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

Abstract

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 highresolution 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.solid-state NMR | cytoskeleton | protein structure | filaments

Solid-state NMR: An emerging technique in structural biology of self-assemblies

“…Given the exact 4.7 Å spacing between β-strands in β-sheets arrangement, MPL can provide the x-foldsymmetry in case of multiple subunits constituting one β-sheet spacing, for instance for x = 2 or 3 (Ab amyloid fibrils [48,171]) or x = 0.5 (HET-s prion domain [172]). SsNMR and STEM MPL data were efficiently combined to determine the 3D atomic arrangement of several diseaserelated amyloid fibrils, such as several Alzheimer's β-amyloid polymorphs and mutants [48,51,173] or the cytoskeletal bactofilin [7].…”

“…Before reaching the atomic structure determination, amyloids can be characterized by several general structural features: the extent and location of residues involved in the amyloid core, the parallel or antiparallel, in-register or out-of-register arrangements of the β-strands in the axial direction and the straight, left-handed or right-handed twisted appearance. Protein assembly into amyloid fibrils can either be pathological such as Aβ and Tau in Alzheimer's, α-synuclein in Parkinson's, the prion protein PrP in Creutzfeldt-Jacob disease and the islet amyloid polypeptide (IAPP) in type 2 diabetes or functional [183], as has been shown for proteins involved in fungal heterokaryon incompatibility [184], mammalian skin pigmentation [185] and hormone storage [186], mammalian and fungal signaling pathways [9][10][11]187], bacterial cell-cell contacts, biofilms and cytoskeleton architecture [7,188,189]. Several prions and prion domains have also been identified as amyloid folds leading to characteristic phenotypes in yeast using ssNMR [53,54,56,[190][191][192].…”

“…The morphologies of the self-assemblies are diverse; however, fibrils, filaments, tubes, capsids, pores, rings or large nanoparticles are commonly observed. Among the wide range of molecular assemblies observed in living cells, self-assemblies made of protein subunits are ubiquitous and constitute complex functional entities such as bacterial secretion systems [1,2], virus capsids [3][4][5], cytoskeleton bactofilin filaments [6,7] or apoptosis signal transducers [8][9][10][11]. Nevertheless, they can also occur as undesired and deleterious side products such as amyloid deposits in neurodegenerative disorders [12,13], or infective material in the propagation of abnormal protein folds in prion diseases [14][15][16].…”

“…Protein samples ranging from micro- [28] to nano-crystals [29], fibrils [30,31], sedimented samples [32][33][34], membrane proteins embedded in lipids [35] to proteins in a physiological cellular environment [36], or attached to cellular components [37,38] can currently be investigated using multidimensional MAS ssNMR methods. Structural investigations on protein self-assemblies are increasingly reported such as on amyloids (functional [9,22,39] and deleterious [40][41][42][43][44][45][46][47][48][49][50][51]), prions [52][53][54][55][56][57][58][59], phages [25,[60][61][62], bacterial appendages [24,26,[63][64][65] and cytoskeleton filaments [7]. As shown in Fig.…”

Bacteriophage Tail‐Tube Assembly Studied by Proton‐Detected 4D Solid‐State NMR

“…Solid-state NMR is as pectroscopic technique that can be applied to investigate the structure and dynamics of insoluble proteins,i ncluding amyloid fibrils, [1,2] functional supramolecular assemblies, [3,4] and membrane-integrated proteins, [5] at atomic resolution. [6][7][8] In contrast to other methods,solid-state NMR does not require long-range order and can operate with proteins under in vivo conditions,f or instance membrane proteins can be studied in lipid bilayers [9,10] or in nanodiscs.…”