Spectroscopic and ITC study of the conformational change upon Ca2+-binding in TnC C-lobe and TnI peptide complex from Akazara scallop striated muscle

Akazara scallop (Chlamys nipponensis akazara) troponin C (TnC) of striated adductor muscle binds only one Ca²⁺ ion at the C-terminal EF-hand motif (Site IV), but it works as the Ca²⁺-dependent regulator in adductor muscle contraction. In addition, the scallop troponin (Tn) has been thought to regulate muscle contraction via activating mechanisms that involve the region spanning from the TnC C-lobe (C-lobe) binding site to the inhibitory region of the TnI, and no alternative binding of the TnI C-terminal region to TnC because of no similarity between second TnC-binding regions of vertebrate and the scallop TnIs. To clarify the Ca²⁺-regulatory mechanism of muscle contraction by scallop Tn, we have analyzed the Ca²⁺-binding properties of the complex of TnC C-lobe and TnI peptide, and their interaction using isothermal titration microcalorimetry, nuclear magnetic resonance, circular dichroism, and gel filtration chromatography. The results showed that single Ca²⁺-binding to the Site IV leads to a structural transition not only in Site IV but also Site III through the structural network in the C-lobe of scallop TnC. We therefore assumed that the effect of Ca²⁺-binding must lead to a change in the interaction mode between the C-lobe of TnC and the TnI peptide. The change should be the first event of the transmission of Ca²⁺ signal to TnI in Tn ternary complex.     

Structural characterization of the catalytic γ and regulatory β subunits of phosphorylase kinase in the context of the hexadecameric enzyme complex

In the tightly regulated glycogenolysis cascade, the breakdown of glycogen to glucose‐1‐phosphate, phosphorylase kinase (PhK) plays a key role in regulating the activity of glycogen phosphorylase. PhK is a 1.3 MDa hexadecamer, with four copies each of four different subunits (α, β, γ and δ), making the study of its structure challenging. Using hydrogen‐deuterium exchange, we have analyzed the regulatory β subunit and the catalytic γ subunit in the context of the intact non‐activated PhK complex to study the structure of these subunits and identify regions of surface exposure. Our data suggest that within the non‐activated complex the γ subunit assumes an activated conformation and are consistent with a previous docking model of the β subunit within the cryoelectron microscopy envelope of PhK.     

Structural basis for the regulation of muscle contraction by troponin and tropomyosin

The molecular switching mechanism governing skeletal and cardiac muscle contraction couples the binding of Ca²⁺ on troponin to the movement of tropomyosin on actin filaments. Despite years of investigation, this mechanism remains unclear because it has not yet been possible to directly assess the structural influence of troponin on tropomyosin that causes actin filaments, and hence myosin-crossbridge cycling and contraction, to switch on and off. A C-terminal domain of troponin I is thought to be intimately involved in inducing tropomyosin movement to an inhibitory position that blocks myosin-crossbridge interaction. Release of this regulatory, latching domain from actin after Ca²⁺ binding to TnC (the Ca²⁺ sensor of troponin that relieves inhibition) presumably allows tropomyosin movement away from the inhibitory position on actin, thus initiating contraction. However, the structural interactions of the regulatory domain of TnI (the “inhibitory” subunit of troponin) with tropomyosin and actin that cause tropomyosin movement are unknown, and thus, the regulatory process is not well defined. Here, thin filaments were labeled with an engineered construct representing C-terminal TnI, and then, 3D electron microscopy was used to resolve where troponin is anchored on actin-tropomyosin. Electron microscopy reconstruction showed how TnI binding to both actin and tropomyosin at low Ca²⁺ competes with tropomyosin for a common site on actin and drives tropomyosin movement to a constrained, relaxing position to inhibit myosin-crossbridge association. Thus, the observations reported reveal the structural mechanism responsible for troponin-tropomyosin-mediated steric interference of actin-myosin interaction that regulates muscle contraction.     

