Coupling between voltage sensor activation,Ca2+ binding and channel opening in large conductance (BK) potassium channels

To determine how intracellular Ca(2+) and membrane voltage regulate the gating of large conductance Ca(2+)-activated K(+) (BK) channels, we examined the steady-state and kinetic properties of mSlo1 ionic and gating currents in the presence and absence of Ca(2+) over a wide range of voltage. The activation of unliganded mSlo1 channels can be accounted for by allosteric coupling between voltage sensor activation and the closed (C) to open (O) conformational change (Horrigan, F.T., and R.W. Aldrich. 1999. J. Gen. Physiol. 114:305-336; Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277-304). In 0 Ca(2+), the steady-state gating charge-voltage (Q(SS)-V) relationship is shallower and shifted to more negative voltages than the conductance-voltage (G(K)-V) relationship. Calcium alters the relationship between Q-V and G-V, shifting both to more negative voltages such that they almost superimpose in 70 microM Ca(2+). This change reflects a differential effect of Ca(2+) on voltage sensor activation and channel opening. Ca(2+) has only a small effect on the fast component of ON gating current, indicating that Ca(2+) binding has little effect on voltage sensor activation when channels are closed. In contrast, open probability measured at very negative voltages (less than -80 mV) increases more than 1,000-fold in 70 microM Ca(2+), demonstrating that Ca(2+) increases the C-O equilibrium constant under conditions where voltage sensors are not activated. Thus, Ca(2+) binding and voltage sensor activation act almost independently, to enhance channel opening. This dual-allosteric mechanism can reproduce the steady-state behavior of mSlo1 over a wide range of conditions, with the assumption that activation of individual Ca(2+) sensors or voltage sensors additively affect the energy of the C-O transition and that a weak interaction between Ca(2+) sensors and voltage sensors occurs independent of channel opening. By contrast, macroscopic I(K) kinetics indicate that Ca(2+) and voltage dependencies of C-O transition rates are complex, leading us to propose that the C-O conformational change may be described by a complex energy landscape.     

Temperature dependence of dynamics and thermodynamics of the regulatory domain of human cardiac troponin C

Binding of Ca(2+) to the regulatory domain of troponin C (TnC) in cardiac muscle initiates a series of protein conformational changes and modified protein-protein interactions that initiate contraction. Cardiac TnC contains two Ca(2+) binding sites, with one site being naturally defunct. Previously, binding of Ca(2+) to the functional site in the regulatory domain of TnC was shown to lead to a decrease in conformational entropy (TDeltaS) of 2 and 0.5 kcal mol(-1) for the functional and nonfunctional sites, respectively, using (15)N nuclear magnetic resonance (NMR) relaxation studies [Spyracopoulos, L., et al. (1998) Biochemistry 37, 18032-18044]. In this study, backbone dynamics of the Ca(2+)-free regulatory domain are investigated by backbone amide (15)N relaxation measurements at eight temperatures from 5 to 45 degrees C. Analysis of the relaxation measurements yields an order parameter (S(2)) indicating the degree of spatial restriction for a backbone amide H-N vector. The temperature dependence of S(2) allows estimation of the contribution to protein heat capacity from pico- to nanosecond time scale conformational fluctuations on a per residue basis. The average heat capacity contribution (C(p,j)) from backbone conformational fluctuations for regions of secondary structure for the regulatory domain of cardiac apo-TnC is 6 cal mol(-1) K(-1). The average heat capacity for Ca(2+) binding site 1 is larger than that for site 2 by 1.3 +/- 0.8 cal mol(-1) K(-1), and likely represents a mechanism where differences in affinity between Ca(2+) binding sites for EF hand proteins can be modulated.     

Conformational changes in sarcoplasmic reticulum Ca(2+)-ATPase mutants: effect of mutations either at Ca(2+)-binding site II or at tryptophan 552 in the cytosolic domain

