Tryptophan 1093 Is Largely Responsible for the Slow Off Rate of Calmodulin from Plasma Membrane Ca2+ Pump 4b (original) (raw)
Related papers
Interaction of calmodulin with the calmodulin binding domain of the plasma membrane calcium pump
Biochemistry, 1990
Peptides corresponding to the calmodulin binding domain of the plasma membrane Ca2+ pump (James et al., 1988) were synthesized, and their interaction with calmodulin was studied with circular dichroism, infrared spectroscopy, nuclear magnetic resonance, and fluorescence techniques. They corresponded to the complete calmodulin binding domain (28 residues), to its first 15 or 20 amino acids, and to its C-terminal 14 amino acids. The first three peptides interacted with calmodulin. The K value was similar to that of the intact enzyme in the 28 and 20 amino acid peptides, but increased substantially in the shorter 15 amino acid peptide. The 14 amino acid peptide corresponding to the C-terminal portion of the domain failed to bind calmodulin. 2D N M R experiments on the 20 amino acid peptides have indicated that the interaction occurred with the C-terminal half of calmodulin. A tryptophan that is conserved in most calmodulin binding 'This work has been made possible by the financial contributions of the Swiss Nationalfonds (Grant 3.53 1.086) and the National Institues of Health (Grant GM28835).
Plasma Membrane Ca2+ Pump Isoforms 2a and 2b Are Unusually Responsive to Calmodulin and Ca2+
Journal of Biological Chemistry, 1997
The full-length a and b variants of the rat plasma membrane calcium pump, isoform 2 (rPMCA2a and rPMCA2b), were constructed and expressed in COS-7 cells. To characterize these isoforms, calcium transport was determined in a microsomal fraction. Both rPMCA2a and rPMCA2b had a much higher affinity for calmodulin than the corresponding forms of hPMCA4, and rPMCA2b had the highest affinity among the isoforms that have been tested so far. When analyzed at a relatively high calmodulin concentration, rPMCA2b and, to a lesser extent, rPMCA2a showed higher apparent calcium affinity; i.e. they were more active at lower Ca 2؉ concentrations than hPMCA4b. This indicates that these two variants of rat isoform 2 will tend to maintain a lower free cytosolic Ca 2؉ level in cells where they are expressed. Both variants also showed a higher level of basal activity (in the complete absence of calmodulin) than hPMCA4b, a property which would reinforce their ability to maintain a low free cytosolic Ca 2؉ concentration. Experiments designed to determine the source of the higher apparent Ca 2؉ affinity of rPMCA2b showed that it came from the properties of the carboxyl terminus, rather than from any difference in the catalytic core.
Journal of Biological Chemistry, 2007
The inhibition by the regulatory domain and the interaction with calmodulin (CaM) vary among plasma membrane calcium pump (PMCA) isoforms. To explore these differences, the kinetics of CaM effects on PMCA4a were investigated and compared with those of PMCA4b. The maximal apparent rate constant for CaM activation of PMCA4a was almost twice that for PMCA4b, whereas the rates of activation for both isoforms showed similar dependence on Ca 2؉. The inactivation of PMCA4a by CaM removal was also faster than for PMCA4b, and Ca 2؉ showed a much smaller effect (2versus 30-fold modification). The rate constants of the individual steps that determine the overall rates were obtained from stopped-flow experiments in which binding of TA-CaM was observed by changes in its fluorescence. TA-CaM binds to two conformations of PMCA4a, an "open" conformation with high activity, and a "closed" one with lower activity. Compared with PMCA4b (
The Biochemical journal, 1998
A mutant of the plasma membrane Ca2+ pump (PMCA) called (nCI)hPMCA4b(ct120), in which the C-terminal regulatory segment including the calmodulin-binding autoinhibitory domains C and I had been relocated near the N-terminus, has been expressed in COS-1 cells. The measurements of Ca2+ transport in microsomal preparations showed that the rearranged enzyme was functional. The activity of the (nCI)hPMCA4b(ct120) mutant was compared with those of the wild-type hPMCA4b and the fully active calmodulin-insensitive mutant hPMCA4b(ct120). In the absence of calmodulin the activity of (nCI)hPMCA4b(ct120) was higher than that of hPMCA4b but only 45% of that of hPMCA4b(ct120). Mutant (nCI)hPMCA4b(ct120) exhibited an apparent affinity for Ca2+ similar to that of hPMCA4b, typical of the inhibited state of the enzyme. Calmodulin at concentrations that fully activated hPMCA4b increased the activity of (nCI)hPMCA4b(ct120) to 68% of that of hPMCA4b(ct120). The lower maximal activity of (nCI)hPMCA4b(ct12...
