Toggling of Diacylglycerol Affinity Correlates with Conformational Plasticity in C1 Domains

Stewart, Mark; Igumenova, Tatyana I.

doi:10.1021/acs.biochem.7b00228

Cited by 9 publications

(9 citation statements)

References 19 publications

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“…the parameter that defines the intrinsic thresholds of DAG-mediated activation (Extended discussion 2). Our structural data (supported by previous NMR work [23][24][25] ) suggest that higher hydrophobicity and lipophilicity of Trp confers thermodynamic advantages and hence higher DAG-sensitivity to the C1B domains of novel PKC isoforms relative to conventional PKC isoforms that carry a Tyr at the equivalent position.…”

Section: Comparative Analyses Of the C1bd-agonist Structuressupporting

confidence: 81%

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Structural anatomy of C1 domain interactions with DAG and other agonists

Katti

Krieger

Ann

et al. 2021

Preprint

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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. DAG effector proteins compete for available lipid 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 first high-resolution crystal structures of a C1 domain (C1B from PKCdelta) 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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Section: Comparative Analyses Of the C1bd-agonist Structuressupporting

confidence: 81%

“…According to solution NMR, this region undergoes conformational exchange on the µs-timescale 23,25 . Apo C1Bd was also crystallized under conditions similar to those for the C1Bd-DAG complex.…”

Section: Structure Of the C1bd-dag Complexmentioning

confidence: 99%

Structural anatomy of C1 domain interactions with DAG and other agonists

Katti

Krieger

Ann

et al. 2021

Preprint

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“…The most variable region is located between helix α1 and the C-terminal Cysteine residue that coordinates a structural Zn 2+ ion. According to solution NMR, this region undergoes conformational exchange on the μs-ms timescale 21 , 23 . We crystallized apo C1Bδ under conditions similar to those used for the C1Bδ-DAG complex (but without detergent) for a direct comparison.…”

Section: Resultsmentioning

confidence: 99%

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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“…A 13 C label was installed at the C12-OAc carbonyl position, and 2 H was incorporated as a CD 3 group on the C13-OAc, where the REDOR-determined distance would extend from the C12 carbonyl to the average position of the three deuterons ( Figure 1 B). Labeled ligands were complexed with a 51-residue peptide corresponding to the mouse PKCδ-C1b domain, 38 allowing comparison of previous structural studies 39 − 50 using this isoform domain, which recapitulates the ligand binding capabilities of the full-length protein. 51 The folding and function of PKC and its C1 domain are sensitive to its environment, and in the case of membrane-associated PKC, we have shown that the C1 domain does not fold properly for binding in water alone but only in association with a PS vesicle.…”

Section: Results and Discussionmentioning

confidence: 99%

REDOR NMR Reveals Multiple Conformers for a Protein Kinase C Ligand in a Membrane Environment

et al. 2018

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Bryostatin 1 (henceforth bryostatin) is in clinical trials for the treatment of Alzheimer’s disease and for HIV/AIDS eradication. It is also a preclinical lead for cancer immunotherapy and other therapeutic indications. Yet nothing is known about the conformation of bryostatin bound to its protein kinase C (PKC) target in a membrane microenvironment. As a result, efforts to design more efficacious, better tolerated, or more synthetically accessible ligands have been limited to structures that do not include PKC or membrane effects known to influence PKC–ligand binding. This problem extends more generally to many membrane-associated proteins in the human proteome. Here, we use rotational-echo double-resonance (REDOR) solid-state NMR to determine the conformations of PKC modulators bound to the PKCδ-C1b domain in the presence of phospholipid vesicles. The conformationally limited PKC modulator phorbol diacetate (PDAc) is used as an initial test substrate. While unanticipated partitioning of PDAc between an immobilized protein-bound state and a mobile state in the phospholipid assembly was observed, a single conformation in the bound state was identified. In striking contrast, a bryostatin analogue (bryolog) was found to exist exclusively in a protein-bound state, but adopts a distribution of conformations as defined by three independent distance measurements. The detection of multiple PKCδ-C1b-bound bryolog conformers in a functionally relevant phospholipid complex reveals the inherent dynamic nature of cellular systems that is not captured with single-conformation static structures. These results indicate that binding, selectivity, and function of PKC modulators, as well as the design of new modulators, are best addressed using a dynamic multistate model, an analysis potentially applicable to other membrane-associated proteins.

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Toggling of Diacylglycerol Affinity Correlates with Conformational Plasticity in C1 Domains

Cited by 9 publications

References 19 publications

Structural anatomy of C1 domain interactions with DAG and other agonists

Structural anatomy of C1 domain interactions with DAG and other agonists

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

REDOR NMR Reveals Multiple Conformers for a Protein Kinase C Ligand in a Membrane Environment

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