930 zyxwvutsrqpo Biochemistry 1995,34, 930-939 Mechanism of Ca-ATPase Inhibition by Melittin in Skeletal Sarcoplasmic Reticulum? John C. Voss, James E. Mahaney, and David D. Thomas* Department zyxwvutsr of Biochemistry, University of Minnesota Medical School, Minneapolis, Minnesota 55455 Received June zyxwvuts 20, 1994; Revised Manuscript Received October 12, 1994@ ABSTRACT: We have previously shown that the basic, amphipathic peptide melittin inhibits the Ca-ATPase of the sarcoplasmic reticulum membrane by inducing large-scale aggregation of the enzyme via electrostatic cross-linking. To better understand the physical mechanism by which melittin-induced Ca-ATPase aggregation inhibits the enzyme, we have performed time-resolved phosphorescence anisotropy (TPA) and steady-state fluorescence experiments in combination with enzyme kinetic assays, utilizing (1) native and charge-modified melittin in order to characterize the peptide charge dependence of the melittin-SR interaction, and (2) various calcium levels in order to define the effect of melittin on the enzyme’s E l and E2 conformational equilibrium. TPA results showed that decreasing melittin’s positive charge dramatically decreases the ability of the peptide to aggregate the enzyme, which correlates with a reduced potency of the modified peptide to inhibit enzymatic activity. Steady-state fluorescence of fluorescein isothiocyanate- labeled Ca-ATPase showed that melittin reduces Ca-ATPase affinity for calcium by shifting the enzyme’s El -E2 conformational equilibrium toward E2, but increasing calcium progressively reverses this shift. Kinetic experiments showed that melittin does not prevent ATP-dependent enzyme phosphorylation, but it completely inhibits Pi-dependent EP formation and substantially slows Pi release during steady-state cycling. We conclude that melittin-induced aggregation of the Ca-ATPase depends on the electrostatic interaction of the peptide with cytoplasmic Ca2+-dependent sites on the enzyme, and that enforced Ca- ATPase protein-protein interactions inhibit the conformational transitions that facilitate phosphoenzyme hydrolysis. The Ca-ATPase in fast-twitch skeletal sarcoplasmic reticu- lum (SR)’ is a transmembrane protein of approximately 110 kD, which couples the transport of 2 mol of Ca2+across the SR membrane per mole of ATP hydrolyzed (Inesi, 1985). A thoroughly tested and widely accepted model for the Ca- ATPase enzymatic cycle is shown in Scheme 1. In this model, the enzyme cycles between two fundamental con- formations, El and E2, which couple ATP hydrolysis to calcium transport via differences in their affinities and vectorial specificities for ATP and Ca2+. In the absence of substrates and/or ligands, the enzyme is in equilibrium between E l and E2 (step 8), but micromolar calcium shifts this conformational equilibrium zyxwvutsr [& x 1 x 10l2 M-2 (Alonso & Hecht, 1990)] strongly toward El, forming Ca2*E1 (Inesi, 1985; Froud & Lee, 1986; Wakabayashi et al., 1990). ATP binds rapidly and with high affinity to Ca2*E1 [step 1, (DuPont, 1980)], and a conformationalchange [CayEl*ATP to CayEl’*ATP (step 2)] activates the enzyme (Coan & Inesi; 1977; Petithory & Jencks, 1986; Obara et al., 1988; Lewis & Thomas, 1992) for ATP-dependent phosphoenzyme formation (Froehlich & Taylor, 1975; Petithory & Jencks, 1986). Calcium is translocated across the membrane (step t This work was supported by a grant to D.D.T. from the National Institutes of Health (GM27906). J.E.M. was supported by a Grant-in- Aid from the American Heart Association, Minnesota Affiliate. * To whom correspondence should be addressed. @ Abstract published in Advance ACS Absfructs, December 1, 1994. Abbreviations: SR, sarcoplasmic reticulum; MOPS, 34N- mor- pholino)propanesulfonic acid; ATP, adenosine triphosphate; EGTA, ethylene glycol bis@-aminoethyl ether)-NJQV”-tetraacetic acid; TPA, transient phosphorescence anisotropy; ErITC, erythrosin-5-isothio- cyanate. 0006-2960/95/0434-930$09 zyxwvut .OO/O Scheme 1 MgATP ADP 2Ca~i\ca~i .ATPL!EI~.ATP~E~ 2 P . 1 4) by a conformational change that isomerizes CayElP to CayE2P (Froehlich & Heller, 1985). Following the release of calcium (step 5) from the enzyme into the SR lumen (Beeler & Keffer, 1984), E2P is hydrolyzed (step 6) to E2Pi (de Meis, 1988; Wictome et al., 1992; Obara et al., 1988), after which Pi and Mg2+are released [step 7 (de Meis, 1988)l. Extensive physical studies of skeletal SR have shown that the rotational dynamics of the Ca-ATPase have a substantial impact on Ca-ATPase activity (Thomas & Mahaney, 1993; Thomas & Karon, 1994), due to a requirement for dynamic changes in the oligomeric state of the enzyme during key steps the enzymatic cycle (Mahaney et al., 1994; Kaon et al., 1994; Karon & Thomas, 1993; Bigelow et al., 1992; Martonosi et al., 1990; Squier & Thomas, 1988; Vanderkooi et al., 1977). Saturation transfer EPR studies of Ca-ATPase rotational dynamics (Thomas & Hidalgo, 1978; Bigelow et al., 1986) have shown that enzymatic activity correlates with the protein’s rotational mobility, such that perturbations that decrease (increase) the Ca-ATPase mobility inhibit (enhance) enzymatic activity (Lewis & Thomas, 1986; Bigelow & 0 1995 American Chemical Society