Alpha7 Helix Plays an Important Role in the Conformational Stability of PTP1B

Abstract

The C-terminus of Protein Tyrosine Phosphatase 1B (PTP1B) includes an α-helix (α7), which forms an allosteric binding site 20 Å away from the active site. This helix is specific to PTP1B and its truncation decreases the catalytic activity significantly. Here, molecular dynamics (MD) simulations in the presence and absence of α7 were performed to investigate the role played by α7. The highly mobile α7 was found to maintain its contacts with loop 11 (L11)- α3 helix throughout the simulations. The interactions of Tyr152 on L11, Tyr176, Thr177 on the catalytically important WPD loop and Ser190 on α3 are important for the conformational stability and the concerted motions of the regions surrounding the WPD loop. In the absence of α7, L11 and WPD loop move away from their crystal structure conformations, resulting in the loss of the interactions in this region, and a decrease in the residue displacement correlations in the vicinity of WPD loop. Therefore, we suggest that one of the functionally important roles of α7 may be to limit the L11 and α3 motions, and, facilitate the WPD loop motions. Truncation of α7 in PTP1B is found to affect distant regions as well, such as the substrate recognition site and the phosphate binding-loop (P-loop), changing the conformations of these regions significantly. Our results show that the PTP1B specific α7 is important for the conformation and dynamics of the WPD loop, and also may play a role in ligand binding.     

Desensitization and Internalization of Endothelin Receptor A: IMPACT OF G PROTEIN-COUPLED RECEPTOR KINASE 2 (GRK2)-MEDIATED PHOSPHORYLATION*

Endothelin receptor A (ET_A), a G protein-coupled receptor, mediates endothelin signaling, which is regulated by GRK2. Three Ser and seven Thr residues recently proven to be phosphoacceptor sites are located in the C-terminal extremity (CTE) of the receptor following its palmitoylation site. We created various phosphorylation-deficient ET_A mutants. The phospholipase C activity of mutant receptors in HEK-293 cells was analyzed during continuous endothelin stimulation to investigate the impact of phosphorylation sites on ET_A desensitization. Total deletion of phosphoacceptor sites in the CTE affected proper receptor regulation. However, proximal and distal phosphoacceptor sites both turned out to be sufficient to induce WT-like desensitization. Overexpression of the Gα_q coupling-deficient mutant GRK2-D110A suppressed ET_A-WT signaling but failed to decrease phospholipase C activity mediated by the phosphorylation-deficient mutant ET_A-6PD. In contrast, GRK2-WT acted on both receptors, whereas the kinase-inactive mutant GRK2-D110A/K220R failed to inhibit signaling of ET_A-WT and ET_A-6PD. This demonstrates that ET_A desensitization involves at least two autonomous GRK2-mediated components: 1) a phosphorylation-independent signal decrease mediated by blocking of Gα_q and 2) a mechanism involving phosphorylation of Ser and Thr residues in the CTE of the receptor in a redundant fashion, able to incorporate either proximal or distal phosphoacceptor sites. High level transfection of GRK2 variants influenced signaling of ET_A-WT and ET_A-6PD and hints at an additional phosphorylation-independent regulatory mechanism. Furthermore, internalization of mRuby-tagged receptors was observed with ET_A-WT and the phosphorylation-deficient mutant ET_A-14PD (lacking 14 phosphoacceptor sites) and turned out to be based on a phosphorylation-independent mechanism.     

Protease-activated Receptor 1 (PAR1) and PAR4 Heterodimers Are Required for PAR1-enhanced Cleavage of PAR4 by α-Thrombin

Thrombin is a potent platelet agonist that activates platelets and other cells of the cardiovascular system by cleaving its G-protein-coupled receptors, protease-activated receptor 1 (PAR1), PAR4, or both. We now show that cleaving PAR1 and PAR4 with α-thrombin induces heterodimer formation. PAR1-PAR4 heterodimers were not detected when unstimulated; however, when the cells were stimulated with 10 nm α-thrombin, we were able to detect a strong interaction between PAR1 and PAR4 by bioluminescence resonance energy transfer. In contrast, activating the receptors without cleavage using PAR1 and PAR4 agonist peptides (TFLLRN and AYPGKF, respectively) did not enhance heterodimer formation. Preventing PAR1 or PAR4 cleavage with point mutations or hirugen also prevented the induction of heterodimers. To further characterize the PAR1-PAR4 interactions, we mapped the heterodimer interface by introducing point mutations in transmembrane helix 4 of PAR1 or PAR4 that prevented heterodimer formation. Finally, we show that mutations in PAR1 or PAR4 at the heterodimer interface prevented PAR1-assisted cleavage of PAR4. These data demonstrate that PAR1 and PAR4 require allosteric changes induced via receptor cleavage by α-thrombin to mediate heterodimer formation, and we have determined the PAR1-PAR4 heterodimer interface. Our findings show that PAR1 and PAR4 have dynamic interactions on the cell surface that should be taken into account when developing and characterizing PAR antagonists.     