Theoretical model of the interactions between Ca2+, calmodulin and myosin light chain kinase

Active Ca²⁺/calmodulin (CaM)-dependent myosin light chain kinase (MLCK) plays an important role in the process of MLC phosphorylation and consecutive smooth muscle contraction. Here, we propose a mathematical model of a detailed kinetic scheme describing interactions among Ca²⁺, CaM and MLCK and taking into account eight different aggregates. The main model result is the prediction of the Ca²⁺ dependent active form of MLCK, which is in the model taken as proportional to the concentration of Ca₄CaM · MLCK complex. Wegscheider’s condition is additionally applied as a constraint enabling the prediction of some parameter values that have not yet been obtained by experiments.     

The regulatory beta subunit of phosphorylase kinase interacts with glyceraldehyde-3-phosphate dehydrogenase

Skeletal muscle phosphorylase kinase (PhK) is an (alphabetagammadelta) 4 hetero-oligomeric enzyme complex that phosphorylates and activates glycogen phosphorylase b (GP b) in a Ca (2+)-dependent reaction that couples muscle contraction with glycogen breakdown. GP b is PhK's only known in vivo substrate; however, given the great size and multiple subunits of the PhK complex, we screened muscle extracts for other potential targets. Extracts of P/J (control) and I/lnJ (PhK deficient) mice were incubated with [gamma- (32)P]ATP with or without Ca (2+) and compared to identify potential substrates. Candidate targets were resolved by two-dimensional polyacrylamide gel electrophoresis, and phosphorylated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified by matrix-assisted laser desorption ionization mass spectroscopy. In vitro studies showed GAPDH to be a Ca (2+)-dependent substrate of PhK, although the rate of phosphorylation is very slow. GAPDH does, however, bind tightly to PhK, inhibiting at low concentrations (IC 50 approximately 0.45 microM) PhK's conversion of GP b. When a short synthetic peptide substrate was substituted for GP b, the inhibition was negligible, suggesting that GAPDH may inhibit predominantly by binding to the PhK complex at a locus distinct from its active site on the gamma subunit. To test this notion, the PhK-GAPDH complex was incubated with a chemical cross-linker, and a dimer between the regulatory beta subunit of PhK and GAPDH was formed. This interaction was confirmed by the fact that a subcomplex of PhK missing the beta subunit, specifically an alphagammadelta subcomplex, was unable to phosphorylate GAPDH, even though it is catalytically active toward GP b. Moreover, GAPDH had no effect on the conversion of GP b by the alphagammadelta subcomplex. The interactions described herein between the beta subunit of PhK and GAPDH provide a possible mechanism for the direct linkage of glycogenolysis and glycolysis in skeletal muscle.     

Structural Basis for the Activation of Muscle Contraction by Troponin and Tropomyosin

The molecular regulation of striated muscle contraction couples the binding and dissociation of Ca²⁺ on troponin (Tn) to the movement of tropomyosin on actin filaments. In turn, this process exposes or blocks myosin binding sites on actin, thereby controlling myosin crossbridge dynamics and consequently muscle contraction. Using 3D electron microscopy, we recently provided structural evidence that a C-terminal extension of TnI is anchored on actin at low Ca²⁺ and competes with tropomyosin for a common site to drive tropomyosin to the B-state location, a constrained, relaxing position on actin that inhibits myosin-crossbridge association. Here, we show that release of this constraint at high Ca²⁺ allows a second segment of troponin, probably representing parts of TnT or the troponin core domain, to promote tropomyosin movement on actin to the Ca²⁺-induced C-state location. With tropomyosin stabilized in this position, myosin binding interactions can begin. Tropomyosin appears to oscillate to a higher degree between respective B- and C-state positions on troponin-free filaments than on fully regulated filaments, suggesting that tropomyosin positioning in both states is troponin-dependent. By biasing tropomyosin to either of these two positions, troponin appears to have two distinct structural functions; in relaxed muscles at low Ca²⁺, troponin operates as an inhibitor, while in activated muscles at high Ca²⁺, it acts as a promoter to initiate contraction.     

