Mycobacterium tuberculosis universal stress protein Rv2623 interacts with the putative ATP binding cassette (ABC) transporter Rv1747 to regulate mycobacterial growth
Fig 1
M. tuberculosis Rv2623 interacts with Rv1747.
(A) The primary structure of Rv1747 with 2 FHA domains (Orange: FHA I & FHA II); elements typical of ABC transporters: NBD (nucleoside-binding domain; 1–559 amino acids; Walker A&B (Yellow; the ATP-binding domain), and transmembrane domain (Blue bars: transmembrane helices). (B) The GAL4-based Matchmaker Gold Yeast Two-Hybrid system was used to identify interacting partners of Rv2623, which was cloned into the pGBKT7 vector as a fusion to the GAL4 DNA-binding domain (pGBKT7::Rv2623). A DNA library of M. tuberculosis Erdman prey proteins were expressed as fusions to the Gal4 activation domain using pGADT7AD. The screen revealed that Rv2623 interacts with the N-terminal FHA I domain of Rv1747. Analysis of a re-cloned full-length Rv1747 FHA I domain validated the interaction (B, bottom panel: pGBKT7::Rv2623/pGADT7AD::FHA I). Rv2623 dimerization was exploited to serve as positive control (B, top panel; pGBKT7::Rv2623/pGADT7AD::Rv2623). pGBKT7::Rv2623/pGADT7AD and pGBKT7/pGADT7AD::FHA I represent negative controls. The interaction was further confirmed by affinity chromatography study (C). Purified histidine (His6)-tagged Rv2623 (Rv2623) and FLAG-tagged FHA I (FHA I: first 100 amino acids of Rv1747) were expressed in M. smegmatis mc2155. Purified FLAG-tagged FHA I was passed over columns with or without Rv2623 immobilized onto the Nickel (Ni)-NTA resin. Western analyses of the appropriate elution fractions using anti-Rv2623 and anti-FLAG antibodies revealed that Rv2623 and Rv1747 FHA I co-eluted—upper and lower panels of lane 3 represent the results of probing eluents from column containing both (Ni)-NTA resin-immobilized (His6)-tagged Rv2623 and FLAG-tagged Rv1747 FHA I with anti-Rv2623 and anti-FLAG antibody, respectively—thus demonstrating interaction of these two mycobacterial components. Lane 1: upper panel and lower panel represent results of reacting eluents from column with only Rv2623 with the appropriate antibody. The upper and lower panels of Lane 2 depict reactivity of eluents from column harboring only FHA I with the appropriate antibody. Lane 4 of upper and lower panels represent recombinant Rv2623 (+ve Rv) and FLAG-FHA I (+FHA); respectively, loaded as positive controls. α-Rv2623 and α-FLAG: anti-Rv2623 and anti-FLAG antibodies; respectively. Arrows indicated the molecular weight of His-tagged Rv2623 (~32.31 kDa)) and FLAG-tagged Rv1747 FHA I (~12 kDa; expressed as the first 100 amino acids of Rv1747).