Browsing by Subject "Protein Conformation"

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    Open Access
    Asparagine 706 and Glutamate 183 at the Catalytic Site of Sarcoplasmic Reticulum Ca 2+ -ATPase Play Critical but Distinct Roles in E 2 States
    (2006) Clausen, Johannes D; McIntosh, David B; Woolley, David G; Anthonisen, Anne Nyholm; Vilsen, Bente; Andersen, Jens Peter
    Mutants with alteration to Asn(706) of the highly conserved (701)TGDGVND(707) motif in domain P of sarcoplasmic reticulum Ca(2+)-ATPase were analyzed for changes in transport cycle kinetics and binding of the inhibitors vanadate, BeF, AlF, and MgF. The fluorides likely mimic the phosphoryl group/P(i) in the respective ground, transition, and product states of phosphoenzyme hydrolysis (Danko, S., Yamasaki, K., Daiho, T., and Suzuki, H. (2004) J. Biol. Chem. 279, 14991-14998). Binding of BeF, AlF, and MgF was also studied for mutant Glu(183) --> Ala, where the glutamate of the (181)TGES(184) motif in domain A is replaced. Mutations of Asn(706) and Glu(183) have in common that they dramatically impede the function of the enzyme in E2 states, but have little effect in E1. Contrary to the Glu(183) mutant, in which E2P slowly accumulates (Clausen, J. D., Vilsen, B., McIntosh, D. B., Einholm, A. P., and Andersen, J. P. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 2776-2781), E2P formation was not detectable with the Asn(706) mutants. Differential sensitivities of the mutants to inhibition by AlF, MgF, and BeF made it possible to distinguish different roles of Asn(706) and Glu(183). Hence, Asn(706) is less important than Glu(183) for gaining the transition state during E2P hydrolysis but plays critical roles in stabilization of E2P ground and E2.P(i) product states and in the major conformational changes associated with the Ca(2)E1P --> E2P and E2 --> Ca(2)E1 transitions, which seem to be facilitated by interaction of Asn(706) with domain A.
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    Open Access
    Structural Studies of a Stabilized Phosphoenzyme Intermediate of Ca 2+ -ATPase
    (2005) Stokes, David L; Delavoie, Franck; Rice, William J; Champeil, Philippe; McIntosh, David B; Lacapère, Jean-Jacques
    Ca(2+)-ATPase belongs to the family of P-type ATPases and maintains low concentrations of intracellular Ca(2+). Its reaction cycle consists of four main intermediates that alternate ion binding in the transmembrane domain with phosphorylation of an aspartate residue in a cytoplasmic domain. Previous work characterized an ultrastable phosphoenzyme produced first by labeling with fluorescein isothiocyanate, then by allowing this labeled enzyme to establish a maximal Ca(2+) gradient, and finally by removing Ca(2+) from the solution. This phosphoenzyme is characterized by very low fluorescence and has specific enzymatic properties suggesting the existence of a high energy phosphoryl bond. To study the structural properties of this phosphoenzyme, we used cryoelectron microscopy of two-dimensional crystals formed in the presence of decavanadate and determined the structure at 8-A resolution. To our surprise we found that at this resolution the low fluorescence phosphoenzyme had a structure similar to that of the native enzyme crystallized under equivalent conditions. We went on to use glutaraldehyde cross-linking and proteolysis for independent structural assessment and concluded that, like the unphosphorylated native enzyme, Ca(2+) and vanadate exert a strong influence over the global structure of this low fluorescence phosphoenzyme. Based on a structural model with fluorescein isothiocyanate bound at the ATP site, we suggest that the stability as well as the low fluorescence of this phosphoenzyme is due to a fluorescein-mediated cross-link between two cytoplasmic domains that prevents hydrolysis of the aspartyl phosphate. Finally, we consider the alternative possibility that phosphate transfer to fluorescein itself could explain the properties of this low fluorescence species.
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    Open Access
    The Functional Microdomain in Transmembrane Helices 2 and 7 Regulates Expression, Activation, and Coupling Pathways of the Gonadotropin-releasing Hormone Receptor
    (1999) Flanagan, Colleen A; Zhou, Wei; Chi, Ling; Yuen, Tony; Rodic, Vladimir; Robertson, Derek; Johnson, Melanie; Holland, Pamela; Millar, Robert P; Weinstein, Harel; Mitchell, Rory; Sealfon, Stuart C
    Structural microdomains of G protein-coupled receptors (GPCRs) consist of spatially related side chains that mediate discrete functions. The conserved helix 2/helix 7 microdomain was identified because the gonadotropin-releasing hormone (GnRH) receptor appears to have interchanged the Asp(2.50) and Asn(7.49) residues which are conserved in transmembrane helices 2 and 7 of rhodopsin-like GPCRs. We now demonstrate that different side chains of this microdomain contribute specifically to receptor expression, heterotrimeric G protein-, and small G protein-mediated signaling. An Asn residue is required in position 2.50(87) for expression of the GnRH receptor at the cell surface, most likely through an interaction with the conserved Asn(1.50(53)) residue, which we also find is required for receptor expression. Most GPCRs require an Asp side chain at either the helix 2 or helix 7 locus of the microdomain for coupling to heterotrimeric G proteins, but the GnRH receptor has transferred the requirement for an acidic residue from helix 2 to 7. However, the presence of Asp at the helix 7 locus precludes small G protein-dependent coupling to phospholipase D. These results implicate specific components of the helix 2/helix 7 microdomain in receptor expression and in determining the ability of the receptor to adopt distinct activated conformations that are optimal for interaction with heterotrimeric and small G proteins.