Ca2+-dependent Muscle Dysfunction Caused by Mutation of the Caenorhabditis elegans Troponin T-1 Gene

We have investigated the functions of troponin T (CeTnT-1) in Caenorhabditis elegans embryonic body wall muscle. TnT tethers troponin I (TnI) and troponin C (TnC) to the thin filament via tropomyosin (Tm), and TnT/Tm regulates the activation and inhibition of myosin-actin interaction in response to changes in intracellular [Ca²⁺]. Loss of CeTnT-1 function causes aberrant muscle trembling and tearing of muscle cells from their exoskeletal attachment sites (Myers, C.D., P.-Y. Goh, T. StC. Allen, E.A. Bucher, and T. Bogaert. 1996. J. Cell Biol. 132:1061–1077). We hypothesized that muscle tearing is a consequence of excessive force generation resulting from defective tethering of Tn complex proteins. Biochemical studies suggest that such defective tethering would result in either (a) Ca²⁺-independent activation, due to lack of Tn complex binding and consequent lack of inhibition, or (b) delayed reestablishment of TnI/TnC binding to the thin filament after Ca²⁺ activation and consequent abnormal duration of force. Analyses of animals doubly mutant for CeTnT-1 and for genes required for Ca²⁺ signaling support that CeTnT-1 phenotypes are dependent on Ca²⁺ signaling, thus supporting the second model and providing new in vivo evidence that full inhibition of thin filaments in low [Ca²⁺] does not require TnT.     

Ca-Dependent Photocrosslinking of Tropomyosin Residue 146 to Residues 157-163 in the C-Terminal Domain of Troponin I in Reconstituted Skeletal Muscle Thin Filaments

The Ca²⁺-dependent interaction of troponin I (TnI) with actin·tropomyosin (Tm) in muscle thin filaments is a critical step in the regulation of muscle contraction. Previous studies have suggested that, in the absence of Ca²⁺, TnI interacts with Tm and actin in reconstituted muscle thin filaments, maintaining Tm at the outer domain of actin and blocking myosin-actin interaction. To obtain direct evidence for this Tm-TnI interaction, we performed photochemical crosslinking studies using Tm labeled with 4-maleimidobenzophenone at position 146 or 174 (Tm*146 or Tm*174, respectively), reconstituted with actin and troponin [composed of TnI, troponin T (TnT), and troponin C] or with actin and TnI. After near-UV irradiation, SDS gels of the Tm*146-containing thin filament showed three new high-molecular-weight bands determined to be crosslinked products Tm*146-TnI, Tm*146-troponin C, and Tm*146-TnT using fluorescence-labeled TnI, mass spectrometry, and Western blot analysis. While Tm*146-TnI was produced only in the absence of Ca²⁺, the production of other crosslinked species did not show Ca²⁺ dependence. Tm*174 mainly crosslinked to TnT. In the absence of actin, a similar crosslinking pattern was obtained with a much lower yield. A tryptic peptide from Tm*146-TnI with a molecular mass of 2601.2 Da that was not present in the tryptic peptides of Tm*146 or TnI was identified using HPLC and matrix-assisted laser desorption/ionization time-of-flight. This was shown, using absorption and fluorescence spectroscopy, to be the 4-maleimidobenzophenone-labeled peptide from Tm crosslinked to TnI peptide 157-163. These data, which show that a region in the C-terminal domain of TnI interacts with Tm in the absence of Ca²⁺, support the hypothesis that a TnI-Tm interaction maintains Tm at the outer domain of actin and will help efforts to localize troponin in actin·Tm muscle thin filaments.     

