Molecular Analysis of the Epidermal Growth Factor-like Short Consensus Repeat Domain-mediated Protein-Protein Interactions: DISSECTION OF THE CD97-CD55 COMPLEX

Hsi-Hsien Lin,Martin Stacey,Claire Saxby,Vroni Knott,Yasmin Chaudhry,David Evans,Siamon Gordon,Andrew J Mcknight,Penny Handford,Susan Lea

doi:10.1074/jbc.m101770200

Abstract

Epidermal growth factor-like (EGF) and short consensus repeat (SCR) domains are commonly found in cell surface and soluble proteins that mediate specific protein-protein recognition events. Unlike the immunoglobulin (Ig) superfamily, very little is known about the general properties of intermolecular interactions encoded by these common modules, and in particular, how specificity of binding is achieved. We have dissected the binding of CD97 (a member of the EGF-TM7 family) to the complement regulator CD55, two cell surface modular proteins that contain EGF and SCR domains, respectively. We demonstrate that the interaction is mediated solely by these domains and is characterized by a low affinity (86 microm) and rapid off-rate (at least 0.6 s(-1)). The interaction is Ca(2+) -dependent but is unaffected by glycosylation of the EGF domains. Using biotinylated multimerized peptides in cell binding assays and surface plasmon resonance, we show that a CD97-related EGF-TM7 molecule (termed EMR2), differing by only three amino acids within the EGF domains, binds CD55 with a K(D) at least an order of magnitude weaker than that of CD97. These results suggest that low affinity cell-cell interactions may be a general feature of highly expressed cell surface proteins and that specificity of SCR-EGF binding can be finely tuned by a small number of amino acid changes on the EGF module surface.

Highlights

Epidermal growth factor-like (EGF) and short consensus repeat (SCR) domains are commonly found in cell surface and soluble proteins that mediate specific protein-protein recognition events
These results suggest that low affinity cell-cell interactions may be a general feature of highly expressed cell surface proteins and that specificity of SCREGF binding can be finely tuned by a small number of amino acid changes on the EGF module surface
CD55 Binding Is Mediated Exclusively by the EGF Domains of CD97—Previous studies have demonstrated that the EGF domains of CD97 are necessary for CD97-CD55 interaction [13] and that EMR2 is unable to interact with CD55 [19]

Summary

EXPERIMENTAL PROCEDURES

Materials—All chemicals and reagents were obtained from Sigma unless otherwise specified. The final constructs contained various EGF-like domains followed by the EMR2 stalk region, a biotinylation signal and a stop codon. The level of cell surface protein expression on transfected cells was determined by FACS analysis using appropriate CD97 mAbs. For cells transfected with EMR2 and CD97 isoforms as well as the domain-swapping and deletion chimeras, CLB-CD97/1 and BL-Ac/F2, which recognize the first EGF-like domain of both EMR2 and CD97, were used. The red blood cell binding ability of transfected cells was represented by the measurement of peroxidase activities normalized by the median level of cell surface protein expression determined by FACS analysis. Production of Biotinylated Proteins—Conditioned medium collected from transfected HEK293T cells was concentrated to ϳ0.5 ml using a 30-kDa cut-off Centriprep tube (Millipore, Bedford, MA), dialyzed with 10 mM Tris-HCl, pH 8 buffer and incubated with 1 ␮l of BirA enzyme (Avidity, Denver, CO) overnight at room temperature. Additional reagents (divalent cations, EGTA, and mAbs) were added to the cells 5–10 min before the introduction of the protein-bead complex

RESULTS

Soluble protein

DISCUSSION