By analyzing, after expression in yeast and purification, the intrinsic fluorescence properties of point mutants of rabbit Ca(2+)-ATPase (SERCA1a) with alterations to amino acid residues in Ca(2+)-binding site I (E(771)), site II (E(309)), in both sites (D(800)), or in the nucleotide-binding domain (W(552)), we were able to follow the conformational changes associated with various steps in the ATPase catalytic cycle. Whereas Ca(2+) binding to purified wild-type (WT) ATPase in the absence of ATP leads to the rise in Trp fluorescence expected for the so-called E2 --> E1Ca(2) transition, the Ca(2+)-induced fluorescence rise is dramatically reduced for the E(309)Q mutant. As this purified E(309)Q mutant retains the ability to bind Ca(2+) at site I (but not at site II), we tentatively conclude that the protein reorganization induced by Ca(2+) binding at site II makes the major contribution to the overall Trp fluorescence changes observed upon Ca(2+) binding to both sites. Judging from the fluorescence response of W(552)F, similar to that of WT, these changes appear to be primarily due to membranous tryptophans, not to W(552). The same holds for the fluorescence rise observed upon phosphorylation from P(i) (the so-called E2 --> E2P transition). As for WT ATPase, Mg(2+) binding in the absence of Ca(2+) affects the fluorescence of the E(309)Q mutant, suggesting that this Mg(2+)-dependent fluorescence rise does not reflect binding of Mg(2+) to Ca(2+) sites; instead, Mg(2+) probably binds close to the catalytic site, or perhaps near transmembrane span M3, at a location recently revealed by Fe(2+)-catalyzed oxidative cleavage. Mutation of W(552) hardly affects ATP-induced fluorescence changes in the absence of Ca(2+), which are therefore mostly due to membranous Trp residues, demonstrating long-range communication between the nucleotide-binding domain and the membranous domain.     

Functional consequences of alterations to Thr247, Pro248, Glu340, Asp813, Arg819, and Arg822 at the interfaces between domain P, M3, and L6-7 of sarcoplasmic reticulum Ca2+-ATPase. Roles in Ca2+ interaction and phosphoenzyme processing

Point mutants with alterations to amino acid residues Thr(247), Pro(248), Glu(340), Asp(813), Arg(819), and Arg(822) of sarcoplasmic reticulum Ca(2+)-ATPase were analyzed by transient kinetic measurements. In the Ca(2+)-ATPase crystal structures, most of these residues participate in a hydrogen-bonding network between the phosphorylation domain (domain P), the third transmembrane helix (M3), and the cytoplasmic loop connecting the sixth and the seventh transmembrane helices (L6-7). In several of the mutants, a pronounced phosphorylation "overshoot" was observed upon reaction of the Ca(2+)-bound enzyme with ATP, because of accumulation of dephosphoenzyme at steady state. Mutations of Glu(340) and its partners, Thr(247) and Arg(822), in the bonding network markedly slowed the Ca(2+) binding transition (E2 --> E1 --> Ca(2)E1) as well as Ca(2+) dissociation from Ca(2+) site II back toward the cytosol but did not affect the apparent affinity for vanadate. These mutations may have caused a slowing, in both directions, of the conformational change associated directly with Ca(2+) interaction at Ca(2+) site II. Because mutation of Asp(813) inhibited the Ca(2+) binding transition, but not Ca(2+) dissociation, and increased the apparent affinity for vanadate, the effect on the Ca(2+) binding transition seems in this case to be exerted by slowing the E2 --> E1 conformational change. Because the rate was not significantly enhanced by a 10-fold increase of the Ca(2+) concentration, the slowing is not the consequence of reduced affinity of any pre-binding site for Ca(2+). Furthermore, the mutations interfered in specific ways with the phosphoenzyme processing steps of the transport cycle; the transition from ADP-sensitive phosphoenzyme to ADP-insensitive phosphoenzyme (Ca(2)E1P --> E2P) was accelerated by mutations perturbing the interactions mediated by Glu(340) and Asp(813) and inhibited by mutation of Pro(248), and mutations of Thr(247) induced charge-specific changes of the rate of dephosphorylation of E2P.     

Loss of a metal-binding site in gelsolin leads to familial amyloidosis-Finnish type.

Mutations in domain 2 (D2, residues 151-266) of the actin-binding protein gelsolin cause familial amyloidosis-Finnish type (FAF). These mutations, D187N or D187Y, lead to abnormal proteolysis of plasma gelsolin at residues 172-173 and a second hydrolysis at residue 243, resulting in an amyloidogenic fragment. Here we present the structure of human gelsolin D2 at 1.65 A and find that Asp 187 is part of a Cd2+ metal-binding site. Two Ca2+ ions are required for a conformational transition of gelsolin to its active form. Differential scanning calorimetry (DSC) and molecular dynamics (MD) simulations suggest that the Cd2+-binding site in D2 is one of these two Ca2+-binding sites and is essential to the stability of D2. Mutation of Asp 187 to Asn disrupts Ca2+ binding in D2, leading to instabilities upon Ca2+ activation. These instabilities make the domain a target for aberrant proteolysis, thereby enacting the first step in the cascade leading to FAF.     

