Atomic structures of a bacteriocin targeting Gram-positive bacteria

Abstract

Due to envelope differences between Gram-positive and Gram-negative bacteria, engineering precision bactericidal contractile nanomachines requires atomic-level understanding of their structures; however, only those killing Gram-negative bacteria are currently known. Here, we report the atomic structures of an engineered diffocin, a contractile syringe-like molecular machine that kills the Gram-positive bacterium Clostridioides difficile. Captured in one pre-contraction and two post-contraction states, each structure fashions six proteins in the bacteria-targeting baseplate, two proteins in the energy-storing trunk, and a collar linking the sheath with the membrane-penetrating tube. Compared to contractile machines targeting Gram-negative bacteria, major differences reside in the baseplate and contraction magnitude, consistent with target envelope differences. The multifunctional hub-hydrolase protein connects the tube and baseplate and is positioned to degrade peptidoglycan during penetration. The full-length tape measure protein forms a coiled-coil helix bundle homotrimer spanning the entire diffocin. Our study offers mechanical insights and principles for designing potent protein-based precision antibiotics.

Fig. 1. CryoEM reconstructions of the diffocin in pre- and post-contraction states.

a Organization of engineered diffocin genes. Gene accession numbers of wild-type diffocin are shown below the corresponding genes. A portion of the gene segment encoding the tail fiber and the genes encoding the receptor-binding protein and two tail fiber chaperones were replaced with those of the phi027 prophage, which targets the epidemic C. difficile BI/NAP1/027 strain. Genes framed in black encode proteins that are resolved in the cryoEM reconstructions. b A representative cryoEM image showing diffocin particles in the pre- and post-contraction states. Scale bar, 100 nm. c Length of diffocin particles in pre-contraction, post-contraction transitional, and post-contraction final states measured from collar to baseplate (detailed in “Methods”). The sample sizes are 1088, 742, and 872 for the particles in the pre-contraction, post-contraction transitional, and post-contraction final states, respectively. Medians shown as black lines. Statistics performed by two-tailed unpaired t-test; P value is 1.4 × 10⁻¹⁵ (****P < 0.0001). Source data of the length of diffocin particles are provided as a Source Data file. d–f Composite cryoEM density maps of the diffocin in the pre-contraction (d), post-contraction transitional (e), and post-contraction final (f) states. A sectional view of the pre-contraction state is presented in (d). Structural subunits are colored as in (a). L_1-32 denotes layers of the sheath.

Fig. 2. Overview of the atomic model of diffocin in the pre-contracted state.

CryoEM densities encasing the corresponding atomic models of individual diffocin proteins in the pre-contraction state are shown on both sides of diffocin complex. Regions of the cryo-EM density map (semi-transparent densities) superimposed with atomic models (sticks) are shown in boxes, demonstrating the agreement between the observed and modeled amino acid side chains. Numbers denote chain termini.

Fig. 3. Molecular organization of the diffocin baseplate.

a Longitudinal (left) and transversal (right) cut views of the cryoEM map of the diffocin baseplate in the pre-contraction state. b Ribbon diagram of the spike trimer. A monomer is colored according to the linear diagram. At the tip of the spike, His72 and His74 from three monomers collectively chelate with an iron ion. Comparison of the central parts of the diffocin (c) and R-type pyocin (d) baseplates. e Linear schematic and ribbon diagram of the hub-hydrolase. The hub (blue), lytic transglycosylase (yellow) and endopeptidase (red) are connected sequentially through linkers (gray). The catalytic centers of two hydrolases are labeled with pentagrams. f Zoom-in views of the catalytic triad of hydrolases. The lytic transglycosylase and endopeptidase cleave glycosidic bonds and peptide bonds of the peptidoglycan mesh, respectively. NAG N-acetylglucosamine, NAM N-acetylmuramic acid.

Fig. 4. Sheath initiator accommodates the symmetry mismatch from the sheath (C6 symmetry) to the baseplate (C3 symmetry).

a Interactions between the sheath initiator and the baseplate shown in perpendicular views. Two conformers of the sheath initiator (shown as pink and blue surfaces) bind to the hub-hydrolase alternatively. b Ribbon diagrams of the two conformers of the sheath initiator. The structures are rainbow colored from N-terminus (blue) to C-terminus (red). Differences between the two conformers are present at the N-terminal loops; C-terminal globular domains are the same. c Two interfaces on the C-terminal domain of the sheath initiator (gray surface) with the sheath and the triplex core bundle. d Close-up view of the handshake β-sheet formed by the C-terminal domain of the sheath initiator and two neighboring sheath subunits. e Close-up view of the interface between the C-terminal domain of the sheath initiator and the triplex core bundle. f Close-up view of the interfaces between sheath initiators and the hub-hydrolase. The N-terminal loops of adjacent sheath initiators adopt different conformations to bind at the junction of hub domains.

Fig. 5. Structure of trunk sheaths in pre- and post-contraction states.

Top and side views of diffocin trunks in the pre- (a) and post-contraction states (b). Only four layers, L_{(n-1)/(n)/(n+1)/(n+2)}, are shown for simplicity. Sheath proteins are presented as ribbon diagrams and tube proteins are presented as gray surfaces. The inner and outer diameters of sheath rings are labeled on the top views. Ribbon diagrams depicting interactions between neighboring sheaths in the pre- (c) and post-contraction states (d). Four β-strands from three neighboring sheath subunits jointly form the conserved handshake β-sheet. Schematic diagrams for diffocin sheath topology of the extended mesh created by the handshake interaction of augmented β-sheet in the pre- (e) and post-contraction states (f).

Fig. 6. Gradual contraction progress of collar-proximal sheaths.

Surface representations of the diffocin collars in the pre-contraction (a), post-contraction transitional (b), and post-contraction final states (c). The three structures are aligned using their collars. Five collar-proximal layers of the sheath (L_32–28) are shown for each structure. Sheath color is coded by different conformations. L_29–28 of sheath in the transitional state and L_30–28 of sheath in the final state are trunk sheathes with the post conformer. d, Plot of outer diameters of sheath L_32–28 in three states. e–g Views showing the interface between the collar and sheath layer 32 (L₃₂) in three states. h Additional interactions between collar and sheath L₃₂ that only appear in the final state, labeled as hexagram in right panel of (g).

Fig. 7. Tape measure protein (TMP) of the diffocin.

a Sectional view of composite cryoEM density map of the diffocin in the pre-contraction state. TMPs are colored in magenta and the other components are in semitransparent colors. b–e Zoom-in views of diffocin TMP densities as indicated in (a). Corresponding top views are shown on the right. TMP models are fitted into the cryoEM densities in side views. f Interface between the TMP trimer and spike trimer. g Interactions between the C-terminal helix of the TMP and the N-terminal helix of the spike. Residues on the interface are labeled and shown in stick representation. h Model of the full-length diffocin TMP. One TMP subunit is rainbow colored from its N-terminus (blue) to C-terminus (red). i Illustration of predicted secondary structures of TMPs from phages and phage tail-like nanomachines. Alpha-helices, transmembrane (TM) domains and disordered regions were predicted by Phyre2. Globular domains were predicted by AlphaFold2. Globular domains in the same color indicate their structural similarity (see details in Supplementary Fig. 9).