Characterization of novel calmodulin binding domains within IQ motifs of IQGAP1

IQ motif-containing GTPase-activating protein 1 (IQGAP1), which is a well-known calmodulin (CaM) binding protein, is involved in a wide range of cellular processes including cell proliferation, tumorigenesis, adhesion, and migration. Interaction of IQGAP1 with CaM is important for its cellular functions. Although each IQ domain of IQGAP1 for CaM binding has been characterized in a Ca²⁺-dependent or -independent manner, it was not clear which IQ motifs are physiologically relevant for CaM binding in the cells. In this study, we performed immunoprecipitation using 3xFLAGhCaM in mammalian cell lines to characterize the domains of IQGAP1 that are key for CaM binding under physiological conditions. Interestingly, using this method, we identified two novel domains, IQ(2.7–3) and IQ(3.5–4.4), within IQGAP1 that were involved in Ca²⁺-independent or -dependent CaM binding, respectively. Mutant analysis clearly showed that the hydrophobic regions within IQ(2.7–3) were mainly involved in apoCaM binding, while the basic amino acids and hydrophobic region of IQ(3.5–4.4) were required for Ca²⁺/CaM binding. Finally, we showed that IQ(2.7–3) was the main apoCaM binding domain and both IQ(2.7–3) and IQ(3.5–4.4) were required for Ca²⁺/CaM binding within IQ(1-2-3-4). Thus, we identified and characterized novel direct CaM binding motifs essential for IQGAP1. This finding indicates that IQGAP1 plays a dynamic role via direct interactions with CaM in a Ca²⁺-dependent or -independent manner.     

Structure–function relationships of molluscan troponin T revealed by limited proteolysis

Molluscan troponin regulates muscle contraction through a novel Ca²⁺-dependent activating mechanism associated with Ca²⁺-binding to the C-terminal domain of troponin C. To elucidate the further details of this regulation, we performed limited chymotryptic digestion of the troponin complex from akazara scallop striated muscle. The results indicated that troponin T is very susceptible to the protease, compared to troponin C or troponin I. The cleavage occurred at the C-terminal extension, producing an N-terminal 33-kDa fragment and a C-terminal 6-kDa fragment. This extension is conserved in various invertebrate troponin T proteins, but not in vertebrate troponin T. A ternary complex composed of the 33-kDa fragment of troponin T, troponin I, and troponin C could be separated from the 6-kDa troponin T fragment by gel filtration. This complex did not show any Ca²⁺-dependent activation of the Mg-ATPase activity of rabbit-actomyosin–scallop-tropomyosin. In addition, the actin–tropomyosin-binding affinity of this complex was significantly decreased with increasing Ca²⁺ concentration. These results indicate that the C-terminal extension of molluscan troponin T plays a role in anchoring the troponin complex to actin–tropomyosin filaments and is essential for regulation.     

Calcium-dependent regulation of glycogen synthase activity in a muscle glycogen particle

The calcium-dependent inactivation of glycogen synthase in an isolated glycogen-protein complex (glycogen pellet) from rabbit skeletal muscle has been investigated. Addition of 1 mm Ca²⁺, 10 mm Mg²⁺, and 1 mm ATP-γ-S to a concentrated suspension of glycogen pellet resulted in a rapid activation of glycogen phosphorylase concomitant with an inactivation of glycogen synthase. These conversion reactions were blocked by ethylene glycol bis(β-aminoethyl ether) N, N′-tetraacetic acid or by pretreatment of the complex with an antiserum to purified phosphorylase kinase. These data suggest that in the glycogen-protein complex, which may be a functional unit of glycogen metabolism in vivo, phosphorylase kinase can catalyze a Ca²⁺-dependent activation of glycogen phosphorylase synchronized with an inactivation of glycogen synthase. If under similar conditions phosphoprotein phosphatase activity was assayed using exogenous [³²P]phosphorylase, there was an apparent inactivation of the phosphatase. Evidence is presented that this apparent inactivation of phosphatase was due to an accumulation of endogenous phosphorylase a which acted as an inhibitor to the exogenous [³²P]-phosphorylase.     