Population Shift Underlies Ca2+-induced Regulatory Transitions in the Sodium-Calcium Exchanger (NCX)

In eukaryotic Na⁺/Ca²⁺ exchangers (NCX) the Ca²⁺ binding CBD1 and CBD2 domains form a two-domain regulatory tandem (CBD12). An allosteric Ca²⁺ sensor (Ca3–Ca4 sites) is located on CBD1, whereas CBD2 contains a splice-variant segment. Recently, a Ca²⁺-driven interdomain switch has been described, albeit how it couples Ca²⁺ binding with signal propagation remains unclear. To resolve the dynamic features of Ca²⁺-induced conformational transitions we analyze here distinct splice variants and mutants of isolated CBD12 at varying temperatures by using small angle x-ray scattering (SAXS) and equilibrium ⁴⁵Ca²⁺ binding assays. The ensemble optimization method SAXS analysis demonstrates that the apo and Mg²⁺-bound forms of CBD12 are highly flexible, whereas Ca²⁺ binding to the Ca3–Ca4 sites results in a population shift of conformational landscape to more rigidified states. Population shift occurs even under conditions in which no effect of Ca²⁺ is observed on the globally derived D_max (maximal interatomic distance), although under comparable conditions a normal [Ca²⁺]-dependent allosteric regulation occurs. Low affinity sites (Ca1–Ca2) of CBD1 do not contribute to Ca²⁺-induced population shift, but the occupancy of these sites by 1 mm Mg²⁺ shifts the Ca²⁺ affinity (K_d) at the neighboring Ca3–Ca4 sites from ∼ 50 nm to ∼ 200 nm and thus, keeps the primary Ca²⁺ sensor (Ca3–Ca4 sites) within a physiological range. Thus, Ca²⁺ binding to the Ca3–Ca4 sites results in a population shift, where more constraint conformational states become highly populated at dynamic equilibrium in the absence of global conformational transitions in CBD alignment.     

Propofol Binding to the Resting State of the Gloeobacter violaceus Ligand-gated Ion Channel (GLIC) Induces Structural Changes in the Inter- and Intrasubunit Transmembrane Domain (TMD) Cavities

General anesthetics exert many of their CNS actions by binding to and modulating membrane-embedded pentameric ligand-gated ion channels (pLGICs). The structural mechanisms underlying how anesthetics modulate pLGIC function remain largely unknown. GLIC, a prokaryotic pLGIC homologue, is inhibited by general anesthetics, suggesting anesthetics stabilize a closed channel state, but in anesthetic-bound GLIC crystal structures the channel appears open. Here, using functional GLIC channels expressed in oocytes, we examined whether propofol induces structural rearrangements in the GLIC transmembrane domain (TMD). Residues in the GLIC TMD that frame intrasubunit and intersubunit water-accessible cavities were individually mutated to cysteine. We measured and compared the rates of modification of the introduced cysteines by sulfhydryl-reactive reagents in the absence and presence of propofol. Propofol slowed the rate of modification of L240C (intersubunit) and increased the rate of modification of T254C (intrasubunit), indicating that propofol binding induces structural rearrangements in these cavities that alter the local environment near these residues. Propofol acceleration of T254C modification suggests that in the resting state propofol does not bind in the TMD intrasubunit cavity as observed in the crystal structure of GLIC with bound propofol (Nury, H., Van Renterghem, C., Weng, Y., Tran, A., Baaden, M., Dufresne, V., Changeux, J. P., Sonner, J. M., Delarue, M., and Corringer, P. J. (2011) Nature 469, 428–431). In silico docking using a GLIC closed channel homology model suggests propofol binds to intersubunit sites in the TMD in the resting state. Propofol-induced motions in the intersubunit cavity were distinct from motions associated with channel activation, indicating propofol stabilizes a novel closed state.