Structural and functional analysis of the Mycobacterium tuberculosis transcriptional regulator FasR

JULIA LARA, LAUTARO DIACOVICH, NICOLE LARRIEUX, ALEJANDRO BUSCIAZZO, GABRIELA GAGO, HUGO GRAMAJOOLE LARRIEUX2, ALEJANDRO BUSCHIAZZO2, GABRIELA GAGO1, HUGO GRAMAJO

Congreso; Microbiology Society, Annual Conference,; 2018

Mycobacteria have two fatty acid synthases (FAS I and FAS II) which work in concert to synthesize fattyacids and mycolic acids. We identified a transcriptional regulator essential for mycobacterial viability:FasR, which binds the fas promoter and controls the de novo fatty acid biosynthesis. The main purposeof our studies was to understand at the molecular level how mycobacteria exert a fine control over thebiosynthesis of their membrane. The characterization of long chain acyl-CoAs that modulates the affinityof FasR for its target DNA was studied using electrophoretic mobility shift assay, SPR and in vitrotranscription.Crystallization screenings using a 40 aa N-terminal mutant of FasR we identified two different sets ofconditions producing crystals of FasR alone and in complex with C20-CoA, both of which diffracted Xrays at better than 1.8 Å resolution.Electrophoretic mobility shift experiments using FasR mutants generated to prevent the binding of theligand into the effector domain, designed from the structural analysis of FasR: C20-CoA complex,confirm the functionality and key role of the ligand in FasR-DNA interaction. In summary, we show thatlong-chain acyl-CoAs are key effector molecules that coordinate the expression of FAS system, bybinding to FasR.The structural and functional characterization of this novel transcriptional regulator will allow us to gainnew insights into the transcriptional regulation to the fatty biosynthesis pathways in M. tuberculosis,and will help as determine if this protein could represent an attractive target for the development ofnew antituberculosis drugs.