Probing Ca2+-induced conformational changes in porcine calmodulin by H/D exchange and ESI-MS: effect of cations and ionic strength

We applied a new method, "protein-ligand interaction using mass spectrometry, titration, and H/D exchange" (PLIMSTEX) [Zhu, M. M. (2003) J. Am. Chem. Soc. 125, 5252-5253], to determine the conformational changes, binding stoichiometry, and binding constants for Ca(2+) interactions with calmodulin (CaM) under varying conditions of electrolyte identity and ionic strength. The outcome shows that CaM becomes less solvent-accessible and more compact upon Ca(2+)-binding, as revealed by the PLIMSTEX curve. The formation of CaM-4Ca species is the biggest contributor to the shape of the titration curve, indicating that the formation of this species accounts for the largest conformational change in the stepwise Ca(2+) binding. The Ca(2+)-binding constants, when comparisons permit, agree with those in the literature within a factor of 3. The binding is influenced by ionic strength and the presence of other cations, although many of these cations do not cause conformational change in apo-CaM. Furthermore, Ca(2+)-saturated CaM exhibits larger protection and higher Ca(2+) affinity in media of low rather than high ionic strength. Both Ca(2+) and Mg(2+) bind to CaM with different affinities, causing different conformational changes. K(+), if it does bind, causes no detectable conformational change, and interactions of Ca(2+) with CaM in the presence of Li(+), Na(+), and K(+) occur with similar affinities and associated changes in solvent accessibility. These metal ion effects point to nonspecific rather than competitive binding of alkali-metal ions. The rates of deuterium uptake by the various CaM-xCa species follow a three-group (fast, intermediate, slow), pseudo-first-order kinetics model. Calcium binding causes the number of amide hydrogens to shift from the fast to the slow group. The results taken together not only provide new insight into CaM but also indicate that both PLIMSTEX and kinetic modeling of H/D exchange data may become general methods for probing protein conformations and quantifying protein-ligand interactions.     

Catalytic and regulatory ATP-binding sites of the red cell Ca2+ pump studied by irreversible modification with fluorescein isothiocyanate

Catalytic and regulatory binding sites for ATP on the red cell Ca2+ pump have been investigated using fluorescein isothiocyanate (FITC). Both (Ca2+ + Mg2+)-ATPase activity and ATP-dependent Ca2+ flux are selectively and irreversibly inactivated by FITC and the pump is protected from FITC by the presence of ATP. The time course of inactivation by FITC is characteristically biphasic. Analysis of the kinetics of inactivation by FITC and protection by ATP reveals the participation of both high and low affinity binding sites for ATP and FITC. The sites binding ATP or reacting with FITC do not, however, appear to co-exist on the same enzyme molecules. Thus, "flip-flop" mechanisms for (Ca2+ + Mg2+)-ATPase, involving negative interactions between high and low affinity ATP sites, are considered unlikely. The two affinities for ATP are most simply explained by assuming that the Ca2+ pump protein exists in alternative conformational forms, E1 having a high affinity for ATP and E2 having a low affinity for ATP. Ca2+ pumping and (Ca2+ + Mg2+)-ATPase involve interconversion between these forms. It is suggested that regulation of Ca2+ pump activity by Mg-ATP reflects acceleration of the conformational transition between the E1 and E2 forms, as well as a previously described acceleration of phosphoenzyme hydrolysis (Muallem, S., and Karlish, S. J. D. (1981) Biochim. Biophys. Acta 647, 73-86; Garrahan, P. J., and Rega, A. F. (1978) Biochim. Biophys. Acta 513, 59-65).     

