Hybrid Structure of the Type 1 Pilus of Uropathogenic Escherichia coli

Abstract

Type 1 pili are filamentous protein assemblies on the surface of Gram‐negative bacteria that mediate adhesion to host cells during the infection process. The molecular structure of type 1 pili remains elusive on the atomic scale owing to their insolubility and noncrystallinity. Herein we describe an approach for hybrid‐structure determination that is based on data from solution‐state NMR spectroscopy on the soluble subunit and solid‐state NMR spectroscopy and STEM data on the assembled pilus. Our approach is based on iterative modeling driven by structural information extracted from different sources and provides a general tool to access pseudo atomic structures of protein assemblies with complex subunit folds. By using this methodology, we determined the local conformation of the FimA pilus subunit in the context of the assembled type 1 pilus, determined the exact helical pilus architecture, and elucidated the intermolecular interfaces contributing to pilus assembly and stability with atomic detail.

Biomolecular solid-state NMR spectroscopy at highest field: the gain in resolution at 1200 MHz

“…Each monomer consists of 150 amino acids. Using 850 MHz data, the 13 C resonances have been assigned to 98% of the sequence (Habenstein et al 2015). A clear improvement in resolution at the higher field is observed in the expanded regions shown in Figure2c.…”

“…As a second system, we show adhesive type 1 pili from E.coli, which assemble in vitro to form long supramolecular protein filaments (Hahn et al 2002;Habenstein et al 2015). Each monomer consists of 150 amino acids.…”

“…In the following, we compare spectra of amyloid fibrils of the fungal prion HET-s(218-289) (Wasmer et al 2008;van Melckebeke et al 2010); sediments of the bacterial helicase DnaB (Gardiennet et al 2012;Wiegand et al 2019); the bacterial RNA helicase and acetyltransferase TmcA (Ikeuchi, Kitahara, and Suzuki 2008;Chimnaronk et al 2009); the Rpo4/7 protein complex of two subunits of archeal RNA polymerase II (Torosyan et al 2019); the filaments of PYRIN domain of mouse ASC (Sborgi et al 2015;Ravotti et al 2016); the viral capsids of the Hepatitis B virus (Lecoq et al 2019) and the African cichlid nackednavirus (Lauber, Seitz at al. 2017); supramolecular protein filaments of type 1 pili (Hahn et al 2002;Habenstein et al 2015); and the nonstructural membrane protein 4B (NS4B) of the Hepatitis C virus. In all figures, spectra colored in blue were recorded at 850 MHz, and spectra in red at 1200 MHz.…”

“…While the type 1 pili and HET-s(218-289) were small enough for assignment and structure determination at 850 MHz (Wasmer et al 2008;van Melckebeke et al 2010) (Habenstein et al 2015), the DnaB helicase from Helicobacter pylori (6 x 59 kDa) with 488 residues per monomer poses a big challenge at 850 MHz, already for assignment. Divide-and-conquer approaches Wiegand, Gardiennet, Ravotti, et al 2016) or segmental isotope labeling of individual protein domains have thus been applied (Wiegand 2018), however without reaching close-to-complete assignment.…”

Solid-State NMR: Methods for Biological Solids

“…These filametous structures are highly symmetrical assemblies formed from a single protein and give rise to high quality NMR spectra due to their homogeneity . The structure of this needle was solved using a combination of cryo-EM and ssNMR techniques, where NMR played a critical role in deciphering the conformation of the monomeric unit in the filament. , The structures of the type-1 Pilus of E. coli , multimeric structures formed by the protein FimA, were also determined using a combination of solution-state NMR, ssNMR, and STEM techniques . The increased sensitivity of DNP has also been used to obtain the ssNMR spectra of Acinetobactor 205 nucleocapsid and refine the 3D structure of the protein in the context of the capsid …”