Conserved Glycines in the C Terminus of MinC Proteins Are Implicated in Their Functionality as Cell Division Inhibitors

Abstract

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.

MinC Mutants Deficient in MinD- and DicB-Mediated Cell Division Inhibition Due to Loss of Interaction with MinD, DicB, or a Septal Component

“…Ramirez-Arcos et al (30) proposed that MinC interacts with hydrophobic residues that are located in helix 7 of MinD. Recent support for this proposal comes from the independent FIG.…”

“…It was suggested that they are in the signal transduction pathway that links the ␥-phosphate binding domain to a region on the surface that directly interacts with MinC. In the proposed dimer model these residues come into close contact with helix 7 (41), which has been suggested to be the site for interaction with MinC (30).…”

“…Recently, Ramirez-Arcos et al (30) examined the effects of replacing highly conserved glycine residues with negatively charged amino acids. They reported that replacing G135, which is also within the conserved RSGQ motif, prevented interaction with MinD.…”

Maintenance of the Cell Morphology by MinC in Helicobacter pylori

“…It has been shown that four conserved glycine residues at the MinC C-terminus are essential for MinC functionality as a cell division inhibitor and for the interaction of MinC with other Min proteins in E. coli [21]. Four glycine residues (G 104 , G 122 , G 129 , and G 139 ) were also found at C-terminus of the H. pylori MinC proteins.…”

The N Terminus of MinD Contains Determinants Which Affect Its Dynamic Localization and Enzymatic Activity

“…Although our laboratory is focused on the study of Min proteins from the coccus N. gonorrhoeae, it has been demonstrated that MinD Ng is functionally similar to E. coli MinD (27,38). Hence, many functional studies with MinD Ng have been facilitated by using the rod E. coli (26,27,28,38), particularly since the gonococcus is much more fastidious and less amenable to genetic manipulation than E. coli. MinD Ng and MinD Ec exhibit high levels of identity (73%) and similarity (85%); therefore, structure-function analyses of one protein can serve as a paradigm for the other.…”