Interactions of Ca(2+) with sphingomyelin and dihydrosphingomyelin

The changes induced by Ca(2+) on human lens sphingolipids, sphingomyelin (SM), and dihydrosphingomyelin were investigated by infrared spectroscopy. Ca(2+)-concentration-dependent studies of the head group region revealed that, for both sphingolipids, Ca(2+) partially dehydrates some of the phosphate groups and binds to others. Ca(2+) affects the interface of each sphingolipid differently. In SM, Ca(2+) shifts the amide I' band to frequencies lower than those in dehydrated samples of SM alone. This could be attributed to the direct binding of Ca(2+) to carbonyl groups and/or strong tightening of interlipid H-bonds to levels beyond those in dehydrated samples of SM only. In contrast, Ca(2+) induces relatively minor dehydration around the amide groups of dihydrosphingomyelin and a slight enhancement of direct lipid-lipid interactions. Temperature-dependent studies reveal that 0.2 M Ca(2+) increases the transition temperature T(m) from 31.6 +/- 1.0 degrees C to 35.7 +/- 1.1 degrees C for SM and from 45.5 +/- 1.1 degrees C to 48.2 +/- 1.0 degrees C for dihydrosphingomyelin. Binding of Ca(2+) to some phosphate groups remains above T(m). The strength of the interaction is, however, weaker. This allows for the partial rehydration of these moieties. Similarly, above T(m), Ca(2+)-lipid and/or direct inter-lipid interactions are weakened and lead to the rehydration of amide groups.     

Solution structure and fluctuation of the Mg(2+)-bound form of calmodulin C-terminal domain

Calmodulin (CaM) is a Ca(2+)-binding protein that functions as a ubiquitous Ca(2+)-signaling molecule, through conformational changes from the "closed" apo conformation to the "open" Ca(2+)-bound conformation. Mg(2+) also binds to CaM and stabilizes its folded structure, but the NMR signals are broadened by slow conformational fluctuations. Using the E104D/E140D mutant, designed to decrease the signal broadening in the presence of Mg(2+) with minimal perturbations of the overall structure, the solution structure of the Mg(2+)-bound form of the CaM C-terminal domain was determined by multidimensional NMR spectroscopy. The Mg(2+)-induced conformational change mainly occurred in EF hand IV, while EF-hand III retained the apo structure. The helix G and helix H sides of the binding sequence undergo conformational changes needed for the Mg(2+) coordination, and thus the helices tilt slightly. The aromatic rings on helix H move to form a new cluster of aromatic rings in the hydrophobic core. Although helix G tilts slightly to the open orientation, the closed conformation is maintained. The fact that the Mg(2+)-induced conformational changes in EF-hand IV and the hydrophobic core are also seen upon Ca(2+) binding suggests that the Ca(2+)-induced conformational changes can be divided into two categories, those specific to Ca(2+) and those common to Ca(2+) and Mg(2+).     

The W105G and W99G sorcin mutants demonstrate the role of the D helix in the Ca(2+)-dependent interaction with annexin VII and the cardiac ryanodine receptor

Sorcin, a 21.6 kDa two-domain penta-EF-hand (PEF) protein, when activated by Ca(2+) binding, interacts with target proteins in a largely uncharacterized process. The two physiological EF-hands EF3 and EF2 do not belong to a structural pair but are connected by the D helix. To establish whether this helix is instrumental in sorcin activation, two D helix residues were mutated: W105, located near EF3 and involved in a network of interactions, and W99, located near EF2 and facing solvent, were substituted with glycine. Neither mutation alters calcium affinity. The interaction of the W105G and W99G mutants with annexin VII and the cardiac ryanodine receptor (RyR2), requiring the sorcin N-terminal and C-terminal domain, respectively, was studied. Surface plasmon resonance experiments show that binding of annexin VII to W99G occurs at the same Ca(2+) concentration as that of the wild type, whereas W105G requires a significantly higher Ca(2+) concentration. Ca(2+) spark activity of isolated heart cells monitors the sorcin-RyR2 interaction and is unaltered by W105G but is reduced equally by W99G and the wild type. Thus, substitution of W105, via disruption of the network of D helix interactions, affects the capacity of sorcin to recognize and interact with either target at physiological Ca(2+) concentrations, while mutation of solvent-facing W99 has little effect. The D helix appears to amplify the localized structural changes that occur at EF3 upon Ca(2+) binding and thereby trigger a structural rearrangement that enables interaction of sorcin with its molecular targets. The same activation process may apply to other PEF proteins in view of the D helix conservation.     