Ca2+ -induced tropomyosin movement in scallop striated muscle thin filaments

Striated muscle contraction in most animals is regulated at least in part by the troponin-tropomyosin (Tn-Tm) switch on the thin (actin-containing) filaments. The only group that has been suggested to lack actin-linked regulation is the mollusks, where contraction is regulated through the myosin heads on the thick filaments. However, molluscan gene sequence data suggest the presence of troponin (Tn) components, consistent with actin-linked regulation, and some biochemical and immunological data also support this idea. The presence of actin-linked (in addition to myosin-linked) regulation in mollusks would simplify our general picture of muscle regulation by extending actin-linked regulation to this phylum as well. We have investigated this question structurally by determining the effect of Ca²⁺ on the position of Tm in native thin filaments from scallop striated adductor muscle. Three-dimensional reconstructions of negatively stained filaments were determined by electron microscopy and single-particle image analysis. At low Ca²⁺, Tm appeared to occupy the “blocking” position, on the outer domain of actin, identified in earlier studies of regulated thin filaments in the low-Ca²⁺ state. In this position, Tm would sterically block myosin binding, switching off filament activity. At high Ca²⁺, Tm appeared to move toward a position on the inner domain, similar to that induced by Ca²⁺ in regulated thin filaments. This Ca²⁺-induced movement of Tm is consistent with the hypothesis that scallop thin filaments are Ca²⁺ regulated.     

Electrostatic changes in phosphorylase kinase induced by its obligatory allosteric activator Ca2+

Skeletal muscle phosphorylase kinase (PhK) is a 1.3-MDa hexadecameric complex that catalyzes the phosphorylation and activation of glycogen phosphorylase b. PhK has an absolute requirement for Ca(2+) ions, which couples the cascade activation of glycogenolysis with muscle contraction. Ca(2+) activates PhK by binding to its nondissociable calmodulin subunits; however, specific changes in the structure of the PhK complex associated with its activation by Ca(2+) have been poorly understood. We present herein the first comparative investigation of the physical characteristics of highly purified hexadecameric PhK in the absence and presence of Ca(2+) ions using a battery of biophysical probes as a function of temperature. Ca(2+)-induced differences in the tertiary and secondary structure of PhK measured by fluorescence, UV absorption, FTIR, and CD spectroscopies as low resolution probes of PhK's structure were subtle. In contrast, the surface electrostatic properties of solvent accessible charged and polar groups were altered upon the binding of Ca(2+) ions to PhK, which substantially affected both its diffusion rate and electrophoretic mobility, as measured by dynamic light scattering and zeta potential analyses, respectively. Overall, the observed physicochemical effects of Ca(2+) binding to PhK were numerous, including a decrease in its electrostatic surface charge that reduced particle mobility without inducing a large alteration in secondary structure content or hydrophobic tertiary interactions. Without exception, for all analyses in which the temperature was varied, the presence of Ca(2+) rendered the enzyme increasingly labile to thermal perturbation.     

Preserved Close Linkage Between the Genes Encoding Troponin I and Troponin T, Reflecting an Evolution of Adapter Proteins Coupling the Ca2+ Signaling of Contractility

Ca²⁺-regulated motility is essential to numerous cellular functions, including muscle contraction. Systems with troponin C, myosin light chain, or calmodulin as the Ca²⁺ receptor have evolved in striated muscle and other types of cells to transduce the cytoplasm Ca²⁺ signals into allosteric conformational changes of contractile proteins. While these Ca²⁺ receptors are homologous proteins, their coupling to the responding elements is quite different in various cell types. The Ca²⁺ regulatory system in vertebrate striated muscle represents a highly specialized such signal transduction pathway consisting of the troponin complex and tropomyosin associated with the actin filament. To understand the molecular mechanism in the Ca²⁺ regulation of muscle contraction and cell motility, we have revealed a preserved ancestral close linkage between the genes encoding two of the troponin subunits, troponin I and troponin T, in the genome of mouse. The data suggest that the troponin I and troponin T genes may have originated from a single locus and evolved in parallel to encode a striated muscle-specific adapter to couple the Ca²⁺ receptor, troponin C, to the actin–myosin contractile machinery. This hypothesis views the three troponin subunits as two structure–function domains: the Ca²⁺ receptor and the signal transducing adapter. This model may help to further our understanding of the Ca²⁺ regulation of muscle contraction and the structure–function relationship of other potential adapter proteins which are converged to constitute the Ca²⁺ signal transduction pathways governing nonmuscle cell motility. Received: 15 April 1999 / Accepted: 15 July 1999     