Journal of Biological Chemistry, 2007
The inhibition by the regulatory domain and the interaction with calmodulin (CaM) vary among plasma membrane calcium pump (PMCA) isoforms. To explore these differences, the kinetics of CaM effects on PMCA4a were investigated and compared with those of PMCA4b. The maximal apparent rate constant for CaM activation of PMCA4a was almost twice that for PMCA4b, whereas the rates of activation for both isoforms showed similar dependence on Ca 2؉. The inactivation of PMCA4a by CaM removal was also faster than for PMCA4b, and Ca 2؉ showed a much smaller effect (2versus 30-fold modification). The rate constants of the individual steps that determine the overall rates were obtained from stopped-flow experiments in which binding of TA-CaM was observed by changes in its fluorescence. TA-CaM binds to two conformations of PMCA4a, an "open" conformation with high activity, and a "closed" one with lower activity. Compared with PMCA4b (
Intra- and Interdomain Effects Due to Mutation of Calcium-binding Sites in Calmodulin
Journal of Biological Chemistry, 2010
The IQ-motif protein PEP-19, binds to the C-domain of calmodulin (CaM) with significantly different k on and k off rates in the presence and absence of Ca 2؉ , which could play a role in defining the levels of free CaM during Ca 2؉ transients. The initial goal of the current study was to determine whether Ca 2؉ binding to sites III or IV in the C-domain of CaM was responsible for affecting the kinetics of binding PEP-19. EF-hand Ca 2؉binding sites were selectively inactivated by the common strategy of changing Asp to Ala at the X-coordination position. Although Ca 2؉ binding to both sites III and IV appeared necessary for native-like interactions with PEP-19, the data also indicated that the mutations caused undesirable structural alterations as evidenced by significant changes in amide chemical shifts for apoCaM. Mutations in the C-domain also affected chemical shifts in the unmodified N-domain, and altered the Ca 2؉ binding properties of the N-domain. Conversion of Asp 93 to Ala caused the greatest structural perturbations, possibly due to the loss of stabilizing hydrogen bonds between the side chain of Asp 93 and backbone amides in apo loop III. Thus, although these mutations inhibit binding of Ca 2؉ , the mutated CaM may not be able to support potentially important native-like activity of the apoprotein. This should be taken into account when designing CaM mutants for expression in cell culture.
Plasma Membrane Ca2+ Pump Isoform 3f Is Weakly Stimulated by Calmodulin
Journal of Biological Chemistry, 2000
Isoform 3f of the plasma membrane Ca 2؉ pump is a major isoform of this pump in rat skeletal muscle. It has an unusual structure, with a short carboxyl-terminal regulatory region of only 33 residues when compared with the 77 to 124 residues found in the other isoforms. Also, whereas the regulatory regions of the other isoforms, downstream of the alternative splice, consist of two homologous groups, the sequence of 3f is not related to either group. A synthetic peptide representing the calmodulin binding domain of isoform 3f had a much lower calmodulin affinity (with a K d of 15 nM) than the corresponding peptide of isoform 2b (K d value was 0.2 nM). The characteristics of this domain were further studied by making chimeras of the 3f regulatory region with the catalytic core of isoform 4 and by making the full-length isoform 3f. Both constructs bound to calmodulin-Sepharose. The chimera was fully active without calmodulin, showing no stimulation of activity when calmodulin was added. The full-length isoform 3f was slightly activated by calmodulin. These data show that the regulatory region of isoform 3f is only a weak autoinhibitor of the enzyme, in contrast to the properties of all the other isoforms studied so far. Rather, this isoform is a special-purpose, constitutively active form of the enzyme, expressed primarily in skeletal muscle and as a minor isoform in brain.
Biochemistry, 1991
Peptide-induced conformational changes in five isofunctional mutants of calmodulin (CaM), each bearing a single tryptophan residue either at the seventh position of each of the four calcium-binding loops (Le., amino acids 26, 62, 99, and 135) or in the central helix (amino acid 81) were studied by using fluorescence spectroscopy. The peptides RS20F and RS20CK correspond to CaM-binding amino acid sequence segments of either nonmuscle myosin light chain kinase (nmMLCK) or calmodulin-dependent protein kinase I1 (CaMPK-11), respectively. Both steady-state and time-resolved fluorescence data were collected from the various peptide-CaM complexes. Steady-state fluorescence intensity measurements indicated that, in the presence of an excess of calcium, both peptides bind to the calmodulin mutants with a 1:l stoichiometry. The tryptophans located in loops I and IV exhibited red-shifted emission maxima (356 nm), high quantum yields (0.3), and long average lifetimes (6 ns). They responded in a similar manner to peptide binding, by only slight changes in their fluorescence features. In contrast, the fluorescence intensity of the tryptophans in loops I1 and I11 decreased markedly, and their fluorescence spectrum was blue-shifted upon peptide binding. Analysis of the tryptophan fluorescence decay of the last mentioned calmodulins supports a model in which the equilibrium between two (Trp-99) or three (Trp-62) states of these tryptophan residues, each characterized by a different lifetime, was altered toward the blue-shifted short lifetime component upon peptide binding. Taken together, these data provide new evidence that both lobes of calmodulin are involved in peptide binding. Both peptides induced similar changes in the fluorescence properties of the tryptophan residues located in the calcium-binding loops, with the exception of calmodulin with Trp-135. For this last mentioned calmodulin, slight differences were observed. Tryptophan in the central helix responded differently to RS2OF and RS20CK binding. RS20F binding induced a red-shift in the emission maximum of Trp-8 1 while RS20CK induced a blue-shift. The quenching rate of Trp-8 1 by iodide was slightly reduced upon RS20CK binding, while RS20F induced a 2-fold increase. These results provide evidence that the environment of Trp-81 is different in each case and are, therefore, consistent with the hypothesis that the central helix can play a differential role in the recognition of, or response to, CaM-binding structures. Calmodulin (CaM)' is a ubiquitous protein involved in the regulation of a wide variety of calcium-dependent intracellular processes as varied as motivity, secretion, glycogen metabolism, synaptic transmission, transport of anions, and cyclic nucleotide metabolism [for reviews, see Rasmussen et al. (1984) and Manalan and Klee (1 984)]. Upon calcium binding, calmodulin apparently undergoes conformational changes, which enable it to bind to and activate target enzymes such as calmodulin-stimulated phosphodiesterase, plasma membrane Ca2+ pump, myosin light chain kinase, and calmodulin-dependent protein kinase 11 (Cox et al., 1984; Stoclet et al., 1987). The interaction of calmodulin with its target enzymes is very little known at the molecular level [for a review, see Blumenthal and Krebs (1 988)]. It is thought that most calmodulin-dependent enzymes contain specific regions that recognize and bind to calmodulin with high affinity. Such putative calmodulin-binding sites have been found in, e.g., skeletal muscle