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Malonate in the nucleotide-binding site traps human AKAP18?/? in a novel conformational state.

ABSTRACT: A-kinase anchoring proteins (AKAPs) are a family of proteins that provide spatiotemporal resolution of protein kinase A (PKA) phosphorylation. In the myocardium, PKA and AKAP18?/? are found in complex with sarcoendoplasmic reticulum Ca(2+)-ATPase 2 (SERCA2) and phospholamban (PLB). This macromolecular complex provides a means by which anchored PKA can dynamically regulate cytoplasmic Ca(2+) release and re-uptake. For this reason, AKAP18?/? presents an interesting drug target with therapeutic potential in cardiovascular disease. The crystal structure of the central domain of human AKAP18? has been determined at the atomic resolution of 1.25?Å. This first structure of human AKAP18? is trapped in a novel conformation by a malonate molecule bridging the important R-loop with the 2H phosphoesterase motif. Although the physiological substrate of AKAP18? is currently unknown, a potential proton wire deep in the central binding crevice has been indentified, leading to bulk solvent below the R-loop. Malonate complexed with AKAP18? at atomic resolution provides an excellent starting point for structure-guided drug design.

SUBMITTER: Bjerregaard-Andersen K

PROVIDER: S-EPMC4973299 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Malonate in the nucleotide-binding site traps human AKAP18γ/δ in a novel conformational state.

Bjerregaard-Andersen Kaare K Østensen Ellen E Scott John D JD Taskén Kjetil K Morth Jens Preben JP

Acta crystallographica. Section F, Structural biology communications 20160713 Pt 8

A-kinase anchoring proteins (AKAPs) are a family of proteins that provide spatiotemporal resolution of protein kinase A (PKA) phosphorylation. In the myocardium, PKA and AKAP18γ/δ are found in complex with sarcoendoplasmic reticulum Ca(2+)-ATPase 2 (SERCA2) and phospholamban (PLB). This macromolecular complex provides a means by which anchored PKA can dynamically regulate cytoplasmic Ca(2+) release and re-uptake. For this reason, AKAP18γ/δ presents an interesting drug target with therapeutic pot ...[more]

PMID: 27487922

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Project description:A central step in the gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the association of its two cytosolic nucleotide-binding domains (NBDs) into a head-to-tail dimer, with two nucleotides bound at the interface. Channel opening and closing, respectively, are coupled to formation and disruption of this tight NBD dimer. CFTR is an asymmetric adenosine triphosphate (ATP)-binding cassette protein in which the two interfacial-binding sites (composite sites 1 and 2) are functionally different. During gating, the canonical, catalytically active nucleotide-binding site (site 2) cycles between dimerized prehydrolytic (state O1), dimerized post-hydrolytic (state O2), and dissociated (state C) forms in a preferential C→O1→O2→C sequence. In contrast, the catalytically inactive nucleotide-binding site (site 1) is believed to remain associated, ATP-bound, for several gating cycles. Here, we have examined the possibility of conformational changes in site 1 during gating, by studying gating effects of perturbations in site 1. Previous work showed that channel closure is slowed, both under hydrolytic and nonhydrolytic conditions, by occupancy of site 1 by N(6)-(2-phenylethyl)-ATP (P-ATP) as well as by the site-1 mutation H1348A (NBD2 signature sequence). Here, we found that P-ATP prolongs wild-type (WT) CFTR burst durations by selectively slowing (>2×) transition O1→O2 and decreases the nonhydrolytic closing rate (transition O1→C) of CFTR mutants K1250A (∼4×) and E1371S (∼3×). Mutation H1348A also slowed (∼3×) the O1→O2 transition in the WT background and decreased the nonhydrolytic closing rate of both K1250A (∼3×) and E1371S (∼3×) background mutants. Neither P-ATP nor the H1348A mutation affected the 1:1 stoichiometry between ATP occlusion and channel burst events characteristic to WT CFTR gating in ATP. The marked effect that different structural perturbations at site 1 have on both steps O1→C and O1→O2 suggests that the overall conformational changes that CFTR undergoes upon opening and coincident with hydrolysis at the active site 2 include significant structural rearrangement at site 1.

Dataset Information

Malonate in the nucleotide-binding site traps human AKAP18?/? in a novel conformational state.

Publications

Malonate in the nucleotide-binding site traps human AKAP18γ/δ in a novel conformational state.

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