Small-angle scattering studies show distinct conformations of calmodulin in its complexes with two peptides based on the regulatory domain of the catalytic subunit of phosphorylase kinase

Small-angle X-ray and neutron scattering have been used to study the solution structures of calmodulin complexed with synthetic peptides corresponding to residues 342-366 and 301-326, designated PhK5 and PhK13, respectively, in the regulatory domain of the catalytic subunit of skeletal muscle phosphorylase kinase. The scattering data show that binding of PhK5 to calmodulin induces a dramatic contraction of calmodulin, similar to that previously observed when calmodulin is complexed with the calmodulin-binding domain peptide from rabbit skeletal muscle myosin light chain kinase. In contrast, calmodulin remains extended upon binding PhK13. In the presence of both peptides, calmodulin also remains extended. Apparently, the presence of PhK13 inhibits calmodulin from undergoing the PhK5-induced contraction. These data indicate that there is a fundamentally different type of calmodulin-target enzyme interaction in the case of the catalytic subunit of phosphorylase kinase compared with that for myosin light chain kinase.     

Structural characterization of Ca2+/CaM in complex with the phosphorylase kinase PhK5 peptide

Phosphorylase kinase (PhK) is a large hexadecameric enzyme consisting of four copies of four subunits: (alphabetagammadelta)4. An intrinsic calmodulin (CaM, the delta subunit) binds directly to the gamma protein kinase chain. The interaction site of CaM on gamma has been localized to a C-terminal extension of the kinase domain. Two 25-mer peptides derived from this region, PhK5 and PhK13, were identified previously as potential CaM-binding sites. Complex formation between Ca2+/CaM with these two peptides was characterized using analytical gel filtration and NMR methods. NMR chemical shift perturbation studies showed that while PhK5 forms a robust complex with Ca2+/CaM, no interactions with PhK13 were observed. 15N relaxation characteristics of Ca2+/CaM and Ca2+/CaM/PhK5 complexes were compared with the experimentally determined structures of several Ca2+/CaM/peptide complexes. Good fits were observed between Ca2+/CaM/PhK5 and three structures: Ca2+/CaM complexes with peptides from endothelial nitric oxide synthase, with smooth muscle myosin light chain kinase and CaM kinase I. We conclude that the PhK5 site is likely to have a direct role in Ca2+-regulated control of PhK activity through the formation of a classical 'compact' CaM complex.     

Ca2+ attenuates nucleation activity of leiomodin

A transient increase in Ca²⁺ concentration in sarcomeres is essential for their proper function. Ca²⁺ drives striated muscle contraction via binding to the troponin complex of the thin filament to activate its interaction with the myosin thick filament. In addition to the troponin complex, the myosin essential light chain and myosin‐binding protein C were also found to be Ca²⁺ sensitive. However, the effects of Ca²⁺ on the function of the tropomodulin family proteins involved in regulating thin filament formation have not yet been studied. Leiomodin, a member of the tropomodulin family, is an actin nucleator and thin filament elongator. Using pyrene‐actin polymerization assay and transmission electron microscopy, we show that the actin nucleation activity of leiomodin is attenuated by Ca²⁺. Using circular dichroism and nuclear magnetic resonance spectroscopy, we demonstrate that the mostly disordered, negatively charged region of leiomodin located between its first two actin‐binding sites binds Ca²⁺. We propose that Ca²⁺ binding to leiomodin results in the attenuation of its nucleation activity. Our data provide further evidence regarding the role of Ca²⁺ as an ultimate regulator of the ensemble of sarcomeric proteins essential for muscle function.Summary StatementCa²⁺ fluctuations in striated muscle sarcomeres modulate contractile activity via binding to several distinct families of sarcomeric proteins. The effects of Ca²⁺ on the activity of leiomodin—an actin nucleator and thin filament length regulator—have remained unknown. In this study, we demonstrate that Ca²⁺ binds directly to leiomodin and attenuates its actin nucleating activity. Our data emphasizes the ultimate role of Ca²⁺ in the regulation of the sarcomeric protein interactions.     