Cloning,overexpression, and characterization of a bacterial Ca2+-dependent phospholipase D

Phospholipase D (PLD), an important enzyme involved in signal transduction in mammals, is also secreted by many microorganisms. A highly conserved HKD motif has been identified in most PLD homologs in the PLD superfamily. However, the Ca(2+)-dependent PLD from Streptomyces chromofuscus exhibits little homology to other PLDs. We have cloned (using DNA isolated from the ATCC type strain), overexpressed in Escherichia coli (two expression systems, pET-23a(+) and pTYB11), and purified the S. chromofuscus PLD. Based on attempts at sequence alignment with other known Ca(2+)-independent PLD enzymes from Streptomyces species, we mutated five histidine residues (His72, His171, His187, His200, His226) that could be part of variants of an HKD motif. Only H187A and H200A showed dramatically reduced activity. However, mutation of these histidine residues to alanine also significantly altered the secondary structure of PLD. Asparagine replacements at these positions yielded enzymes with structure and activity similar to the recombinant wild-type PLD. The extent of phosphatidic acid (PA) activation of PC hydrolysis by the recombinant PLD enzymes differed in magnitude from PLD purified from S. chromofuscus culture medium (a 2-fold activation rather than 4-5-fold). One of the His mutants, H226A, showed a 12-fold enhancement by PA, suggesting this residue is involved in the kinetic activation. Another notable difference of this bacterial PLD from others is that it has a single cysteine (Cys123); other Streptomyces Ca(2+)-independent PLDs have eight Cys involved in intramolecular disulfide bonds. Both C123A and C123S, with secondary structure and stability similar to recombinant wild-type PLD, exhibited specific activity reduced by 10(-5) and 10(-4). The Cys mutants still bound Ca(2+), so that it is likely that this residue is part of the active site of the Ca(2+)-dependent PLD. This would suggest that S. chromofuscus PLD is a member of a new class of PLD enzymes.     

Dynamics of the transition between open and closed conformations in a calmodulin C-terminal domain mutant

BACKGROUND: Calmodulin is a ubiquitous Ca(2+)-activated regulator of cellular processes in eukaryotes. The structures of the Ca(2+)-free (apo) and Ca(2+)-loaded states of calmodulin have revealed that Ca(2+) binding is associated with a transition in each of the two domains from a closed to an open conformation that is central to target recognition. However, little is known about the dynamics of this conformational switch. RESULTS: The dynamics of the transition between closed and open conformations in the Ca(2+)-loaded state of the E140Q mutant of the calmodulin C-terminal domain were characterized under equilibrium conditions. The exchange time constants (tau(ex)) measured for 42 residues range from 13 to 46 micros, with a mean of 21 +/- 3 micros. The results suggest that tau(ex) varies significantly between different groups of residues and that residues with similar values exhibit spatial proximity in the structures of apo and/or Ca(2+)-saturated wild-type calmodulin. Using data for one of these groups, we obtained an open population of p(o) = 0.50 +/- 0.17 and a closed --> open rate constant of k(o) = x 10(4) s(-1). CONCLUSIONS: The conformational exchange dynamics appear to involve locally collective processes that depend on the structural topology. Comparisons with previous results indicate that similar processes occur in the wild-type protein. The measured rates match the estimated Ca(2+) off rate, suggesting that Ca(2+) release may be gated by the conformational dynamics. Structural interpretation of estimated chemical shifts suggests a mechanism for ion release.     

Ca(2+)- and H(+)-dependent conformational changes of calbindin D(28k)

Calbindin D(28k) is a member of a large family of intracellular Ca(2+) binding proteins characterized by EF-hand structural motifs. Some of these proteins are classified as Ca(2+)-sensor proteins, since they are involved in transducing intracellular Ca(2+) signals by exposing a hydrophobic patch on the protein surface in response to Ca(2+) binding. The hydrophobic patch serves as an interaction site for target enzymes. Other members of this group are classified as Ca(2+)-buffering proteins, because they remain closed after Ca(2+) binding and participate in Ca(2+) buffering and transport functions. ANS (8-anilinonaphthalene-1-sulfonic acid) binding and affinity chromatography on a hydrophobic column suggested that both the Ca(2+)-free and Ca(2+)-loaded form of calbindin D(28k) have exposed hydrophobic surfaces. Since exposure of hydrophobic surface is unfavorable in the aqueous intracellular milieu, calbindin D(28k) most likely interacts with other cellular components in vivo. A Ca(2+)-induced conformational change was readily detected by several optical spectroscopic methods. Thus, calbindin D(28k) shares some of the properties of Ca(2+)-sensor proteins. However, the Ca(2+)-induced change in exposed hydrophobic surface was considerably less pronounced than that in calmodulin. The data also shows that calbindin D(28k) undergoes a rapid and reversible conformational change in response to a H(+) concentration increase within the physiological pH range. The pH-dependent conformational change was shown to reside mainly in EF-hands 1-3. Urea-induced unfolding of the protein at pH 6, 7, and 8 showed that the stability of calbindin D(28k) was increased in response to H(+) in the range examined. The results suggest that calbindin D(28k) may interact with targets in a Ca(2+)- and H(+)-dependent manner.     

