title: Cyclic AMP-induced conformational changes in mycobacterial protein acetyltransferases.
abstract: The activities of a number of proteins are regulated by the binding of cAMP and cGMP to cyclic nucleotide binding (CNB) domains that are found associated with one or more effector domains with diverse functions. Although the conserved architecture of CNB domains has been extensively studied by x-ray crystallography, the key to unraveling the mechanisms of cAMP action has been protein dynamics analyses. Recently, we have identified a novel cAMP-binding protein from mycobacteria, where cAMP regulates the activity of an associated protein acetyltransferase domain. In the current study, we have monitored the conformational changes that occur upon cAMP binding to the CNB domain in these proteins, using a combination of bioluminescence resonance energy transfer and amide hydrogen/deuterium exchange mass spectrometry. Coupled with mutational analyses, our studies reveal the critical role of the linker region (positioned between the CNB domain and the acetyltransferase domain) in allosteric coupling of cAMP binding to activation of acetyltransferase catalysis. Importantly, major differences in conformational change upon cAMP binding were accompanied by stabilization of the CNB and linker domain alone. This is in contrast to other cAMP-binding proteins, where cyclic nucleotide binding has been shown to involve intricate and parallel allosteric relays. Finally, this powerful convergence of results from bioluminescence resonance energy transfer and hydrogen/deuterium exchange mass spectrometry reaffirms the power of solution biophysical tools in unraveling mechanistic bases of regulation of proteins in the absence of high resolution structural information.
authors.affiliation: Department of Molecular Reproduction, Development, and Genetics, Indian Institute of Science, Bangalore, India.
authors.forename: Subhalaxmi
authors.initials: S
authors.lastname: Nambi
authors.forename: Suguna
authors.initials: S
authors.lastname: Badireddy
authors.forename: Sandhya S
authors.initials: SS
authors.lastname: Visweswariah
authors.forename: Ganesh S
authors.initials: GS
authors.lastname: Anand
mesh_terms: D000107:Acetylation; D000123:Acetyltransferases / Q000737:chemistry* / Q000378:metabolism; D000495:Allosteric Site; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism / Q000502:physiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000201:enzymology*; D011485:Protein Binding; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D000242:Cyclic AMP; D000123:Acetyltransferases