Research ArticleSTRUCTURAL BIOLOGY

Architecture and regulation of an enterobacterial cellulose secretion system

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Science Advances  27 Jan 2021:
Vol. 7, no. 5, eabd8049
DOI: 10.1126/sciadv.abd8049
  • Fig. 1 The E. coli–like cellulose secretion system and architecture of the Bcs macrocomplex.

    (A) Left: Topology and functional roles of Bcs subunits (NS-EM, negative-stain electron microscopy; CDG, c-di-GMP; REC, receiver; NTPase, nucleoside triphosphatase; TAC, transcription antitermination complex; NTD, N-terminal domain; SIMIBI, signal recognition particle, MinD, and BioD; IM, inner membrane; TMD, transmembrane domain; TA, tail-anchor; CBD, carbohydrate-binding domains; pEtN, phosphoethanolamine; TPR, tetratricopeptide repeats; OM, outer membrane; CTD, C-terminal domain). Right: Structure of an assembled Bcs macrocomplex encompassing most IM and cytosolic subunits as obtained by single-particle NS-EM (1). (B) Cryo-EM 2D class averages of detergent-extracted and affinity-purified Bcs macrocomplex. (C) Density assignments within the assembled Bcs macrocomplex. A hexameric BcsB crown derived from the locally refined BcsBperi pentamer (see below) was fitted in the periplasmic densities. A model of a CDG-bound BcsA was generated by homology modeling in Robetta and was then fitted and refined against the densities corresponding to the BcsRQAB subcomplex, themselves improved by local refinement. The crystal structure of a sandwich BcsR2Q2 dimer was fitted in the apical densities, with BcsR adopting an extended four-turn α1 helix, as observed in some of the crystallized states (see below). A Robetta-derived model of dimeric BcsENTD was fitted in the membrane-proximal densities opposite BcsA. The BcsENTD copies are fitted in head-to-tail orientation consistent with recently reported interaction data (10). Regions corresponding to BcsERec*-GGDEF* did not yield interpretable densities even after local refinement, likely due to conformational heterogeneity (see below). Most BcsB tail-anchors as well as the BcsE-interacting BcsF chains (predicted to fold into a single transmembrane helix each) were not visible in the 3D-reconstructed micelle.

  • Fig. 2 Structure, polymerization, and asymmetry of the periplasmic crown and noncanonical BcsAB synthase tandem assembly.

    (A) Structural comparison of BcsBFL monomers from E. coli (left) and R. sphaeroides (right) shown in surface (top) and cartoon (bottom) representation in PyMOL. pdb, Protein Data Bank. (B) Top: A model of a hexameric BcsB crown derived from the refined BcsBperi pentamer in top and side views. Bottom: A close-up view of the nine-stranded β-sheet formed by β-sheet complementation between adjacent BcsB subunits. (C) Coomassie-stained SDS-PAGE of purified, detergent-extracted, full-length E. coli BcsBHis. (D) Cryo-EM 2D class averages of full-length BcsBHis. (E) Left: Cryo-EM density reconstruction of purified full-length BcsBHis with density-fitted BcsBperi octamers. Right: A composite atomic model of the BcsBFL hexadecamer with visualized IM tail-anchors as observed in the BcsRQAB assembly. Color coding is as in (A). (F) Structure of the BcsRQAB assembly following particle subtraction and local refinement of the cryo–electron density. A single BcsBperi copy was fitted in the periplasmic region, and the amphipathic and transmembrane C-terminal helices were built in the refined density. A CDG-bound BcsA homology model was fitted in the transmembrane and membrane-proximal regions, and the polypeptide backbone was refined against the experimental density. A BcsR2Q2 crystal structure was fitted in the apical region as in Fig. 1. Separate structural modules and ligand molecules are indicated.

  • Fig. 3 Structural and functional analyses of the essential for cellulose secretion subunit BcsQ.

    (A) Structure of a BcsRQHis heterodimer showing the conserved SIMIBI fold (in spectrum-colored cartoon), bound ATP (in black sticks), and Mg++ ion (as a purple sphere). (B) Crystal structure of the ATP-bound BcsRQHis heterocomplex. Right: Coomassie-stained SDS-PAGE of the purified heterocomplex. (C) A close-up view of the ATP-binding pocket. BcsQ subunits are shown as cartoon in blue and lime, with ATP-coordinating residues colored in cyan and tan, respectively. Water molecules and the Mg++ ion are shown as gray and purple spheres, respectively. ATP and key surrounding residues are shown as sticks. (D) Primary sequence comparison of key functional motifs in canonical SIMIBI NTPases and enterobacterial BcsQ. (E) Functional complementation assay examining the role of key BcsQ residues in vivo. Colony cellulose secretion is evaluated by fluorescent calcofluor binding. Results are representative of at least six biological replicates. All BcsQ mutants were tested for protein expression and interactions with the BcsR and BcsE partners (E).

  • Fig. 4 Structural and functional analyses of the essential for cellulose secretion subunit BcsR.

    (A) Comparison of the BcsRQ complex with two-component complexes of SIMIBI superfamily members. (B) Overlay of BcsR subunits found in the asymmetric units of all structures resolved in this study. (C) Size exclusion chromatography and SDS-PAGE analysis of purified BcsRQ and BcsΔNTDRQ. Molecular weights for the tag-free BcsR and BcsΔNTDR are 7.4 and 3 kDa, respectively. (D) Functional complementation assay of the role of BcsR domains in cellulose secretion in vivo. Results are representative of at least six biological replicates. (E) Western blot detection of cellular BcsA upon Bcs macrocomplex expression in the context of BcsRNTD deletion [GAPDH, glyceraldehyde-3-phosphate dehydrogenase (loading control)]. The results are representative of at least three replicates.

  • Fig. 5 Multisite c-di-GMP recognition and structure of the BcsRQR156E-BcsEREC*-GGDEF* complex.

    (A) Crystal structure of untagged BcsRQR156E-E217–523 assembly in complex with ATP/ACP (β,γ-methyleneadenosine triphosphate or AppCp) and c-di-GMP. (B) Full-length BcsE domain architecture and functionally validated c-di-GMP–binding motifs (RxxD I-sites I and II) as identified in this study. (C) Captured conformational changes in the BcsEREC*-GGDEF* tandem between the BcsE217–523 [“Splayed” (10)] and BcsRQR156EE217–523 (“Closed”) crystal structures. (D) Close-up views of c-di-GMP complexation in the three-component complex. Left: An |Fo|-|Fc| partial electron density map calculated from a model before inclusion of the dinucleotide and contoured at 2.5 σ. Right: A cartoon-and-stick representation of c-di-GMP coordination. Canonical I-site I residues are colored in cyan; secondary I-site II residues are colored in teal. (E) Functional validation of the secondary I-site on the BcsEREC* module (R306ATD) by isothermal titration calorimetry. Kd values were calculated using a two-site binding model.

Supplementary Materials

  • Supplementary Materials

    Architecture and regulation of an enterobacterial cellulose secretion system

    Wiem Abidi, Samira Zouhir, Meryem Caleechurn, Stéphane Roche, Petya Violinova Krasteva

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