Solution structure of the sortase required for efficient production of infectious Bacillus anthracis spores

Abstract

Bacillus anthracis forms metabolically dormant endospores that upon germination can cause lethal anthrax disease in humans. Efficient sporulation requires the activity of the SrtC sortase (BaSrtC), a cysteine transpeptidase that covalently attaches the BasH and BasI proteins to the peptidoglycan of the forespore and predivisional cell, respectively. To gain insight into the molecular basis of protein display, we used nuclear magnetic resonance to determine the structure and backbone dynamics of the catalytic domain of BaSrtC (residues Ser(56)-Lys(198)). The backbone and heavy atom coordinates of structurally ordered amino acids have coordinate precision of 0.42 ± 0.07 and 0.82 ± 0.05 Å, respectively. BaSrtC(Δ55) adopts an eight-stranded β-barrel fold that contains two short helices positioned on opposite sides of the protein. Surprisingly, the protein dimerizes and contains an extensive, structurally disordered surface that is positioned adjacent to the active site. The surface is formed by two loops (β2-β3 and β4-H1 loops) that surround the active site histidine, suggesting that they may play a key role in associating BaSrtC with its lipid II substrate. BaSrtC anchors proteins bearing a noncanonical LPNTA sorting signal. Modeling studies suggest that the enzyme recognizes this substrate using a rigid binding pocket and reveals the presence of a conserved subsite for the signal. This first structure of a class D member of the sortase superfamily unveils class-specific features that may facilitate ongoing efforts to discover sortase inhibitors for the treatment of bacterial infections.

Figure 1

NMR spectra and amino acid sequence of BaSrtC. (A) Sequence alignment of B. anthracis (Ames strain) class A sortase (BaSrtA), class B sortase (BaSrtB), and class D sortase (BaSrtC). The sequence alignment was performed by ClustalW. Conserved residues are colored orange, while identical residues are colored red. The predicted transmembrane region (TM) is indicated by a cylinder labeled TM. Secondary structure features of BaSrtC are indicated by cylinders or arrows above the sequence. (B) ¹H–¹⁵N HSQC spectra of BaSrtC_Δ19 (left) and BaSrtC_Δ55 (right). BaSrtC_Δ19 contains approximately 30 more peaks, the majority of which reside toward the center of the spectrum. The BaSrtC_Δ55 HSQC spectrum has its residue assignments indicated.

Figure 2

MALDI-TOF demonstrating BaSrtC_Δ55 can cleave the LPNTA-containing peptide. A peptide substrate, VQGEKLPNTASNN, was incubated with BaSrtC_Δ55 for 24 h at room temperature. MALDI spectra of the peptide were taken immediately upon addition of the enzyme (A) and after incubation with BaSrtC_Δ55 for 24 h (B).

Figure 3

Structures of B. anthracis class D sortase. (A) Stereoview of the overlay of the ensemble of NMR structures of BaSrtC_Δ55 (PDB entry 2LN7). The unassigned β2–β3 and β4–H1 loops are colored red. The assigned but poorly ordered β7–β8 loop is colored orange. (B) Ribbon diagram with secondary structure elements labeled. Loop structures are labeled with text. Active site residue side chains for Arg¹⁸⁵, Cys¹⁷³, and His⁸⁶ are colored green.

Figure 4

Dynamics and ultracentrifugation analysis. (A) Plot of experimentally determined T₂ vs T₁ data (black dots) for 1 mM BaSrtC_Δ55 (assignable backbone ¹⁵N resonances). Also plotted are lines that correspond to calculated T₂ and T₁ values for various S² order parameter values and correlation times (correlation times increase along each line in a clockwise fashion). (B) Same as panel A, but T₂ and T₁ data were collected at a protein concentration of 0.125 mM. Residues with T₂ times of <0.07 s are colored orange. (C) Analytical ultracentrifugation equilibrium data demonstrating that BaSrtC is a dimer with a K_D of 89 µM. (D) Structure of BaSrtC with unassigned residue backbone nitrogens colored red. Residue backbone nitrogens with short T₂ times identified in panel B are colored orange.

Figure 5

Comparison of representative structures from class A–D sortases. B. anthracis class D enzymes are structurally similar to class A enzymes. BaSrtC lacks the long, structured, β6–β7 loop (yellow) present in class B enzymes, but the appearance of a short 3₁₀-helix in this region likely indicates the existence of a rigid substrate-binding pocket, similar to BaSrtA. BaSrtC also lacks the structured N-terminal lid present in class C enzymes (green).

Figure 6

(A) Model of the CWSS peptide structure from the SaSrtA–LPAT* complex on the substrate binding surface of BaSrtC. The peptide (green) is taken from the SaSrtA–LPAT* structure without modification. Active site residues are indicated along with the potential Ser¹¹⁴ binding site. The hydroxyl oxygen of Ser¹¹⁴ (Ser¹¹⁴ is colored gray, while its OH group is colored white) comes into close contact with the side chain of position X. (B) Close-up overlay of the BaSrtC structure (green) with apo-SaSrtA (red, left), holo-SaSrtA (blue, center), and apo-BaSrtA (orange, right). Active site arginine, cysteine, and histidine residues are denoted. The active site residues of apo-BaSrtC overlay best with those of holo-SaSrtA (SaSrtA–LPAT*).