G-protein-coupled receptors (GPCRs) are important membrane proteins that mediate cellular signaling and represent primary targets for about one-third of currently marketed drugs. Recent x-ray crystallographic studies identified distinct conformations of GPCRs in the active and inactive states. An allosteric sodium ion was found bound to a highly conserved D2.50 residue in inactive GPCRs, whereas the D2.50 allosteric pocket became collapsed in active GPCR structures. However, the dynamic mechanisms underlying these observations remain elusive. In this study, we aimed to understand the mechanistic effects of sodium ion binding on dynamic activation of the M3 muscarinic GPCR through long-timescale accelerated molecular dynamics (aMD) simulations. Results showed that with the D2.50 residue deprotonated, the M3 receptor is bound by an allosteric sodium ion and confined mostly in the inactive state with remarkably reduced flexibility. In contrast, the D2.50-protonated receptor does not exhibit sodium ion binding to the D2.50 allosteric site and samples a significantly larger conformational space. The receptor activation is captured and characterized by large-scale structural rearrangements of the transmembrane helices via dynamic hydrogen bond and salt bridge interactions. The residue motions are highly correlated during receptor activation. Further network analysis revealed that the allosteric signaling between residue D2.50 and key residues in the intracellular, extracellular, and orthosteric pockets is significantly weakened upon sodium ion binding.

The activation/deactivation processes for G-protein coupled receptors (GPCRs) have been computationally studied for several different classes, including rhodopsin, the b2 adrenergic receptor, and the M2 muscarinic receptor. Despite determined cocrystal structures of the adenosine A 2A receptor (A 2A AR) in complex with antagonists, agonists and an antibody, the deactivation process of this GPCR is not completely understood. In this study, we investigate the convergence of two apo simulations, one starting with an agonist-bound conformation (PDB: 3QAK) 14 and the other starting with an antagonist-bound conformation (PDB: 3EML) 11. Despite the two simulations not completely converging, we were able to identify distinct intermediate steps of the deactivation process characterized by the movement of Y288 7.53 in the NPxxY motif. We find that Y288 7.53 contributes to the process by forming hydrogen bonds to residues in transmembrane helices 2 and 7 and losing these interactions upon full deactivation. Y197 5.58 also plays a role in the process by forming a hydrogen bond only once the side chain moves from the lipid interface to the middle of the helical bundle.

Atypical protein kinase C (aPKC) isozymes are unique in the protein kinase C (PKC) superfamily in that they are not regulated by the lipid second messenger diacylglycerol. Whether a different second messenger acutely controls their function is unknown. Here we show that the lipid mediator, sphingosine 1-phosphate (S1P), controls the cellular activity of aPKC. Using a genetically-encoded reporter we designed, aPKC-specific C Kinase Activity Reporter (aCKAR), we demonstrate that intracellular S1P activates aPKC. Biochemical studies reveal that S1P directly binds to the kinase domain of aPKC to relieve autoinhibitory constraints. In silico studies identify potential binding sites on the kinase domain, one of which was validated biochemically. Lastly, functional studies reveal that S1P-dependent activation of aPKC suppresses apoptosis in HeLa cells. Taken together, our data reveal a previously undescribed molecular mechanism for controlling the cellular activity of atypical PKC and identify a new molecular target for S1P.

The A adenosine receptor (A AR) is a G protein-coupled receptor that is pharmacologically targeted for the treatment of inflammation, sepsis, cancer, neurodegeneration, and Parkinson's disease. Recently, we applied long-timescale molecular dynamics simulations on two ligand-free receptor conformations, starting from the agonist-bound (PDB ID: 3QAK) and antagonist-bound (PDB ID: 3EML) X-ray structures. This analysis revealed four distinct conformers of the A AR: the active, intermediate 1, intermediate 2, and inactive. In this study, we apply the fragment-based mapping algorithm, FTMap, on these receptor conformations to uncover five non-orthosteric sites on the A AR. Two sites that are identified in the active conformation are located in the intracellular region of the transmembrane helices (TM) 3/TM4 and the G protein-binding site in the intracellular region between TM2/TM3/TM6/TM7. Three sites are identified in the intermediate 1 and intermediate 2 conformations, annexing a site in the lipid interface of TM5/TM6. Five sites are identified in the inactive conformation, comprising a site in the intracellular region of TM1/TM7 and in the extracellular region of TM3/TM4 of the A AR. We postulate that these sites on the A AR be screened for allosteric modulators for the treatment of inflammatory and neurological diseases.