Calcium-modulated S100 protein-phospholipid interactions. An NMR study of calbindin D9k and DPC

The cellular functions of several S100 proteins involve specific interactions with phospholipids and the cell membrane. The interactions between calbindin D(9k) (S100D) and the detergent dodecyl phosphocholine (DPC) were studied using NMR spectroscopy. In the absence of Ca(2+), the protein associates with DPC micelles. The micelle-associated state has intact helical secondary structures but no apparent tertiary fold. At neutral pH, Ca(2+)-loaded calbindin D(9k) does not associate with DPC micelles. However, a specific interaction is observed with individual DPC molecules at a site close to the linker between the two EF-hands. Binding to this site occurs only when Ca(2+) is bound to the protein. A reduction in pH in the absence of Ca(2+) increases the stability of the micelle-associated state. This along with the corresponding reduction in Ca(2+) affinity causes a transition to the micelle-associated state also in the presence of Ca(2+) when the pH is lowered. Site-specific analysis of the data indicates that calbindin D(9k) has a core of three tightly packed helices (A, B, and D), with a dynamic fourth helix (C) more loosely associated. Evidence is presented that the Ca(2+)-binding characteristics of the two EF-hands are distinctly different in a micelle environment. The role of calbindin D(9k) in the cell is discussed, along with the broader implications for the function of the S100 protein family.     

Conformational substates of calmodulin revealed by single-pair fluorescence resonance energy transfer: influence of solution conditions and oxidative modification

A calmodulin (CaM) mutant (T34,110C-CaM) doubly labeled with fluorescence probes AlexaFluor 488 and Texas Red in opposing domains (CaM-DA) has been used to examine conformational heterogeneity in CaM by single-pair fluorescence resonance energy transfer (spFRET). Burst-integrated FRET efficiencies of freely diffusing CaM-DA single molecules yielded distributions of distance between domains of CaM-DA. We recently reported distinct conformational substates of Ca(2+)-CaM-DA and apoCaM-DA, with peaks in the distance distributions centered at approximately 28 A, 34-38 A, and 55 A [Slaughter et al. (2004) J. Phys. Chem. B 108, 10388-10397]. In the present study, shifts in the amplitudes and center distances of the conformational substates were detected with variation in solution conditions. The amplitude of an extended conformation was observed to change as a function of Ca(2+) over a free Ca(2+) range that is consistent with binding to the high affinity, C-terminal Ca(2+) binding sites, suggesting the existence of communication between lobes of CaM. Lowering pH shifted the relative amplitudes of the conformations, with a marked increase in the presence of the compact conformations and an almost complete absence of the extended conformation. In addition, the single-molecule distance distribution of apoCaM-DA at reduced ionic strength was shifted to longer distance and showed evidence of an increase in conformational heterogeneity relative to apoCaM-DA at physiological ionic strength. Oxidation of methionine residues in CaM-DA produced a substantial increase in the amplitude of the extended conformation relative to the more compact conformation. The results are considered in light of a hypothesis that suggests that electrostatic interactions between charged amino acid side chains play an important role in determining the most stable CaM conformation under varying solution conditions.     

