RecA-NT homology motif in ImuB is essential for mycobacterial ImuA'-ImuB protein interaction and mutasome function

Abstract

The mycobacterial mutasome - minimally comprising ImuA', ImuB, and DnaE2 proteins - has been implicated in DNA damage-induced mutagenesis in Mycobacterium tuberculosis. ImuB, predicted to enable mutasome function via its interaction with the β clamp, is a catalytically inactive member of the Y-family of DNA polymerases. Like other members of the Y family, ImuB features a recently identified amino acid motif with homology to the RecA-N-terminus (RecA-NT). In RecA, the motif mediates oligomerization of RecA monomers into RecA filaments. Given the role of ImuB in the mycobacterial mutasome, we hypothesized that the ImuB RecA-NT motif might mediate its interaction with ImuA', a RecA homolog of unknown function. To investigate this possibility, we constructed a panel of imuB alleles in which RecA-NT was removed, or mutated. Results from microbiological and biochemical assays indicate that RecA-NT is critical for the interaction of ImuB with ImuA'. A region downstream of RecA-NT (ImuB-C) also appears to stabilize the ImuB-ImuA' interaction, but its removal does not prevent complex formation. In contrast, replacing two key hydrophobic residues of RecA-NT, L378 and V383, is sufficient to disrupt ImuA'-ImuB interaction. To our knowledge, this constitutes the first experimental evidence showing the role of the RecA-NT motif in mediating the interaction between a Y-family member and a RecA homolog.

Figure 1.. Computational model of mutasome interactions.

A. Conservation logo of RecA-NT with the corresponding M. smegmatis ImuB sequence in black below. Residue positions are shown for the start and end of the motif, as well as the individual residues selected for site-directed mutagenesis. B. Interaction model between M. smegmatis ImuA’ (yellow) and ImuB (green). Displayed here are ImuA’ without the unstructured N-terminal tail (residues 1–69) in yellow and the RecA-NT-like motif of ImuB. Key residues predicted for the ImuA’-ImuB interaction are labelled and represented in sticks for both ImuA’ (H99) and ImuB (D363, V374, L378, V383). C. M. smegmatis imuA’imuB operon with predicted interaction sites. In ImuA’, the predicted ImuB interaction site stretches from G97 to M144. In ImuB, the β clamp-binding motif (Q352-W356) precedes RecA-NT (I359-V387) and is followed by ImuB-C (remaining 138 residues). The panels below indicate the site-directed mutations and ImuB C-terminal deletion mutants constructed in this study.

Figure 2.. AlphaFold model of the ImuA’-ImuB complex.

In yellow is the surface of ImuA’ in contact with ImuB ; green represents the RecA-NT motif (residues 360–387), pink represents the ImuB-C (residues 422–525) and orange indicates the linker between the two ImuB domains (residues 389–421). The residues where C-terminal truncations were made are highlighted with spheres.

Figure 3.. Microbiological assessment of the impact of the mutant alleles.

Evaluation of functional complementation by means of A. Mitomycin C (MMC) damage sensitivity and B. UV mutagenesis assays.

Figure 4.. Biochemical confirmation of disruption of ImuA’-ImuB interaction in ImuBΔC380 and L378A+V383A.

A. Expression samples correspond to the total expression after growth for 3h following IPTG induction. Bands corresponding to individual induced proteins are marked l (His-ImuB), (Strep-ImuA’) and n (His-ImuBΔC380). B. SDS-PAGE analysis of the co-expression of His-ImuB mutants with Strep-ImuA’ and the two subsequent pull-downs with HisTag and StrepTag. Expression samples correspond to the total expression after growth for 3h after IPTG induction. Bands corresponding to individual induced proteins are marked l (His-ImuB mutants) and (Strep-ImuA’).