Electron cryo-microscopy reveals the structure of the archaeal thread filament

Abstract

Pili are filamentous surface extensions that play roles in bacterial and archaeal cellular processes such as adhesion, biofilm formation, motility, cell-cell communication, DNA uptake and horizontal gene transfer. The model archaeaon Sulfolobus acidocaldarius assembles three filaments of the type-IV pilus superfamily (archaella, archaeal adhesion pili and UV-inducible pili), as well as a so-far uncharacterised fourth filament, named "thread". Here, we report on the cryo-EM structure of the archaeal thread. The filament is highly glycosylated and consists of subunits of the protein Saci_0406, arranged in a head-to-tail manner. Saci_0406 displays structural similarity, but low sequence homology, to bacterial type-I pilins. Thread subunits are interconnected via donor strand complementation, a feature reminiscent of bacterial chaperone-usher pili. However, despite these similarities in overall architecture, archaeal threads appear to have evolved independently and are likely assembled by a distinct mechanism.

Fig. 1. Helical reconstruction and atomic model of the thread.

a Segmented surface representation of the cryoEM map showing each subunit in a different colour. Glycan densities are shown in white (white arrowheads). b The atomic model of one Saci_0406 subunit (rainbow) fitted into the cryoEM map (transparent grey) N-terminus, blue; C-terminus, red. Glycans not shown for simplicity. c Intermolecular isopeptide bond is shown as a dashed purple line. The N-terminal residue Asp24 of a subunit (n + 2; carbons in ice blue) appears to be covalently bound to Asn57 two subunits along the chain (n; carbons in gold). d Atomic model of 5 consecutive Saci_0406 subunits in ribbon representation. Glycans are not shown for simplicity. A white arrow indicates the location of the donor strand complementation between the N-terminal tail domain of subunit (n) and the C-terminal head domain of the previous subunit in the chain (n − 1). Red arrows indicate the location of isopeptide bonds. The tail domain of each subunit n is partially buried in the two consecutive subunits n − 1 and n − 2. e Atomic model of the thread filament with glycans in stick representation. f–j Closeups of the five glycosylation sites found in Saci_0406. Atomic models of the glycans are in stick, the polypeptide backbone in ribbon representation. The CryoEM density map is shown as grey mesh. k, l Closeups of map and fitted atomic model demonstrating the quality of the data. The colour scheme is as in C, chain (n-1) shows carbons in coral. Scale bar 20 Å.

Fig. 2. Threads form cables of parallel or antiparallel filaments.

a CryoEM map of a thread cable at 13 Å resolution. Atomic models of the thread (in cartoon representation) can be fitted into the map in parallel (b, c) or antiparallel orientation (d). Shape complementarity between parallel and antiparallel threads is very similar (c, d). Glycans (sticks) mediate the interaction between the threads in the cable. A and B are in the same orientation. c and d are rotated by 150° with respect to a and b. Scale bar 40 Å.

Fig. 3. Threads are conserved in crenarchaeal relatives.

a Phylogenetic tree of species where Saci_0406 homologues have been identified. b Saci_0406 (purple) compared with Alphafold2 predictions of homologues found in V. moutnovskia (blue) A. hospitalis (pink). c Superimposing the structures from B shows their similarity.Pairwise RMSD values calculated between Saci_0406 and the thread homologues from V. moutnovskia (d) and A. hospitalis (e). Blue indicates low and red high values. Average value RMSD is 2.18 Å for D and 2.63 Å for E. Scale bar 20 Å.

Fig. 4. Hypothetical models for thread assembly.

Saci_0406 precursor proteins are initially secreted through the SEC translocon (1). Signal peptidase 1 (SP1) cleaves the N-terminal signal peptide (2). The protein is glycosylated by AglB (3; glycans shown as black sticks). Soluble, mature Saci_0406 proteins assemble into the thread via donor strand complementation (4, 5). This process may be spontaneous or catalysed by an assembly machinery. The putative cap protein Saci_0405 is not predicted to be secreted through SEC. Its location in the thread depends on the polarity of the filament, which is currently unknown. If the Saci_0406 tails point away from the cell, Saci_0405 forms a cell proximal cap that terminates thread assembly (hypothesis 1). If the Saci_0406 tails point toward the cell, Saci_0405 forms a cell distal cap, which initiates thread assembly (hypothesis 2).