Mutational analyses reveal that the staphylococcal immune evasion molecule Sbi and complement receptor 2 (CR2) share overlapping contact residues on C3d: implications for the controversy regarding the CR2/C3d cocrystal structure.

We recently characterized an interaction between the Staphylococcus aureus immune evasion molecule Staphylococcus aureus binder of Ig (Sbi) and complement C3, an interaction mediated primarily through the binding of C3d(g) to Sbi domain IV. Events related to these studies prompted us to investigate via mutagenesis the binding interface of C3d for Sbi domain IV (Sbi-IV), as well as to revisit the controversial issue of the complement receptor 2 (CR2) binding site of C3d. Specifically, we had shown that Sbi domains III and IV fragment binding to C3dg inhibited the latter's binding to CR2. Moreover, a published cocrystal structure of C3d bound to complement inhibitory C-terminal domain of extracellular fibrinogen-binding protein (Efb-C), a structural and functional homolog of Sbi-IV, showed Efb-C binding to a region on the concave face of C3d previously implicated in CR2 binding by our mutagenesis data but not confirmed in the CR2(short consensus repeat [SCR]1-2):C3d cocrystal structure. We have now analyzed by surface plasmon resonance the binding of a series of variant C3dg molecules to biosensor-bound Sbi-IV or CR2(SCR1-2). We found that mutations to the concave face acidic pocket of C3d significantly affected binding to both Sbi-IV and CR2, although there was divergence in which residues were most important in each case. By contrast, no binding defects were seen for mutations made to the sideface of C3d implicated from the cocrystal structure to be involved in binding CR2(SCR1-2). The results with Sbi-IV suggest a mode of binding highly similar to that visualized in the Efb-C:C3d complex. The results with CR2 confirm our earlier mapping studies and cast even further doubt on the physiologic relevance of the complex visualized in the C3d:CR2 cocrystal.

Analysis of protein glycation using phenylboronate acrylamide gel electrophoresis.

The incorporation of the specialized carbohydrate affinity ligand methacrylamido phenylboronic acid in polyacrylamide gels for SDS-PAGE analysis has been successful for the separation of carbohydrates and has here been adapted for the analysis of post-translationally modified proteins. While conventional SDS-PAGE analysis cannot distinguish between glycated and unglycated proteins, methacrylamido phenylboronate acrylamide gel electrophoresis (mP-AGE) in low loading shows dramatic retention of delta-gluconolactone modified proteins, while the mobility of the unmodified proteins remains unchanged. With gels containing 1% methacrylamido phenylboronate, mP-AGE analysis of gluconoylated recombinant protein Sbi results in the retention of the modified protein at a position expected for a protein that has quadrupled its expected molecular size. Subsequently, mP-AGE was tested on HSA, a protein that is known to undergo glycation under physiological conditions. mP-AGE could distinguish between various carbohydrate-protein adducts, using in vitro glycated HSA, and discriminate early from late glycation states of the protein. Enzymatically glycosylated proteins show no altered retention in the phenylboronate-incorporated gels, rendering this method highly selective for glycated proteins. We reveal that a trident interaction between phenylboronate and the Amadori cis 1,2 diol and amine group provides the molecular basis of this specificity. These results epitomize mP-AGE as an important new proteomics tool for the detection, separation, visualization and identification of protein glycation. This method will aid the design of inhibitors of unwanted carbohydrate modifications in recombinant protein production, ageing, diabetes, cardiovascular diseases and Alzheimer's disease.

A structural basis for Staphylococcal complement subversion: X-ray structure of the complement-binding domain of Staphylococcus aureus protein Sbi in complex with ligand C3d.

The structure of the complement-binding domain of Staphylococcus aureus protein Sbi (Sbi-IV) in complex with ligand C3d is presented. The 1.7Å resolution structure reveals the molecular details of the recognition of thioester-containing fragment C3d of the central complement component C3, involving interactions between residues of Sbi-IV helix α2 and the acidic concave surface of C3d. The complex provides a structural basis for the binding preference of Sbi for native C3 over C3b and explains how Sbi-IV inhibits the interaction between C3d and complement receptor 2. A second C3d binding site on Sbi-IV is identified in the crystal structure that is not observed in related S. aureus C3 inhibitors Efb-C and Ehp. This binding mode perhaps hints as to how Sbi-IV, as part of Sbi, forms a C3b-Sbi adduct and causes futile consumption of C3, an extraordinary aspect of Sbi function that is not shared by any other known Staphylococcal complement inhibitor.