Crystal structure of ACE19, the collagen binding subdomain of Enterococus faecalis surface protein ACE

Abstract

Enterococus faecalis is an extracellular pathogen known to cause many clinical infections, such as septicemia, bacteremia, and urinary tract infections.1 The bacterial pathogenesis is a complex process at the molecular level and the bacterial adherence to extracellular matrix components like collagen, fibrinogen, and fibronectin is a prerequisite for pathogenesis. Gram-positive bacteria are endowed with adhesive proteins termed as MSCRAMMs (Microbial Surface Component Recognizing Adhesive Matrix Molecules), located on the surface of the microbe to mediate such attachment. Characterization of several MSCRAMMs from various bacterial species revealed that they are structurally similar.2 In general, they contain an N-terminal signal peptide followed by (i) a ligand binding A region that is composed of two or more subdomains, each adopting a novel immunoglobulin-like (DEv-IgG) fold3; (ii) a B region that is made up of short repeat sequences; and (iii) the C-terminal end that contains the cell wall anchoring LPXTG motif, a hydrophobic transmembrane region (M), and a positively charged cytoplasmic tail (c), features that are essential for sorting these proteins to the cell wall.2 ACE is a collagen binding MSCRAMM of E. faecalis.4,5 The structural characteristics of ACE are similar to that of collagen binding protein CNA of Staphylococcus aureus [Fig. 1(A)], which has been studied in significant detail both in structural and biochemical aspects.6–10 The 55kDa ligand binding A region of CNA (termed CNA55) contains three subdomains namely N1, N2, and N3 and the minimum collagen binding region was localized to a 19-kDa N2 subdomain (termed CNA19) and its crystal structure was solved earlier in our laboratory.8 Recently, we determined the crystal structure of N1N2 subdomain of CNA (termed CNA35) both as an apo-protein and in complex with a synthetic collagen triple helix peptide. On the basis of these two crystal structures, we proposed a ‘‘Collagen Hug’’ binding mechanism for the association of CNA with collagen.11 The crystal structure of CNA35collagen complex revealed that collagen-like triple helical peptide mainly interacts with residues present in the ‘‘trench’’ region of the N2 domain, however, N1 domain also contributes critical residues for the stabilization of complex by sequestering critical regions of the ligand, explaining the 10-fold higher ligand binding affinity of CNA35 (N1 and N2) compared with CNA19. The 40-kDa A-region of ACE (termed ACE40) was predicted to have two subdomains namely N1 and N2. CNA55 and ACE40 share significant sequence similarity and particularly the minimum ligand binding region of CNA (CNA19) exhibits high degree of similarity (about 95%) with the corresponding region of ACE40 (i.e. ACE19). Previous modeling and spectroscopic studies predicted that the backbone folding of ACE19 is highly