements solid-state NMR data (which provide local information on secondary structure, subunit tertiary structure and subunit-subunit intermolecular restraints) by providing global information on the assembly (rigid body positioning of the subunits, symmetry, diameter etc.).

Importantly, we determined the structures of bactofilin and of the type i pilus rod and thereby demonstrated the power of solid-state NMR for the study of supramolecular protein assemblies. In both cases we complemented the data from solid-state NMR with data from other structural methods (solution NMR, electron microscopy). It is now increasingly recognized that hybrid approaches are important tools to address the structures of complex molecular machines and with our studies we could increase the knowledge in this growing research field.

The structure of the type i pilus yielded valuable information on the atomic scale, but in general corroborated/refined existing earlier coarse models. In contrast, the bactofilin structure revealed a very surprising beta-helical fold with six windings. Such a beta-helical architecture had never been observed before for other cytoskeletal filaments. Interestingly, however, the structure bears similarities to that of the fungal prion protein HET-s.