期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：2
页码：E127-E136
DOI：10.1073/pnas.1418450112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceBactofilins are a new class of cytoskeletal proteins that are involved in key cellular processes. For instance, in the human pathogen Helicobacter pylori, they are responsible for maintaining its characteristic helical cell shape, a feature required for cells to efficiently colonize the gastric mucus. So far the atomic structure of bactofilin filaments has remained elusive, as the large bactofilin assemblies are not amenable to standard methods for 3D structure determination. Here, we have applied a combination of solid-state NMR and electron microscopy and discovered that bactofilins adopt a {beta}-helical architecture, which has not been observed before for other cytoskeletal filaments. Interestingly, however, the structure bears similarities to that of the fungal prion protein HET-s. Bactofilins are a widespread class of bacterial filament-forming proteins, which serve as cytoskeletal scaffolds in various cellular pathways. They are characterized by a conserved architecture, featuring a central conserved domain (DUF583) that is flanked by variable terminal regions. Here, we present a detailed investigation of bactofilin filaments from Caulobacter crescentus by high-resolution solid-state NMR spectroscopy. De novo sequential resonance assignments were obtained for residues Ala39 to Phe137, spanning the conserved DUF583 domain. Analysis of the secondary chemical shifts shows that this core region adopts predominantly {beta}-sheet secondary structure. Mutational studies of conserved hydrophobic residues located in the identified {beta}-strand segments suggest that bactofilin folding and polymerization is mediated by an extensive and redundant network of hydrophobic interactions, consistent with the high intrinsic stability of bactofilin polymers. Transmission electron microscopy revealed a propensity of bactofilin to form filament bundles as well as sheet-like, 2D crystalline assemblies, which may represent the supramolecular arrangement of bactofilin in the native context. Based on the diffraction pattern of these 2D crystalline assemblies, scanning transmission electron microscopy measurements of the mass per length of BacA filaments, and the distribution of {beta}-strand segments identified by solid-state NMR, we propose that the DUF583 domain adopts a {beta}-helical architecture, in which 18 {beta}-strand segments are arranged in six consecutive windings of a {beta}-helix.
关键词：solid-state NMR ; cytoskeleton ; protein structure ; filaments