Activation of the first sphingosine-1-phosphate receptor (S1PR ) promotes permeability of the blood brain barrier, astrocyte and neuronal protection, and lymphocyte egress from secondary lymphoid tissues. Although an agonist often activates the S1PR , the receptor exhibits high levels of basal activity. In this study, we performed long-timescale molecular dynamics and accelerated molecular dynamics (aMD) simulations to investigate activation mechanisms of the ligand-free (apo) S1PR . In the aMD enhanced sampling simulations, we observed four independent events of activation, which is characterized by close interaction between Y311 and Y221 and increased distance between the intracellular ends of transmembrane (TM) helices 3 and 6. Although TM helices TM3, TM6, TM5 and, TM7 are associated with GPCR activation, we discovered that their movements are not necessarily correlated during activation. Instead, TM5 showed a decreased correlation with each of these regions during activation. During activation of the apo receptor, Y221 and Y311 became more solvated, because a water channel formed in the intracellular pocket. Additionally, a lipid molecule repeatedly entered the receptor between the extracellular ends of TM1 and TM7, providing important insights into the pathway of ligand entry into the S1PR .

It is well known that cells, especially cancer cells, have an ability of evasion of apoptosis by cellular stress like nutrient starvation. And the balance between apoptosis signal and apoptosis‐resistant signal will determine the fate of cells, dead or alive. Here we found new cell signaling system that plays a role in applying the brakes to cell death that in case cancer cells avoid apoptosis by cellular stress like starvation using newly developed biosensor and in silico docking simulation technique. The new cell signaling system is that second messenger, sphingosine 1‐phosphate (S1P), directly activates a key cell signaling protein, atypical protein kinase C (aPKC). First, we found that the inhibition of aPKC induces apoptosis of cancer cell lines. Next, for making clear the molecular mechanism of the aPKC‐induced apoptosis resistance, we generated a genetically encoded reporter with the same modular structure as the original C kinase activity reporter (CKAR) but with a unique substrate sequence that allows specific visualization of atypical PKC activity in cells. Using the atypical PKC‐specific CKAR (aCKAR) we found that intracellular S1P induces the activation of atypical PKC in an S1P receptor‐independent manner. Biochemical studies revealed that S1P directly binds to the kinase domain of atypical PKC isozymes, relieving autoinhibitory constraints to activate the enzyme. In silico docking studies were used to identify potential binding sites for aPKC, one of which was validated by biochemical and aCKAR imaging techniques. Now we got new insights about the player of evasion of apoptosis in cancer at the molecular level, and it has potential for development of new molecular‐targeted agents to release brakes against cell death. Support or Funding Information This study was supported by NIH to A.C.N., JSPS KAKENHI, Kobe University Grant for Japan‐US Collaboration, Nakatani Foundation Grant for Technology Development Research to T.K., NIH, HHMI, NBCR, NSF to J.A.M., JSPS KAKENHI to S.N. and T.O. A.D.C. was supported in part by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology.

The protein kinase C (PKC) family comprises 9 members; conventional PKC isozymes (α, β γ), novel PKC isozymes (δ, ɛ, θ, η), and atypical PKC isozymes (ζ, ι/λ). It is well established that conventional and novel PKC isozymes are activated by second messengers, diacylglycerol and, for the conventional PKC isozymes, calcium ion. However the activation mechanism and physiological function of atypical PKC isozymes are less well understood. Here we show that a lipid mediator, sphingosine 1‐phosphate (S1P) controls the cellular activity of atypical PKC isozymes. First, we generated a genetically encoded reporter with the same modular structure as the original C kinase activity reporter (CKAR) but with a unique substrate sequence that allows specific visualization of atypical PKC activity in cells. Using the atypical PKC‐specific CKAR (aCKAR) we found that intracellular S1P induces the activation of atypical PKC in an S1P receptor‐independent manner. Biochemical studies reveal that S1P directly binds to the kinase domain of atypical PKC isozymes, relieving autoinhibitory constraints to activate the enzyme. In silico docking studies were used to identify potential binding sites for aPKC, one of which was validated biochemically. Our results are consistent with a model in which S1P binds the kinase domain of atypical PKC isozymes, an event that releases autoinhibitory constraints to lock the PKC in an active conformation. Our results provide a new molecular mechanism for controlling the cellular activity of atypical PKC.Support or Funding InformationThis study was supported by NIH to A.C.N., NIH, HHMI, NBCR, NSF to J.A.M., JSPS KAKENHI, Kobe University Grant for Japan‐US Collaboration, Nakatani Foundation Grant for Technology Development Research to T.K., JSPS KAKENHI to S.N. A.D.C. was supported in part by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.