Structural basis for rifamycin recognition and sequential substrate binding by the mycobacterial ADP-ribosyltransferase ARR

The mycobacterial ADP-ribosyltransferase (ARR) mediates intrinsic antibiotic resistance by modifying the ansa-bridge of rifamycins with an ADP-ribose moiety derived from nicotinamide adenine dinucleotide (NAD+). Here, we elucidate the substrate binding mechanism of M. smegmatis ARR using X-ray crystallography and size-exclusion chromatography. We report the first apo structure of ARR, together with complexes involving rifampicin, rifabutin, and rifaximin. Biochemical data demonstrate an ordered sequential binding mechanism in which NAD+ requires prior rifamycin occupancy for recruitment to ARR. Structural comparison of apo ARR to rifamycin bound ARR, reveals that it undergoes significant conformational ordering upon rifamycin binding, characterized by a transition from highly flexible regions in the C-terminus and 2-helix to a more rigid state. Specifically, we identify a 2.9 [A] shift in the loop between {beta}-sheets 2 and 3, involving Phe39, which suggests a gating mechanism that organizes the NAD+ binding pocket. This experimental evidence is complemented by computational modelling of the NAD+ binding mode, which suggests a structurally plausible ternary arrangement. Together, these results establish that rifamycin binding is a prerequisite for NAD+ recruitment and provide a structural basis for the catalytic cycle of rifamycin ADP-ribosylation.