Chaowei Shi

β-Helical architecture of cytoskeletal bactofilin filaments revealed by solid-state NMR

Significance Bactofilins 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 β-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 β-sheet secondary structure. Mutational studies of conserved hydrophobic residues located in the identified β-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 β-strand segments identified by solid-state NMR, we propose that the DUF583 domain adopts a β-helical architecture, in which 18 β-strand segments are arranged in six consecutive windings of a β-helix.

Atomic-resolution structure of cytoskeletal bactofilin by solid-state NMR

The first atomic structure of a member of a recently discovered class of cytoskeletal proteins was revealed. Bactofilins are a recently discovered class of cytoskeletal proteins of which no atomic-resolution structure has been reported thus far. The bacterial cytoskeleton plays an essential role in a wide range of processes, including morphogenesis, cell division, and motility. Among the cytoskeletal proteins, the bactofilins are bacteria-specific and do not have a eukaryotic counterpart. The bactofilin BacA of the species Caulobacter crescentus is not amenable to study by x-ray crystallography or solution nuclear magnetic resonance (NMR) because of its inherent noncrystallinity and insolubility. We present the atomic structure of BacA calculated from solid-state NMR–derived distance restraints. We show that the core domain of BacA forms a right-handed β helix with six windings and a triangular hydrophobic core. The BacA structure was determined to 1.0 Å precision (heavy-atom root mean square deviation) on the basis of unambiguous restraints derived from four-dimensional (4D) HN-HN and 2D C-C NMR spectra.

Structural heterogeneity in microcrystalline ubiquitin studied by solid-state NMR

By applying [1‐13C]‐ and [2‐13C]‐glucose labeling schemes to the folded globular protein ubiquitin, a strong reduction of spectral crowding and increase in resolution in solid‐state NMR (ssNMR) spectra could be achieved. This allowed spectral resonance assignment in a straightforward manner and the collection of a wealth of long‐range distance information. A high precision solid‐state NMR structure of microcrystalline ubiquitin was calculated with a backbone rmsd of 1.57 to the X‐ray structure and 1.32 Å to the solution NMR structure. Interestingly, we can resolve structural heterogeneity as the presence of three slightly different conformations. Structural heterogeneity is most significant for the loop region β1‐β2 but also for β‐strands β1, β2, β3, and β5 as well as for the loop connecting α1 and β3. This structural polymorphism observed in the solid‐state NMR spectra coincides with regions that showed dynamics in solution NMR experiments on different timescales.

A single NaK channel conformation is not enough for non-selective ion conduction.

NaK and other non-selective channels are able to conduct both sodium (Na+) and potassium (K+) with equally high efficiency. In contrast to previous crystallographic results, we show that the selectivity filter (SF) of NaK in native-like lipid membranes adopts two distinct conformations that are stabilized by either Na+ or K+ ions. The atomic differences of these conformations are resolved by solid-state NMR (ssNMR) spectroscopy and molecular dynamics (MD) simulations. Besides the canonical K+ permeation pathway, we identify a side entry ion-conduction pathway for Na+ permeation unique to NaK. Moreover, under otherwise identical conditions ssNMR spectra of the K+ selective NaK mutant (NaK2K) reveal only a single conformational state. Therefore, we propose that structural plasticity within the SF and the selection of these conformations by different ions are key molecular determinants for highly efficient conduction of different ions in non-selective cation channels.NaK is a non-selective cation channel that conducts sodium (Na+) and potassium (K+) equally well. Here authors use ssNMR and MD simulations to show that the selectivity filter of NaK adopts two conformations in the absence of ions, one of which is preferred by Na+ and the other by K+.

Structure determination of supra-molecular assemblies by solid-state NMR: Practical considerations

In the cellular environment, biomolecules assemble in large complexes which can act as molecular machines. Determining the structure of intact assemblies can reveal conformations and inter-molecular interactions that are only present in the context of the full assembly. Solid-state NMR (ssNMR) spectroscopy is a technique suitable for the study of samples with high molecular weight that allows the atomic structure determination of such large protein assemblies under nearly physiological conditions. This review provides a practical guide for the first steps of studying biological supra-molecular assemblies using ssNMR. The production of isotope-labeled samples is achievable via several means, which include recombinant expression, cell-free protein synthesis, extraction of assemblies directly from cells, or even the study of assemblies in whole cells in situ. Specialized isotope labeling schemes greatly facilitate the assignment of chemical shifts and the collection of structural data. Advanced strategies such as mixed, diluted, or segmental subunit labeling offer the possibility to study inter-molecular interfaces. Detailed and practical considerations are presented with respect to first setting up magic-angle spinning (MAS) ssNMR experiments, including the selection of the ssNMR rotor, different methods to best transfer the sample and prepare the rotor, as well as common and robust procedures for the calibration of the instrument. Diagnostic spectra to evaluate the resolution and sensitivity of the sample are presented. Possible improvements that can reduce sample heterogeneity and improve the quality of ssNMR spectra are reviewed.