Fold and function of the InlB B-repeat

Abstract

Host cell invasion by the facultative intracellular pathogen Listeria monocytogenes requires the invasion protein InlB in many cell types. InlB consists of an N-terminal internalin domain that binds the host cell receptor tyrosine kinase Met and C-terminal GW domains that bind to glycosaminoglycans (GAGs). Met binding and activation is required for host cell invasion, while the interaction between GW domains and GAGs enhances this effect. Soluble InlB elicits the same cellular phenotypes as the natural Met ligand hepatocyte growth factor/scatter factor (HGF/SF), e.g. cell scatter. So far, little is known about the central part of InlB, the B-repeat. Here we present a structural and functional characterization of the InlB B-repeat. The crystal structure reveals a variation of the β-grasp fold that is most similar to small ubiquitin-like modifiers (SUMOs). However, structural similarity also suggests a potential evolutionary relation to bacterial mucin-binding proteins. The B-repeat defines the prototype structure of a hitherto uncharacterized domain present in over a thousand bacterial proteins. Generally, this domain probably acts as a spacer or a receptor-binding domain in extracellular multi-domain proteins. In cellular assays the B-repeat acts synergistically with the internalin domain conferring to it the ability to stimulate cell motility. Thus, the B-repeat probably binds a further host cell receptor and thereby enhances signaling downstream of Met.

FIGURE 1.

Domain structure of InlB and crystal structure of the B-repeat overlaid with structurally similar proteins. A, domain structure of InlB and the constructs employed in this study. SP: signal peptide. B, schematic representation of the B-repeat with backbone H-bonds at the connector-arm/shelf and between β3 and β3′ shown as blue dashes. C–E, overlay of the B-repeat (blue) with C, SUMO3 (red; PDB ID 2io1); D, Mth1743 from Methanobacterium thermoautotrophicum (green, PDB ID 1ryj); E, a variant of protein L (cyan, PDB ID 1kh0).

FIGURE 2.

Conserved residues and hydrophobic core of the B-repeat. A, ribbon representation of the B-repeat in two orientations rotated by 180° (axis indicated as arrow). Coloring as in Fig. 1B. Residues that form the hydrophobic core and are conserved among homolgous bacterial proteins are shown as sticks. Conserved glycines are shown as spheres. Further conserved residues are shown as thin lines. B, sequence of the B-repeat and secondary structure elements indicated above. Colored residues are those that are shown in A as lines or, if in bold face type, as sticks and spheres. C, HMM logo without gaps generated from an alignment of over 300 homologous sequences with a length between 63 and 73 residues. The conservation score and the consensus sequence calculated with Jalview from the same alignment are shown below.

FIGURE 3.

InlB₃₉₂ stimulates wound healing and scatter in primate cells. A and B, closure of scratch wounds in A549 or Vero cells upon incubation with various ligands. For normalization, wound closure of cells incubated without ligand was subtracted. Full-length InlB and InlB₃₉₂ stimulated wound healing comparable to HGF/SF, while all other InlB constructs were inactive. The asterisk marks statistical significance at a level of 0.01. Note that for the sake of clarity, statistical significance is only shown for the two most relevant pairs of proteins (InlB₃₉₂ versus InlB₃₂₁ and InlB₃₉₂ versus B-rep+GW) at 24 h and 1 nm. C, closure of scratch wounds in Vero cells upon incubation with 1 nm InlB₃₉₂ (top) and 1 nm InlB₃₂₁ (bottom). D, full-length InlB and InlB₃₉₂ at 1 nm stimulate scattering of HT29 cells, while 1 nm InlB₃₂₁ shows little or no activity.

FIGURE 4.

InlB₃₉₂ stimulates wound healing and scatter in canine cells. A, full-length InlB stimulates scattering of MDCK cells (clone 20) at 1 nm. InlB₃₉₂ is inactive at 1 nm but induces scattering at 10 nm. InlB₃₂₁ and a construct consisting of the InlB B-repeat and the GW domains are inactive even at 10 nm. B, closure of scratch wounds in MDCK cells upon incubation with various ligands. InlB₃₉₂ is inactive at 1 nm but shows activity at 10 nm. InlB₃₂₁ and the construct B-repeat+GW domains are both inactive at 1 nm and 10 nm. The asterisk marks statistical significance at a level of 0.01. Note that for the sake of clarity, statistical significance is only shown for the two most relevant pairs of proteins (InlB₃₉₂ versus InlB₃₂₁ and InlB₃₉₂ versus B-rep+GW) at 24 h and 10 nm.

FIGURE 5.

Surface plasmon resonance shows no binding of the InlB B-repeat to Met. Sensorgrams depicting the binding of different InlB constructs to the complete extracellular domain of Met are shown. Varying concentrations of InlB were injected over a chip surface coated with the Met ectodomain. The solid lines represent the measured response in resonance units (RU). A and B, InlB₃₂₁ and InlB₃₉₂ were injected at concentrations of 0, 100, 200, 400, 600, 800, and 1000 nm. Dashed lines represent fits using a 1:1 Langmuir interaction with drifting baseline. C, overlay of 24 sensograms of the B-repeat in the concentration range of 0.6 nm to 55.8 μm.

FIGURE 6.

Native mass spectrometry shows that InlB₃₉₂, just like InlB₃₂₁, forms 1:1 complexes with Met₉₂₈. LILBID mass spectrum of Met₉₂₈ and of the Met₉₂₈ + InlB₃₂₁ and Met₉₂₈ + InlB₃₉₂ complexes. Under soft desorption conditions the intact Met₉₂₈ + InlB₃₂₁ and Met₉₂₈ + InlB₃₉₂ complexes appear at four different overall charge states (the black sticks indicate the theoretical mass/charge positions).

FIGURE 7.

Pairwise overlay of the InlB B-repeat and structurally related domains from other bacterial surface proteins. All structures are shown in schematic representation. The InlB B-repeat (gray) is overlaid with A, the B1 (dark blue) and B, the B2 (cyan) domain of repeat 5 of mucus-binding protein (Mub-R5) from Lactobacillus reuteri (PDB ID 3i57); C, residues 34–128 (red, PDB ID 2kt7) and D, residues 161–235 (green, PDB ID 2kvz) of lmo0835, a putative peptidoglycan-bound protein from Listeria monocytogenes; E, the adhesion protein PEPE_0118 from Pediococcus pentosaceus (pink, PDB ID 3lyy).