Cysteine Residues Are Required for the Sliding Clamp-Dependent Endonuclease Activity of Neisseria gonorrhoeae MutL.

Background

Neisseria gonorrhoeae is a human pathogen that causes gonorrhea and employs a methylation-independent mismatch repair (MMR) pathway to regulate genomic stability and pilin antigenic variation. A key effector of this pathway, MutL, possesses ATPase, DNA-binding, and endonuclease activities, yet the molecular determinants governing its catalytic activity remain poorly understood. In particular, the functional relevance of conserved cysteine (Cys) residues located in the vicinity of the active site is unclear.

Methods

We characterized a Cys free-variant of Neisseria gonorrhoeae MutL, in which all Cys residues were replaced with evolutionarily conserved substitutes, in comparison with the wild-type enzyme. ATPase activity, DNA-binding capacity, and endonuclease activity on supercoiled plasmid and linear DNA duplex substrates were assessed, and molecular dynamics simulations were performed to examine the conformational behavior of the C-terminal domain (CTD).

Results

Cys substitutions do not markedly affect ATPase activity, DNA-binding capacity, or endonuclease activity on supercoiled plasmid substrates. However, the same amino acid substitutions led to an almost complete loss of sliding clamp-dependent endonuclease activity on linear DNA duplexes. Molecular dynamics simulations revealed that the cysteine substitutions alter the conformational dynamics of the CTD, displacing the catalytically active monomer from its productive orientation relative to the sliding clamp and thereby impairing sliding clamp-dependent endonuclease activity.

Conclusions

These results indicate that Cys residues are not directly involved in catalysis but provide the conformational rigidity of the C-terminal domain required for productive engagement with the sliding clamp.