(A) Two LapD constructs, one genetically fused to sfGFP and one non-fluorescent, both harboring the A602C mutation, were engineered and expressed separately. In previously proposed models, EAL domain dimerization was thought to occur within a single LapD dimer (left). Under this model, one would only expect a single band shift corresponding to c-di-GMP- and LapG-activated LapD–sfGFP dimers upon oxidation, even if the sfGFP-fused and non-fluorescent variants were present in the membrane. Alternatively, the EAL domains could dimerize across two LapD dimers (right) to form a dimer-of-dimers. Under the latter model, when the two constructs are mixed, activated with c-di-GMP and LapG, oxidized with a disulfide-promoting copper catalyst, denatured in SDS-PAGE, and imaged by in-gel fluorescence, a faster migrating covalent heterodimeric adduct consisting of LapD–sfGFP and LapD (dark) should be observed in addition to the slower migrating complex containing just LapD–sfGFP homodimeric adduct. (B) Complex formation is mediated by dimerization of EAL domains across two LapD dimers rather than within the same LapD dimer. The two LapD variants shown in (A) were expressed separately. Crosslinking via corresponding A602C residues was induced after incubation with c-di-GMP and LapG, either in detergent-solubilized samples (left panel) or upon fusing membrane fractions from the two cultures (right panel). In both detergent and membranes, SDS-PAGE analysis of this crosslinking experiment shows that a heterodimeric adduct containing both LapD–sfGFP and non-fluorescent LapD (lighter blue triangle; asterisk denotes residue from a non-fluorescent LapD) is observed in addition to a species containing only LapD–sfGFP (darker blue triangle). In detergent, non-fluorescent LapD lacking the A602C mutation serves as a competitor, reducing LapD-A602C–sfGFP crosslinking efficiency. Representative data from two independent, biological replicates are shown.