1H, 13C and 15N NMR assignments of the C1A and C1B subdomains of PKC-delta

Ziemba, Brian P.; Jamie, C. Booth; David, Jones N. M.

doi:10.1007/s12104-010-9283-0

Cited by 4 publications

References 21 publications

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Structural and Functional Characterization of an Anesthetic Binding Site in the Second Cysteine-Rich Domain of Protein Kinase Cδ∗

Shanmugasundararaj

Das

Sandberg

et al. 2012

Biophysical Journal

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Elucidating the principles governing anesthetic-protein interactions requires structural determinations at high resolutions not yet achieved with ion channels. Protein kinase C (PKC) activity is modulated by general anesthetics. We solved the structure of the phorbol-binding domain (C1B) of PKCδ complexed with an ether (methoxymethylcycloprane) and with an alcohol (cyclopropylmethanol) at 1.36-Å resolution. The cyclopropane rings of both agents displace a single water molecule in a surface pocket adjacent to the phorbol-binding site, making van der Waals contacts with the backbone and/or side chains of residues Asn-237 to Ser-240. Surprisingly, two water molecules anchored in a hydrogen-bonded chain between Thr-242 and Lys-260 impart elasticity to one side of the binding pocket. The cyclopropane ring takes part in π-acceptor hydrogen bonds with the amide of Met-239. There is a crucial hydrogen bond between the oxygen atoms of the anesthetics and the hydroxyl of Tyr-236. A Tyr-236-Phe mutation results in loss of binding. Thus, both van der Waals interactions and hydrogen-bonding are essential for binding to occur. Ethanol failed to bind because it is too short to benefit from both interactions. Cyclopropylmethanol inhibited phorbol-ester-induced PKCδ activity, but failed to do so in PKCδ containing the Tyr-236-Phe mutation.

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Structural and Functional Characterization of an Anesthetic Binding Site in the Second Cysteine-Rich Domain of Protein Kinase Cδ∗

Shanmugasundararaj

Das

Sandberg

et al. 2012

Biophysical Journal

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Toward a Biorelevant Structure of Protein Kinase C Bound Modulators: Design, Synthesis, and Evaluation of Labeled Bryostatin Analogues for Analysis with Rotational Echo Double Resonance NMR Spectroscopy

et al. 2015

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Protein kinase C (PKC) modulators are currently of great importance in preclinical and clinical studies directed at cancer, immunotherapy, HIV eradication, and Alzheimer’s disease. However, the bound conformation of PKC modulators in a membrane environment is not known. Rotational Echo Double Resonance (REDOR) NMR spectroscopy could uniquely address this challenge. However, REDOR NMR requires strategically-labeled, high affinity ligands to determine inter-label distances from which the conformation of the bound ligand in the PKC-ligand complex could be identified. Here we report the first computer-guided design and syntheses of three bryostatin analogs strategically-labeled for REDOR NMR analysis. Extensive computer analyses of energetically-accessible analog conformations suggested preferred labeling sites for the identification of the PKC-bound conformers. Significantly, three labeled analogs were synthesized, and, as required for REDOR analysis, all proved highly potent with PKC affinities (~1 nM) on par with bryostatin. These potent and strategically-labeled bryostatin analogs are new structural leads and provide the necessary starting point for projected efforts to determine the PKC-bound conformation of such analogs in a membrane environment, as needed to design new PKC modulators and understand PKC-ligand–membrane structure and dynamics.

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Structural anatomy of Protein Kinase C C1 domain interactions with diacylglycerol and other agonists

et al. 2022

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Diacylglycerol (DAG) is a versatile lipid whose 1,2-sn-stereoisomer serves both as second messenger in signal transduction pathways that control vital cellular processes, and as metabolic precursor for downstream signaling lipids such as phosphatidic acid. Effector proteins translocate to available DAG pools in the membranes by using conserved homology 1 (C1) domains as DAG-sensing modules. Yet, how C1 domains recognize and capture DAG in the complex environment of a biological membrane has remained unresolved for the 40 years since the discovery of Protein Kinase C (PKC) as the first member of the DAG effector cohort. Herein, we report the high-resolution crystal structures of a C1 domain (C1B from PKCδ) complexed to DAG and to each of four potent PKC agonists that produce different biological readouts and that command intense therapeutic interest. This structural information details the mechanisms of stereospecific recognition of DAG by the C1 domains, the functional properties of the lipid-binding site, and the identities of the key residues required for the recognition and capture of DAG and exogenous agonists. Moreover, the structures of the five C1 domain complexes provide the high-resolution guides for the design of agents that modulate the activities of DAG effector proteins.

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1H, 13C and 15N NMR assignments of the C1A and C1B subdomains of PKC-delta

Cited by 4 publications

References 21 publications

Structural and Functional Characterization of an Anesthetic Binding Site in the Second Cysteine-Rich Domain of Protein Kinase Cδ∗

Structural and Functional Characterization of an Anesthetic Binding Site in the Second Cysteine-Rich Domain of Protein Kinase Cδ∗

Toward a Biorelevant Structure of Protein Kinase C Bound Modulators: Design, Synthesis, and Evaluation of Labeled Bryostatin Analogues for Analysis with Rotational Echo Double Resonance NMR Spectroscopy

Structural anatomy of Protein Kinase C C1 domain interactions with diacylglycerol and other agonists

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