Rv1915 and Rv1916 from Mycobacterium tuberculosis H37Rv form in vitro protein-protein complex

• Published in: Biochimica et biophysica acta. General subjects Vol. 1866; no. 6; p. 130130
• Main Authors: Antil, Monika, Gupta, Vibha
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.06.2022
• Subjects: Acetyl Coenzyme A; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; carbon dioxide fixation; computer simulation; drugs; genes; ICL1; ICL2; Isocitrate lyase; Isocitrate Lyase - chemistry; Isocitrate Lyase - genetics; Isocitrate Lyase - metabolism; mass spectrometry; Mycobacterium tuberculosis; Mycobacterium tuberculosis - enzymology; Mycobacterium tuberculosis - genetics; pathogens; Rv1915; Rv1916; site-directed mutagenesis; ICL2; Rv1916; Rv1915; Mycobacterium tuberculosis; Isocitrate lyase; ICL1
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2022.130130

Mycobacterium tuberculosis (Mtb) isocitrate lyase (ICL) is an established drug target that facilitates Mtb persistence. Unlike other mycobacterial strains, where ICL2 is a single gene product, H37Rv has a split event, resulting in two tandemly coded icls - rv1915 and rv1916. Our recent report on functionality of individual Rv1915 and Rv1916, led to postulate the cooperative role of these proteins in pathogen's survival under nutrient-limiting conditions. This study investigates the possibility of Rv1915 and Rv1916 interacting and forming a complex. Pull down assay, activity assay, mass spectrometry and site directed mutagenesis was employed to investigate and validate Rv1915-Rv1916 complex formation. Rv1915 and Rv1916 form a stable complex in vitro, with enhanced ICL/MICL activities as opposed to individual proteins. Further, activities monitored in the presence of acetyl-CoA show significant increase for Rv1916 and the complex but not of Rv0467 and Rv1915Δ90CT. Both full length and truncated Rv1915Δ90CT can form complex, implying the absence of its C-terminal disordered region in complex formation. Further, in silico analysis and site-directed mutagenesis studies reveal Y64 and Y65 to be crucial residues for Rv1915-Rv1916 complex formation. This study uncovers the association between Rv1915 and Rv1916 and supports the role of acetyl-CoA in escalating the ICL/MICL activities of Rv1916 and Rv1915Δ90CT-Rv1916 complex. Partitioning of ICL2 into Rv1915 and Rv1916 that associates to form a complex in Mtb H37Rv, suggests its importance in signaling and regulation of metabolic pathway particularly in carbon assimilation. •Rv1915 and Rv1916 of Mtb H37Rv associate together to form in vitro protein-protein complex.•Deletion of disordered C-terminal of Rv1915 does not abrogate complex formation with Rv1916.•Site-directed mutagenesis studies show Y64 and Y65 residues of Rv1916 to be crucial for complex formation.•The presence of acetyl-CoA increases the ICL and MICL activities of Rv1916 and the complex.