FIG 2.

Successful covalent CDV H-SLAM complex engineering. (A) Structural homology model of the cSLAM V domain in complex with one CDV H head. The inset highlights the putative distances between residue P541of CDV H and amino acids G71 and N72 of cSLAM. (B) Atomic structure of the human SLAM V domain homodimer. The two critical E71 residues are shown. (C and D) Biochemical assessment of the disulfide bond formation linking CDV H to cSLAM. His-tagged soluble versions of CDV H and cSLAM were coexpressed in 293T cells, and the protein complex immunoprecipitated (IP) with an anti-His MAb. Proteins were then run in Tris-acetate gels (nonreducing conditions) (C) or SDS-PAGE gels (reducing conditions) (D) and detected by immunoblotting (IB) using either anti-H (top) or anti-SLAM (bottom) polyclonal antibodies. H, H monomers; H2, H dimers; H4, H tetramers; H2-SLAM (1×), H dimers bound to one SLAM unit; H2-SLAM (2×), H dimers bound to two SLAM units.