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

Abstract

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.

Laboratory-Based X-ray Absorption Spectroscopy on a Working Pouch Cell Battery at Industrially-Relevant Charging Rates

“…39 By tracking the redox behavior of the Ni atoms, the state-of-charge of the battery may be directly assessed and, if desired, be constrained to a range which avoids the formation of undesirable Ni species. In the present instance, comparison to previously reported empirical standards 3,40,41 suggest that the present Ni oxidation state is between +2 and +3 in the discharged state and between +3 and +4 at full charge. Moreover, a strictly lateral displacement devoid of any isosbestic points suggests that the entirety of the Ni atoms evolve in concert rather than forming a linear combination, indicating a solid solution behavior rather than a mixed-phase behavior.…”

Synthesis and evaluation of designed PKC modulators for enhanced cancer immunotherapy

“…In a separate study, a combination of REDOR NMR and molecular dynamics simulations identified a distribution of PKC-bound bryostatin conformers (Fig. 1c), a feature that was unique to the bryostatin scaffold and not observed in a phorbol ester derivative 56 . Together, these studies suggest that the multiple PKC-bound conformers available to the bryostatin scaffold could generate differential orientations of the activated PKC-ligand complex and influence PKC's interactions with downstream effector proteins, thereby explaining bryostatin's unique biological activity.…”

“…Our computational studies revealed that hydrogen-bonding functionalities around the C-ring subunit of the bryostatin scaffold, specifically the C1 carbonyl, C26 hydroxyl and C19 hemiketal, are spatially preorganized by the A and B rings into a binding conformation that mimics PKC's endogenous ligand, DAG ( Fig. 1a) [54][55][56]61 . Indeed, seminal studies by our group showed that modification or deletion of any of the C1, C26 and C19 functionalities reduced or eliminated PKC binding while changes in the A-and B-rings generally had little or no effect on binding, suggesting that changes to these subunits could be used to change physical properties, biological function and biodistribution and thereby potentially improve efficacy and tolerability 20,54,[61][62][63][64] .…”

“…Design and synthesis of bryostatin analogs. Our FOS design strategy for bryostatin analogs was guided by our previously proposed bryostatin pharmacophore model 54 and further supported by recent molecular dynamics simulations 55 and REDOR NMR structural studies 56 . PKC is a family of seven homologous signaling kinases classified as conventional (α, β, γ) or novel (δ, ε, η, θ) based on their subdomain architectures 57 .…”

“…Cell-free ligand binding to PKC is a necessary but not sufficient prerequisite to elicit function, as a ligand must enter the cell and engage PKC to form a functional PKC-ligand membrane complex. Both experimental studies and molecular dynamics simulations suggest that the PKC-ligand complex can assume multiple bound states, putatively influencing the differential association of scaffolding proteins and phosphorylation of downstream effector proteins, thereby resulting in diverse biological outcomes 18,[55][56][57]60 . Therefore, our next goal was to determine whether the most potent PKC binders enter cells and show similar or different dynamic interactions with PKC.…”