Autoinhibitory mechanism and activity-related structural changes in a mycobacterial adenylyl cyclase

Barathy, D.V.; Bharambe, Nikhil; Syed, Wajeed; Zaveri, Anisha; Visweswariah, Sandhya S.; Colaςo, Melwin; Misquith, Sandra; Suguna, K.

doi:10.1016/j.jsb.2015.04.013

Cited by 4 publications

(9 citation statements)

References 27 publications

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“…Our electron density maps are most consistent with a water molecule occupying the ion A position, which explains our observation that calcium does not support catalytic activity. Despite the absence of low-affinity catalytical ion A, the formation of NC dimers in its fully active conformation is well supported [4,11,36].…”

Section: Cagcágtpáca 2+ Active Sitementioning

confidence: 94%

Molecular basis for GTP recognition by light‐activated guanylate cyclase RhGC

et al. 2019

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Cyclic guanosine 3 0 ,5 0-monophosphate (cGMP) is an intracellular signalling molecule involved in many sensory and developmental processes. Synthesis of cGMP from GTP is catalysed by guanylate cyclase (GC) in a reaction analogous to cAMP formation by adenylate cyclase (AC). Although detailed structural information is available on the catalytic region of nucleotidyl cyclases (NCs) in various states, these atomic models do not provide a sufficient explanation for the substrate selectivity between GC and AC family members. Detailed structural information on the GC domain in its active conformation is largely missing, and no crystal structure of a GTP-bound wild-type GC domain has been published to date. Here, we describe the crystal structure of the catalytic domain of rhodopsin-GC (RhGC) from Catenaria anguillulae in complex with GTP at 1.7 A resolution. Our study reveals the organization of a eukaryotic GC domain in its active conformation. We observe that the binding mode of the substrate GTP is similar to that of AC-ATP interaction, although surprisingly not all of the interactions predicted to be responsible for base recognition are present. The structure provides insights into potential mechanisms of substrate discrimination and activity regulation that may be common to all class III purine NCs. Database Structural data are available in Protein Data Bank database under the accession number 6SIR. Enzymes EC 4.6.1.2. Abbreviations AC, adenylate cyclase; ATP, adenosine-5 0-triphosphate; cAMP, cyclic 3 0 ,5 0-adenosine monophosphate; cGMP, cyclic 3 0 ,5 0-guanosine monophosphate; GC, guanylate cyclase; GTP, guanosine-5 0-triphosphate; NC, nucleotidyl cyclase; sGC, soluble guanylate cyclase.

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Section: Cagcágtpáca 2+ Active Sitementioning

confidence: 94%

Molecular basis for GTP recognition by light‐activated guanylate cyclase RhGC

et al. 2019

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“…Ma1120 CHD and its triple mutant (Ma1120 CHD (KDA→EGY)) were cloned in pPROEX‐HT vector using inverse PCR as described previously . Both the proteins were expressed in high levels (~ 15–20 mg·L −1 ) in E. coli after induction by 0.5 m m isopropyl β‐ d ‐1‐thiogalactopyranoside (Calbiochem) and were purified using nickel‐nitrilotriacetic acid resin (GE Healthcare Life Science) . Eluted fractions of the proteins were desalted using HiTrap Desalting Columns (GE Healthcare Life Science) with desalting buffer (50 m m Tris/Cl at pH 8.2, 500 m m NaCl, 10% glycerol, 5 m m β‐mercaptoethanol).…”

Section: Methodsmentioning

confidence: 99%

“…Cyclase assays were carried out with 800 n m proteins in 50 m m Tris/Cl buffer at pH 8.0 containing 50 m m NaCl, 10% glycerol and 10 m m free Mn 2+ by the method described previously , in the presence of varied concentrations of the substrate (0–2 m m ATP/GTP). cAMP and cGMP produced by the enzyme were detected using the EIA reagents from Cayman Chemicals (Ann Arbor, MI, USA).…”

Section: Methodsmentioning

confidence: 99%

“…Cloning, protein expression and purification Ma1120 CHD and its triple mutant (Ma1120 CHD (KDA? EGY)) were cloned in pPROEX-HT vector using inverse PCR as described previously [21]. Both the proteins were expressed in high levels (~15-20 mgÁL À1 ) in E. coli after induction by 0.5 mM isopropyl b-D-1-thiogalactopyranoside (Calbiochem) and were purified using nickel-nitrilotriacetic acid resin (GE Healthcare Life Science) [21].…”

Section: Methodsmentioning

confidence: 99%

“…This mutant shows about fourfold increase in GC activity and a threefold decrease in AC activity (Table ). Previously, we reported the structures of Ma1120‐∆N52 in a monomeric form (residues 53–275) and the complexes of the active dimeric form of the cyclase homology domain of the enzyme (Ma1120 CHD : residues 106–275) with 2′,5′‐dideoxy‐3′‐ATP and pyrophosphate along with metal ions . To understand the mechanism of discrimination and mode of binding of ATP or GTP by Ma1120 enzyme, we determined the crystal structures of Ma1120 CHD and its mutant Ma1120 CHD (KDA→EGY) with ATP and GTP.…”

Section: Introductionmentioning

confidence: 99%

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