The role of N157 of A-ATP synthase subunit B in nucleotide binding and expression, production and structural and mechanistic features of subunits α and ε of Mycobacterium tuberculosis F-ATP synthase

The ATP synthases/ATPases arose from a common ancestor and, therefore, A1AO ATP synthases share properties with the F1FO ATP synthase as present in bacteria, chloroplasts and mitochondria. Although the overall subunit composition and the primary sequence of the major subunits A and B of A1AO ATP synthases is fairly different to F1FO ATP synthases, their function similar to F1FO ATP synthase is to synthesize ATP. The proposed subunit stoichiometry of A1 is A3:B3:C:D:F:(EG)2. The integral AO domain contains subunits a and c. Subunit B is a nucleotide binding regulatory subunit. In the present study, the crystal structure for N157T mutant of P-loop of subunit B has been solved to investigate the role of N157 in nucleotide binding. FCS studies of N157T along with the other mutants H156A, N157A and WT-B revealed that the binding affinities of H156A and N156A for ATP are stronger than those of N157T and WT-B. This feature could be attributed to the fact that the size of the binding cavity has increased on substitution with a residue containing a smaller side chain. Similar binding studies with ADP showed that in comparison to the WT-B, the mutants lost the ability to distinguish between the nucleotides, validating the importance of the histidine and asparagine in the P-loop of subunit B. Subunit α of F-ATP synthase, the homologous subunit of subunit B, in spite of being considered as a non-catalytic subunit does bind nucleotides. Interestingly, subunit α of Mycobacterium tuberculosis (Mtα) contains a unique stretch of polar residues in its C-terminus and this is exclusive only to the species belonging to the Mycobacterium genus. Focus has been directed towards obtaining structural information on Mtα. To understand the role of its C-terminus, a chimeric subunit of α from M. tuberculosis and Bacillus PS3 (PS3) has been generated with residues 1-502 of Bacillus PS3 and residues 515-549 of M. tuberculosis. The same chimeric protein of subunit α is generated in a complex of PS3α3β3γ to give αchi3β3γ and is studied to understand the role of the fused C-terminus of Mtα in the ATP hydrolysing activity. The elongated shape and length of Mtα led to the speculation that this is an adaptation to compensate for the shorter length of subunit e in Mycobacteria. Subunit e of the F-ATP synthases is essential for the coupling of F1 and FO domains. This subunit exists in two conformations, compact and extended. In the extended state subunit e functions as the intrinsic inhibitor for ATP hydrolysis and hence importantly, prevents wasteful hydrolysis to conserve cellular ATP levels. The same is observed for the F-ATP synthase of M. tuberculosis, where ATP hydrolysis is reduced during the latent phase in the course of infection of the pathogen. Encouraging ATP hydrolysis to occur will eventually result in cell death and therefore, subunit e presents an interesting drug target for TB. The disease is difficult to tackle, given the emergence of multi drug resistant strains and hence there is an urgent need for new drug leads and targets.…