title: Novel pppGpp binding site at the C-terminal region of the Rel enzyme from Mycobacterium smegmatis.
abstract: Mycobacterium tuberculosis elicits the stringent response under unfavorable growth conditions, such as those encountered by the pathogen inside the host. The hallmark of this response is production of guanosine tetra- and pentaphosphates, collectively termed (p)ppGpp, which have pleiotropic effects on the bacterial physiology. As the stringent response is connected to survival under stress, it is now being targeted for developing inhibitors against bacterial persistence. The Rel enzyme in mycobacteria has two catalytic domains at its N-terminus that are involved in the synthesis and hydrolysis of (p)ppGpp, respectively. However, the function of the C-terminal region of the protein remained unknown. Here, we have identified a binding site for pppGpp in the C-terminal region of Rel. The binding affinity of pppGpp was quantified by isothermal titration calorimetry. The binding site was determined by crosslinking using the nucleotide analog azido-pppGpp, and examining the crosslink product by mass spectrometry. Additionally, mutations in the Rel protein were created to confirm the site of pppGpp binding by isothermal titration calorimetry. These mutants showed increased pppGpp synthesis and reduced hydrolytic activity. We believe that binding of pppGpp to Rel provides a feedback mechanism that allows the protein to detect and adjust the (p)ppGpp level in the cell. Our work suggests that such sites should also be considered while designing inhibitors to target the stringent response.
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
authors.forename: Kirtimaan
authors.initials: K
authors.lastname: Syal
authors.affiliation: Microbiology and Molecular Biology Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, India.
authors.forename: Himanshu
authors.initials: H
authors.lastname: Joshi
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
authors.forename: Dipankar
authors.initials: D
authors.lastname: Chatterji
authors.affiliation: Microbiology and Molecular Biology Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, India.
authors.forename: Vikas
authors.initials: V
authors.lastname: Jain
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002151:Calorimetry / Q000379:methods; D003432:Cross-Linking Reagents / Q000737:chemistry; D006158:Guanosine Pentaphosphate / Q000378:metabolism*; D008025:Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D020102:Mycobacterium smegmatis / Q000201:enzymology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D006158:Guanosine Pentaphosphate; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases
keywords: (p)ppGpp; mass spectrometry; nucleoside/nucleotide analog; photoaffinity labeling; protein-ligand interaction