Alignment of 36 MinC sequences revealed four completely conserved C-terminal glycines. As MinC inhibits cytokinesis in Neisseria gonorrhoeae and Escherichia coli, the functional importance of these glycines in N. gonorrhoeae MinC (MinC Ng ) and E. coli MinC (MinC Ec ) was investigated through amino acid substitution by using site-directed mutagenesis. Each mutant was evaluated for its ability to arrest cell division and to interact with itself and MinD. In contrast to overexpression of wild-type MinC, overexpression of mutant proteins in E. coli did not induce filamentation, indicating that they lost functionality. Yeast two-hybrid studies showed that MinC Ec interacts with itself and MinD Ec ; however, no interactions involving MinC Ng were detected. Therefore, a recombinant MinC protein, with the N terminus of MinC Ec and the C terminus of MinC Ng , was designed to test for a MinC Ng -MinD Ng interaction. Each MinC mutant interacted with either MinC or MinD but not both, indicating the specificity of glycine residues for particular protein-protein interactions. Each glycine was mapped on the C-terminal surfaces (A, B, and C) of the solved Thermotoga maritima MinC structure. We found that MinC Ec G161, residing in close proximity to the A surface, is involved in homodimerization, which is essential for MinC function. Glycines corresponding to MinC Ec G135, G154, and G171, located within or adjacent to the B-C surface junction, are critical for MinC-MinD interactions. Circular dichroism revealed no gross structural perturbations of the mutant proteins, although the contribution of glycines to protein flexibility and stability cannot be discounted. Using molecular modeling, we propose that exposed conserved MinC glycines interact with exposed residues of the ␣-7 helix of MinD.

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