Novel pppGpp binding site at the C-terminal region of the Rel enzyme from Mycobacterium smegmatis

Syal, Kirtimaan and Joshi, Himanshu and Chatterji, Dipankar and Jain, Vikas (2015) Novel pppGpp binding site at the C-terminal region of the Rel enzyme from Mycobacterium smegmatis. In: FEBS JOURNAL, 282 (19). pp. 3773-3785.

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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.

Item Type:	Journal Article
Publication:	FEBS JOURNAL
Publisher:	WILEY-BLACKWELL
Additional Information:	Copy right for this article belongs to the WILEY-BLACKWELL, 111 RIVER ST, HOBOKEN 07030-5774, NJ USA
Keywords:	(p)ppGpp; mass spectrometry; nucleoside/nucleotide analog; photoaffinity labeling; protein-ligand interaction
Department/Centre:	Division of Biological Sciences > Molecular Biophysics Unit
Date Deposited:	05 Nov 2015 07:10
Last Modified:	23 Oct 2018 11:23
URI:	http://eprints.iisc.ac.in/id/eprint/52684

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