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. 2017 Feb 23;8(1):6.
doi: 10.1038/s41467-016-0015-8.

Molecular Dynamics Simulations Reveal Ligand-Controlled Positioning of a Peripheral Protein Complex in Membranes

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Free PMC article

Molecular Dynamics Simulations Reveal Ligand-Controlled Positioning of a Peripheral Protein Complex in Membranes

Steven M Ryckbosch et al. Nat Commun. .
Free PMC article

Abstract

Bryostatin is in clinical trials for Alzheimer's disease, cancer, and HIV/AIDS eradication. It binds to protein kinase C competitively with diacylglycerol, the endogenous protein kinase C regulator, and plant-derived phorbol esters, but each ligand induces different activities. Determination of the structural origin for these differing activities by X-ray analysis has not succeeded due to difficulties in co-crystallizing protein kinase C with relevant ligands. More importantly, static, crystal-lattice bound complexes do not address the influence of the membrane on the structure and dynamics of membrane-associated proteins. To address this general problem, we performed long-timescale (400-500 µs aggregate) all-atom molecular dynamics simulations of protein kinase C-ligand-membrane complexes and observed that different protein kinase C activators differentially position the complex in the membrane due in part to their differing interactions with waters at the membrane inner leaf. These new findings enable new strategies for the design of simpler, more effective protein kinase C analogs and could also prove relevant to other peripheral protein complexes.Natural supplies of bryostatin, a compound in clinical trials for Alzheimer's disease, cancer, and HIV, are scarce. Here, the authors perform molecular dynamics simulations to understand how bryostatin interacts with membrane-bound protein kinase C, offering insights for the design of bryostatin analogs.

Figures

Fig. 1
Fig. 1
Structures of compounds simulated. Clockwise from top left: bryostatin (1), PDBu (2), prostratin (4), and a bryolog (3). Atom numberings of bryostatin are labeled, as well as bryostatin’s A, B, and C rings. Bryostatin is in the clinic for the treatment of Alzheimer’s disease and for HIV/AIDS eradication. Prostratin shows PKC selectivities similar to bryostatin (albeit with lower affinities) and is a preclinical lead for HIV eradication. PDBu is similar in structure to prostratin, but unlike it is representative of the tumor-promoting phorbol diesters. Bryolog 3, synthesized by Keck and coworkers, is structurally similar to bryostatin itself but has been shown to behave quite differently in biological assays
Fig. 2
Fig. 2
Representative snapshots of PKCδ C1b–bryostatin–membrane complex. Green, blue, and red surfaces correspond to hydrophobic, basic, and acidic residues, respectively. In the more deeply embedded structure a, many hydrophobic residues are able to reside in the hydrocarbon region of the membrane, while the vertical orientation allows the cationic residues along the side of the protein to interact with the anionic headgroups. In the shallower orientation b, fewer hydrophobic residues reside in the membrane, but more cationic residues are able to interact with the anionic headgroups. Such snapshots are similar for the PDBu and ligand-free systems
Fig. 3
Fig. 3
Free energy as a function of depth and angle of peptide in membrane. The panels represent the free energies of the peptide in the presence of various ligands (ad) and without a ligand (e), reported in kcal mol−1. Note that bryostatin c and (to a lesser extent) prostratin b possess two low-free energy wells, one shallow and one deep, while PDBu a and bryolog 3 d only show one free energy minimum, deeply embedded in the membrane. The dashed line indicates the pseudo plane of phosphorus atoms in the bottom leaflet, and distance 0 is the plane through the center of the membrane. Distance is measured from an average of the positions of the alpha carbons of M9, T12, L21, and V25, all atoms near the binding site. The angle is found by first creating a line through this point and an average of the positions of the alpha carbons of F3, G35, N48, and the zinc ion coordinated by H1, C31, C34, and C50, and calculating the angle of this line with the membrane plane. This line goes approximately through the middle of the relatively cylindrical peptide. The range of the 95% confidence interval as determined by bootstrapping simulations was less than 0.1 kcal mol−1 for all configurations and all ligands. See Supplementary Fig. 4 and the Supplementary Discussion for further discussion of error bars for these free energies
Fig. 4
Fig. 4
Structured and unstructured waters coordinating bryostatin in the shallow binding mode. Waters are highlighted for clarity. Note the coordination of a water with C9 and C3 OH groups, as well as SER10 side chain and a lipid carbonyl oxygen. The C3 OH is also hydrogen bonding with the SER10 backbone amide proton. A structured water also coordinates with C19 OH. Unstructured waters from bulk solvent hydrogen bond as well with the C13 Z-enoate. Solvation of both of these moieties provides stabilization of shallow orientation and a barrier to insertion more deeply into the membrane. Other PKC-binding ligands lack such water-coordinating moieties, and therefore only favor the deeply inserted state. Conversely, altering substitution at deeply embedded C7 and C8 can substantially abrogate binding activity altogether (see Supplementary Fig. 5). The conformation shown of bryostatin is the predominant one of the simulations; see Supplementary Figs. 6 and 7 and Supplementary Discussion for more discussion of bryostatin’s conformations

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