CAMP is an allosteric modulator of DNA-binding specificity in the cAMP receptor protein from Mycobacterium tuberculosis

Fernanda Gárate,Stephen Dokas,Maria Fe Lanfranco,Clare Canavan,Irina Wang,John J Correia,Rodrigo A Maillard

doi:10.1016/j.jbc.2021.100480

Fernanda Gárate, Stephen Dokas + Show 5 more

Open Access

https://doi.org/10.1016/j.jbc.2021.100480

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Abstract

Allosteric proteins with multiple subunits and ligand-binding sites are central in regulating biological signals. The cAMP receptor protein from Mycobacterium tuberculosis (CRPMTB) is a global regulator of transcription composed of two identical subunits, each one harboring structurally conserved cAMP- and DNA-binding sites. The mechanisms by which these four binding sites are allosterically coupled in CRPMTB remain unclear. Here, we investigate the binding mechanism between CRPMTB and cAMP, and the linkage between cAMP and DNA interactions. Using calorimetric and fluorescence-based assays, we find that cAMP binding is entropically driven and displays negative cooperativity. Fluorescence anisotropy experiments show that apo-CRPMTB forms high-order CRPMTB–DNA oligomers through interactions with nonspecific DNA sequences or preformed CRPMTB–DNA complexes. Moreover, we find that cAMP prevents and reverses the formation of CRPMTB–DNA oligomers, reduces the affinity of CRPMTB for nonspecific DNA sequences, and stabilizes a 1-to-1 CRPMTB–DNA complex, but does not increase the affinity for DNA like in the canonical CRP from Escherichia coli (CRPEcoli). DNA-binding assays as a function of cAMP concentration indicate that one cAMP molecule per homodimer dissociates high-order CRPMTB–DNA oligomers into 1-to-1 complexes. These cAMP-mediated allosteric effects are lost in the double-mutant L47P/E178K found in CRP from Mycobacterium bovis Bacille Calmette-Guérin (CRPBCG). The functional behavior, thermodynamic stability, and dimerization constant of CRPBCG are not due to additive effects of L47P and E178K, indicating long-range interactions between these two sites. Altogether, we provide a previously undescribed archetype of cAMP-mediated allosteric regulation that differs from CRPEcoli, illustrating that structural homology does not imply allosteric homology.

Highlights

Present address for Fernanda Gárate: Unidad de Microscopia Avanzanda, Pontificia Universidad Catolica de Chile, Santiago, Chile
We investigated the double-mutant L47P/E178K, which is found in the cAMP receptor protein (CRP) from the attenuated Mycobacterium bovis Bacille Calmette-Guérin strain (CRPBCG) and only differs from the CRP from Mycobacterium tuberculosis (CRPMTB) sequence in those two amino acid residues [28,29,30]
The control titration, where only the buffer was added instead of the protein, shows negligible changes in anisotropy. These results suggest that the formation of high-order CRPMTB–DNA oligomers in the absence of cAMP can be driven by interactions with nonspecific DNA sequences

Summary

Introduction

Solution biophysical and structural studies have shown that cAMP binding to the CRP from Escherichia coli (CRPEcoli) stimulates a large conformational change in the DNA-binding domains (Fig. 1, A and B, top) [10, 13, 15,16,17]. The CRPEcoli and CRPMTB display structural differences in both the apo-state and cAMP-bound state, most notably in their DNA-binding domain orientations relative to the cAMP-binding domains (Fig. 1C). In the CRPEcoli, the two subunits in the apo-state are symmetric, but the cAMP-bound state shows asymmetry between the DNA-binding domains’ conformation [18] (Fig. 1D, top). The subunits in the CRPMTB in the apo-state are asymmetric at the dimer interface helix (c-helix) and the DNA-binding domains, but the cAMP-bound state is highly symmetric (Fig. 1D, bottom).

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