Ca- and Mg-dependent association of phosphorylase kinase with human erythrocyte membranes

The interaction of rabbit muscle phosphorylase kinase (EC 2.7.1.38) with human erythrocyte membranes was investigated. It was found that at pH 7.0 the kinase binds to the inner face of the erythrocyte membrane (inside-out vesicles) and that this binding is Ca²⁺- and Mg²⁺-dependent. The sharpest increase in the binding reaction occurs at concentrations between 70 and 550 nM free Ca²⁺. Erythrocyte ghost or right-side out erythrocyte vesicles showed a significantly lower capacity to interact with phosphorylase kinase. Autophosphorylated phosphorylase kinase shows a similar Ca²⁺-dependent binding profile, while trypsin activation of the kinase and calmodulin decrease the original binding capacity by about 50%. Heparin (200 μg/ml) and high ionic strength (50 mM NaCl) almost completely blocks enzyme-membrane interaction; glycogen does not affect the interaction.     

Structural features contributing to complex formation between glycogen phosphorylase and phosphorylase kinase.

A polyclonal antibody was generated against a peptide corresponding to a region opposite the regulatory face of glycogen phosphorylase b (P-b), providing a probe for detecting and quantifying P-b when it is bound to its activating kinase, phosphorylase kinase (PhK). Using both direct and competition enzyme-linked immunosorbent assays (ELISAs), we have measured the extent of direct binding to PhK of various forms of phosphorylase, including different conformers induced by allosteric effectors as well as forms differing at the N-terminal site phosphorylated by PhK. Strong interactions with PhK were observed for both P-b', a truncated form lacking the site for phosphorylation, and P-a, the phosphorylated form of P-b. Further, the binding of P-b, P-b', and P-a was stimulated a similar amount by Mg(2+), or by Ca(2+) (both being activators of PhK). Our results suggest that the presence and conformation of P-b's N-terminal phosphorylation site do not fully account for the protein's affinity for PhK and that regions distinct from that site may also interact with PhK. Direct ELISAs detected the binding of P-b by a truncated form of the catalytic gamma subunit of PhK, consistent with the necessary interaction of PhK's catalytic subunit with its substrate P-b. In contrast, P-b' bound very poorly to the truncated gamma subunit, suggesting that the N-terminal phosphorylatable region of P-b may be critical in directing P-b to PhK's catalytic subunit and that the binding of P-b' by the PhK holoenzyme may involve more than just its catalytic core. The sum of our results suggests that structural features outside the catalytic domain of PhK and outside the phosphorylatable region of P-b may both be necessary for the maximal interaction of these two proteins.     

Ca2+–Calmodulin regulates SNARE assembly and spontaneous neurotransmitter release via v-ATPase subunit V0a1

Most chemical neurotransmission occurs through Ca²⁺-dependent evoked or spontaneous vesicle exocytosis. In both cases, Ca²⁺ sensing is thought to occur shortly before exocytosis. In this paper, we provide evidence that the Ca²⁺ dependence of spontaneous vesicle release may partly result from an earlier requirement of Ca²⁺ for the assembly of soluble N-ethylmaleimide–sensitive fusion attachment protein receptor (SNARE) complexes. We show that the neuronal vacuolar-type H⁺-adenosine triphosphatase V0 subunit a1 (V100) can regulate the formation of SNARE complexes in a Ca²⁺–Calmodulin (CaM)-dependent manner. Ca²⁺–CaM regulation of V100 is not required for vesicle acidification. Specific disruption of the Ca²⁺-dependent regulation of V100 by CaM led to a >90% loss of spontaneous release but only had a mild effect on evoked release at Drosophila melanogaster embryo neuromuscular junctions. Our data suggest that Ca²⁺–CaM regulation of V100 may control SNARE complex assembly for a subset of synaptic vesicles that sustain spontaneous release.