The Enterococcus faecalis MSCRAMM ACE Binds Its Ligand by the Collagen Hug Model

Qing Liu,Karthe Ponnuraj,Yi Xu,Vannakambadi K Ganesh,Jouko Sillanpää,Barbara E Murray,Sthanam V.L Narayana,Magnus Höök

doi:10.1074/jbc.m611137200

Abstract

We have determined the crystal structure of the ligand binding segment of the Enterococcus faecalis collagen binding MSCRAMM ACE (microbial surface components recognizing adhesive matrix molecules adhesin of collagen from enterococci). This segment is composed of two subdomains, N(1) and N(2), each adopting an IgG-like fold and forming a putative collagen binding surface at the interface between the two subdomains. This structure is very similar to that recently reported for CNA, the collagen binding MSCRAMM of Staphylococcus aureus, for which a unique ligand binding mechanism called the Collagen Hug was proposed. We suggest that ACE binds collagen by a similar mechanism and present the first biochemical evidence for this binding model. Replacing residues in the putative collagen binding trench of ACE N(2) with Ala residues affected collagen binding. A closed conformation of ACE stabilized by an engineered disulfide bond is unable to bind collagen. Finally, the importance of the residues in the N(2) extension in stabilizing the MSCRAMM-ligand complex is demonstrated by selected point and truncation mutations.

Highlights

EXPERIMENTAL PROCEDURESBacterial and Growth Conditions—Escherichia coli strains JM101 and XL1-Blue (Stratagene, La Jolla, CA) were used as the bacterial host for plasmid cloning and protein expression
Enterococci are part of the normal human intestinal flora but have emerged as important opportunistic pathogens, ranked third among the organisms isolated from nosocomial infections
ACE is the first MSCRAMM identified on E. faecalis

Summary

EXPERIMENTAL PROCEDURES

Bacterial and Growth Conditions—Escherichia coli strains JM101 and XL1-Blue (Stratagene, La Jolla, CA) were used as the bacterial host for plasmid cloning and protein expression. Recombinant proteins expressed from this vector contain an N-terminal tail of 6 histidine residues. Which the introduced Cys residues had not formed disulfide bonds, the protein sample was passed through a 5-ml activated thiol-Sepharose 4B column previously equilibrated with 50 mM NaH2PO4, 0.5 M NaCl, 1 mM EDTA (pH 7.5). The domain structure of ACE is based on the sequence from E. faecalis strain V583. The different ACE fragments constructed as N-terminal His tag fusion proteins are illustrated below. The KD values for the binding of recombinant MSCRAMMs to the immobilized collagen were calculated by Scatchard analysis based on TABLE 1 the responses at the steady state portion of the sensorgrams. Crystallographic and refinement data of ACE32–367 using a one-site binding non-linear regression model (19)

Rsym No molecules in the asymmetric unit

Protein Data Bank ID

RESULTS

Examining the Putative Ligand

DISCUSSION