The C2 domain of protein kinase calpha is directly involved in the diacylglycerol-dependent binding of the C1 domain to the membrane

Protein kinase Calpha (PKCalpha), which is known to be critical for the control of many cellular processes, was submitted to site-directed mutagenesis in order to test the functionality of several amino acidic residues. Thus, D187, D246 and D248, all of which are located at the Ca(2+) binding site of the C2 domain, were substituted by N. Subcellular fractionation experiments demonstrated that these mutations are important for both Ca(2+)-dependent and diacylglycerol-dependent membrane binding. The mutants are not able to phosphorylate typical PKC substrates, such as histone and myelin basic protein. Furthermore, using increasing concentrations of dioleylglycerol, one of the mutants (D246/248N) was able to recover total activity although the amounts of dioleylglycerol it required were larger than those required by wild type protein. On the other hand, the other mutants (D187N and D187/246/248) only recovered 50% of their activity. These data suggest that there is a relationship between the C1 domain, where dioleylglycerol binds, and the C2 domain, and that this relationship is very important for enzyme activation. These findings led us to propose a mechanism for PKCalpha activation, where C1 and C2 domains cannot be considered independent membrane binding modules.     

Gelsolin domain 2 Ca2+ affinity determines susceptibility to furin proteolysis and familial amyloidosis of finnish type

Mutation of aspartic acid 187 to asparagine (D187N) or tyrosine (D187Y) in domain 2 of the actin-modulating protein gelsolin causes the neurodegenerative disease familial amyloidosis of Finnish type (FAF). These mutations render plasma gelsolin susceptible to aberrant proteolysis by furin in the trans-Golgi network, the initial proteolytic event in the formation of 71 and 53 residue fragments that assemble into amyloid fibrils. Ca(2+) binding stabilizes wild-type domain 2 gelsolin against denaturation and proteolysis, but the FAF variants are unable to bind and be stabilized by Ca(2+). Though the chain of events initiating FAF has been elucidated recently, uncertainty remains about the mechanistic details that allow the FAF variants to be processed. To test the hypothesis that impaired Ca(2+) binding in the D187 variants, but not other factors specific to residue 187, increases susceptibility to aberrant proteolysis and subsequent amyloidogenesis, we designed the gelsolin variant E209Q to remove a different Ca(2+) ligand from the same Ca(2+) site that is affected in the FAF variants. Here, we show that E209Q domain 2 does not bind Ca(2+) and is not stabilized against denaturation or furin proteolysis, analogous to the behavior exhibited by the FAF variants. Transfection of full-length E209Q into COS cells results in secretion of both the full-length and furin-processed fragments, as observed with D187N and D187Y. Mutation of the furin consensus sequence in D187N and E209Q gelsolin prevents cleavage during secretion, indicating that inhibition of proprotein convertases (furin) represents a viable therapeutic approach for the treatment of FAF. Mutations that diminish domain 2 Ca(2+) binding allow furin access to an otherwise protected cleavage site, initiating the proteolytic cascade that leads to gelsolin amyloidogenesis and FAF.     

Functional role of Ca(2+)-binding site IV of scallop troponin C

Scallop troponin C (TnC) binds only one Ca(2+)/mol and the single Ca(2+)-binding site has been suggested to be site IV on the basis of the primary structure [K. Nishita, H. Tanaka, and T. Ojima (1994) J. Biol. Chem. 269, 3464-3468; T. Ojima, H. Tanaka, and K. Nishita (1994) Arch. Biochem. Biophys. 311, 272-276]. In the present study, the functional role of Ca(2+)-binding site IV of akazara scallop (Chlamys nipponensis akazara) TnC in Ca(2+)-regulation was investigated using a site-directed mutant with an inactivated site IV (TnC-ZEQ), N- and C-terminal half molecule mutants (TnC(N) and TnC(C)), and wild-type TnC (TnC(W)). Equilibrium dialysis using (45)Ca(2+) demonstrated that TnC(W) and TnC(C) bind 0.6-0.8 mol of Ca(2+)/mol, but that TnC-ZEQ and TnC(N) bind virtually no Ca(2+). The UV difference spectra of TnC(W) and TnC(C) showed bands at around 280-290 nm due to the perturbation of Tyr and Trp upon Ca(2+)-binding, while TnC-ZEQ and TnC(N) did not show these bands. In addition, TnC(W) and TnC(C) showed retardation of elution from Sephacryl S-200 upon the addition of 1 mM CaCl(2), unlike TnC-ZEQ and TnC(N). These results indicate that Ca(2+) binds only to site IV and that Ca(2+)-binding causes structural changes in both the whole TnC molecule and the C-terminal half molecule. In addition, TnC(W), TnC-ZEQ, and TnC(C), but not TnC(N), were shown to form soluble complexes with scallop TnI at physiological ionic strength. On the other hand, the Mg-ATPase activity of reconstituted rabbit actomyosin in the presence of scallop tropomyosin was inhibited by scallop TnI and recovered by the addition of an equimolar amount of TnC(W), TnC-ZEQ, or TnC(C), but not TnC(N). These results imply that the site responsible for the association with TnI is located in the C-terminal half domain of TnC. Ternary complex constructed from scallop TnT, TnI, and TnC(W) conferred Ca(2+)-sensitivity to the Mg-ATPase of rabbit actomyosin to the same extent as native troponin, but the TnC(N)-TnT-TnI and TnC-ZEQ-TnT-TnI complexes conferred no Ca(2+)-sensitivity, while the TnC(C)-TnT-TnI complex conferred weak Ca(2+)-sensitivity. Thus, the major functions of scallop TnC, such as Ca(2+)-binding and interaction with TnI, are located in the C-terminal domain, however, the full Ca(2+)-regulatory function requires the presence of the N-terminal domain.     

