Mutational studies to characterize the interaction between the GTPase McrB and the endonuclease McrC

Abstract

Modification dependent restriction (MDR) enzymes evolved as a defense mechanism in bacteria against the attack of phages with modified genome. McrBC is a type IV MDR which binds to a RmC and cleaves DNA having two such sites using energy derived from GTP hydrolysis. The functional complex of McrBC is a tetradecamer formed by two hexamers of McrB bridged together by a dimer of McrC. McrB, the GTPase, on its own has a very poor GTPase activity, while McrC, the endonuclease, on its own does not cleave DNA. The GTPase activity of McrB and the endonuclease activity of McrC is stimulated when they together form a complex in presence of GTP. The interaction between McrB and McrC is crucial for the functioning of the enzyme, however the mode of the interaction between the two proteins is unknown.

A series of McrC deletion were generated based on the secondary structure prediction from Phyre2. The mutants hence obtained were subjected to biochemical characterization using analytical size exclusion chromatography, GTP hydrolysis and nucleolytic cleavage. We identified that the first 192 residues in McrC form an independent domain that can interact with McrB and is sufficient to stimulate GTP hydrolysis. The residues in the region 60-100 which forms an extended loop was found to be crucial for the formation of the complex as well as its activities.