Crystal structures of the outer membrane domain of intimin and invasin from enterohemorrhagic E. coli and enteropathogenic Y. pseudotuberculosis

Abstract

Intimins and invasins are virulence factors produced by pathogenic Gram-negative bacteria. They contain C-terminal extracellular passenger domains that are involved in adhesion to host cells and N-terminal β domains that are embedded in the outer membrane. Here, we identify the domain boundaries of an E. coli intimin β domain and use this information to solve its structure and the β domain structure of a Y. pseudotuberculosis invasin. Both β domain structures crystallized as monomers and reveal that the previous range of residues assigned to the β domain also includes a protease-resistant domain that is part of the passenger. Additionally, we identify 146 nonredundant representative members of the intimin/invasin family based on the boundaries of the highly conserved intimin and invasin β domains. We then use this set of sequences along with our structural data to find and map the evolutionarily constrained residues within the β domain.

Figure 1. Domains of intimin and invasin

The β-domains of intimins (red barrel) and invasins (blue barrel) are predicted to contain a β-barrel and linker that would be embedded in the outer membrane. The linker likely passes through the barrel pore and connects the β-barrel to the passenger domain. The passenger domain is exposed to the extracellular space and is comprised of repeating BIG domains and capped by a C-type lectin-like domain. Regions of known structure are shown as ribbon diagrams and domain boundaries are indicated by amino position. Additionally, some Int/Inv contain a predicted peptidoglycan-binding domain (LysM) between their signal peptide (SP) and β-domain; this domain is shown only in panel B and is much more prevalent among intimins. (A and B) E. coli intimin. The D0–D2 domains are BIG domains. The D3 domain is C-type lectin-like domain. In panel B, the domain boundaries defined by Touze et al. for the β-domain (189–550) are shown above and the actual domain boundaries for the β-domain (210–449) and D00 domain (450–550) determined in this study are shown below. (C and D) Y. pseudotuberculosis invasin. The D1–D4 domains are BIG domains. The D5 domain is C-type lectin-like domain. The boundaries of invasin’s β-domain were determined by aligning invasin with residues 189–550 of E. coli intimin.

Figure 2. Residues 210–450 define the boundaries of the intimin β-domain

(A) The HA-Int construct is a full-length version of HA tagged intimin. The C-terminus of this construct was truncated at residue 550 and progressive N-terminal deletions were then made between the HA tag and β-domain. (B) Heat-modifiable mobility assays for the N-terminal deletion constructs. Unfolded (UF) and folded (F) intimin are indicated. (C) C-terminal truncation constructs of intimin. (D) Heat-modifiable mobility assays for the C-terminal truncation constructs. (E) Whole cell PK-sensitivity assays of the C-terminal truncation constructs. Surface exposure of the passenger is indicated by the appearance of a lower molecular weight band compared to the full-length construct when PK was added to the sample.

Figure 3. Structure of the intimin β-domain

(A) Stereo ribbon diagram of the intimin construct 10xHis-TEV-Int_208–449. Strands, salmon; loops, limon; helices, light-orange. The extended portion of the linker inside the barrel pore is highlighted in cyan. (B) Two-dimensional representation of the β-domain imposed onto its secondary structure. Loop residues, green circles; extended linker residues, cyan circles; α-helix residues, orange hexagons; β-strand residues, red (central pore-facing) or blue (outward-facing) arrows. (C) Stereo diagram of β-strands 5, 6, 7, and 8 of intimin in stick form with the 2F_o-F_c electron density (blue mesh) shown at a contour level of 1σ. See also Figure S2.

Figure 4. Intimin’s linker contacts one side of the barrel wall

(A) The intimin β-barrel is shown in stereo ribbon format. Strands, salmon; loops, limon. Hydrogen bonds between residues of the barrel wall (salmon sticks) and residues of the linker (cyan sticks) are shown as dashed lines. (B) Cavity within the β-barrel of intimin is shown as a black surface. (C) Same as panel B but rotated by 270 degrees about the x-axis. See also Figure S3.

Figure 5. Deletion analysis of Intimin’s linker and periplasmic α-helix

(A) Heat-modifiable mobility assays for the linker deletion constructs. C-terminal truncations were made starting with residue 450 of the HA-Int_1–450 construct. Each truncation removed two residues. Unfolded (UF) and folded (F) forms of the β-domain are indicated. (B) Heat-modifiable mobility assays for the constructs HA-Int_1–530 and HA-Int_1–530Δ414–433. In HA-Int_1–530Δ414–433, the periplasmic α-helix was replaced with two glycines. This construct retains heat-modifiable mobility. (C) Whole cell PK sensitivity assays for the constructs in panel B. HA-Int_1–530Δ414–433 translocates its passenger to the cell surface when the periplasmic α-helix is replaced by two glycines. See also Figure S4.

Figure 6. Structure of the invasin β-domain

(A) Ribbon diagram of the invasin β-domain structure with light-blue strands, yellow helices, and violet loops. The extended portion of the linker inside the barrel pore is highlighted green. (B) Superposition of the invasin and intimin β-domains. Invasin is colored as in panel A and intimin is colored as in figure 3A. Extracellular loop 1 for intimin and invasin is marked with an asterisk. See also Figure S1.

Figure 7. Co-evolving residues for the Int/Inv family

(A) A patch of co-evolving residues for the Int/Inv family mapped onto intimin and shown as sticks. Acidic residues are colored salmon, basic residues are colored blue, and all others are colored yellow. Portions of the intimin structure have been removed to more clearly show the patch. (B) Selected co-evolving pairs of residues for the Int/Inv family mapped onto intimin and shown as sticks. Only co-evolving pairs near to each other in the structure are shown. The pairs are color-coordinated and have their side-chains shown as sticks. (C) Circle diagram for the co-evolving patch showing connectivity of the network. Colors are coordinated with panel A. Only connections between co-evolving residues less than 10 Å apart are shown. (D) Circle diagram for co-evolving pairs. Colors for pairs are coordinated with panel B. See also Figure S7 and Table S3.