Conformational fluctuations of the Ca2+-ATPase in the native membrane environment. Effects of pH, temperature, catalytic substrates, and thapsigargin

Digestion with proteinase K or trypsin yields complementary information on conformational transitions of the Ca(2+)-ATPase (SERCA) in the native membrane environment. Distinct digestion patterns are obtained with proteinase K, revealing interconversion of E1 and E2 or E1 approximately P and E2-P states. The pH dependence of digestion patterns shows that, in the presence of Mg(2+), conversion of E2 to E1 pattern occurs (even when Ca(2+) is absent) as H(+) dissociates from acidic residues. Mutational analysis demonstrates that the Glu(309) and Glu(771) acidic residues (empty Ca(2+)-binding sites I and II) are required for stabilization of E2. Glu(309) ionization is most important to yield E1. However, a further transition produced by Ca(2+) binding to E1 (i.e. E1.2Ca(2+)) is still needed for catalytic activation. Following ATP utilization, H(+)/Ca(2+) exchange is involved in the transition from the E1 approximately P.2Ca(2+) to the E2-P pattern, whereby alkaline pH will limit this conformational transition. Complementary experiments on digestion with trypsin exhibit high temperature dependence, indicating that, in the E1 and E2 ground states, the ATPase conformation undergoes strong fluctuations related to internal protein dynamics. The fluctuations are tightly constrained by ATP binding and phosphoenzyme formation, and this constraint must be overcome by thermal activation and substrate-free energy to allow enzyme turnover. In fact, a substantial portion of ATP free energy is utilized for conformational work related to the E1 approximately P.2Ca(2+) to E2-P transition, thereby disrupting high affinity binding and allowing luminal diffusion of Ca(2+). The E2 state and luminal path closure follow removal of conformational constraint by phosphate.     

Calcium-sensitive activity and conformation of Caenorhabditis elegans gelsolin-like protein 1 are altered by mutations in the first gelsolin-like domain

The gelsolin family of actin regulatory proteins is activated by Ca(2+) to sever and cap actin filaments. Gelsolin has six homologous gelsolin-like domains (G1-G6), and Ca(2+)-dependent conformational changes regulate its accessibility to actin. Caenorhabditis elegans gelsolin-like protein-1 (GSNL-1) has only four gelsolin-like domains (G1-G4) and still exhibits Ca(2+)-dependent actin filament-severing and -capping activities. We found that acidic residues (Asp-83 and Asp-84) in G1 of GSNL-1 are important for its Ca(2+) activation. These residues are conserved in GSNL-1 and gelsolin and previously implicated in actin-severing activity of the gelsolin family. We found that alanine mutations at Asp-83 and Asp-84 (D83A/D84A mutation) did not disrupt actin-severing or -capping activity. Instead, the mutants exhibited altered Ca(2+) sensitivity when compared with wild-type GSNL-1. The D83A/D84A mutation enhanced Ca(2+) sensitivity for actin severing and capping and its susceptibility to proteolytic digestion, suggesting a conformational change. Single mutations caused minimal changes in its activity, whereas Asp-83 and Asp-84 were required to stabilize Ca(2+)-free and Ca(2+)-bound conformations, respectively. On the other hand, the D83A/D84A mutation suppressed sensitivity of GSNL-1 to phosphatidylinositol 4,5-bisphosphate inhibition. The structure of an inactive form of gelsolin shows that the equivalent acidic residues are in close contact with G3, which may maintain an inactive conformation of the gelsolin family.