Alpha Loop Research Articles

Functional split and crosslinking of the membrane domain of the beta subunit of proton-translocating transhydrogenase from Escherichia coli.

Proton pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an alpha subunit with the NAD(H)-binding domain I and a beta subunit with the NADP(H)-binding domain III. The membrane domain (domain II) harbors the proton channel and is made up of the hydrophobic parts of the alpha and beta subunits. The interface in domain II between the alpha and the beta subunits has previously been investigated by cross-linking loops connecting the four transmembrane helices in the alpha subunit and loops connecting the nine transmembrane helices in the beta subunit. However, to investigate the organization of the nine transmembrane helices in the beta subunit, a split was introduced by creating a stop codon in the loop connecting transmembrane helices 9 and 10 by a single mutagenesis step, utilizing an existing downstream start codon. The resulting enzyme was composed of the wild-type alpha subunit and the two new peptides beta1 and beta2. As compared to other split membrane proteins, the new transhydrogenase was remarkably active and catalyzed activities for the reduction of 3-acetylpyridine-NAD(+) by NADPH, the cyclic reduction of 3-acetylpyridine-NAD(+) by NADH (mediated by bound NADP(H)), and proton pumping, amounting to about 50-107% of the corresponding wild-type activities. These high activities suggest that the alpha subunit was normally folded, followed by a concerted folding of beta1 + beta2. Cross-linking of a betaS105C-betaS237C double cysteine mutant in the functional split cysteine-free background, followed by SDS-PAGE analysis, showed that helices 9, 13, and 14 were in close proximity. This is the first time that cross-linking between helices in the same beta subunit has been demonstrated.

Solution structure of a chemosensory protein from the moth Mamestra brassicae.

Chemosensory proteins (CSPs) are believed to be involved in chemical communication and perception. A number of such proteins, of molecular mass approximately 13 kDa, have been isolated from different sensory organs of a wide range of insect species. Several CSPs have been identified in the antennae and proboscis of the moth Mamestra brassicae. CSPMbraA6, a 112-amino-acid antennal protein, has been expressed in a soluble form in large quantities in the Escherichi coli periplasm. NMR structure determination of CSPMbraA6 has been performed with 1H- and 15N-labelled samples. The calculated structures present an average root mean square deviation about the mean structure of 0.63 A for backbone atoms and 1.27 A for all non-hydrogen atoms except the 12 N-terminal residues. The protein is well folded from residue 12 to residue 110, and consists of a non-bundle alpha-helical structure with six helices connected by alpha alpha loops. It has a globular shape, with overall dimensions of 32 A x 28 A x 24 A. A channel is visible in the hydrophobic core, with dimensions of 3 A x 9 A x 21 A. In some of the 20 solution structures calculated, this channel is closed either by Trp-94 at one end or by Tyr-26 at the other end; in some other solutions, this channel is closed at both ends. Binding experiments with 12-bromododecanol indicate that the CSPMbraA6 structure is modified upon ligand binding.

Peptide Substrate Specificities and Protein Cleavage Sites of Human Endometase/Matrilysin-2/Matrix Metalloproteinase-26

Human endometase/matrilysin-2/matrix metalloproteinase-26 (MMP-26) is a novel epithelial and cancer-specific metalloproteinase. Peptide libraries were used to profile the substrate specificity of MMP-26 from the P4-P4' sites. The optimal cleavage motifs for MMP-26 were Lys-Pro-Ile/Leu-Ser(P1)-Leu/Met(P1')-Ile/Thr-Ser/Ala-Ser. The strongest preference was observed at the P1' and P2 sites where hydrophobic residues were favored. Proline was preferred at P3, and Serine was preferred at P1. The overall specificity was similar to that of other MMPs with the exception that more flexibility was observed at P1, P2', and P3'. Accordingly, synthetic inhibitors of gelatinases and collagenases inhibited MMP-26 with similar efficacy. A pair of stereoisomers had only a 40-fold difference in K(i)(app) values against MMP-26 compared with a 250-fold difference against neutrophil collagenase, indicating that MMP-26 is less stereoselective for its inhibitors. MMP-26 autodigested itself during the folding process. Two of the major autolytic sites were Leu(49)-Thr(50) and Ala(75)-Leu(76), which still left the cysteine switch sequence (PHC(82)GVPD) intact. This suggests that Cys(82) may not play a role in the latency of the zymogen. Interestingly, inhibitor titration studies revealed that only approximately 5% of the total MMP-26 molecules was catalytically active, indicating that the thiol groups of Cys(82) in the active molecules may be dissociated or removed from the active site zinc ions. MMP-26 cleaved Phe(352)-Leu(353) and Pro(357)-Met(358) in the reactive loop of alpha(1)-proteinase inhibitor and His(140)-Val(141) in insulin-like growth factor-binding protein-1, probably rendering these substrates inactive. Among the fluorescent peptide substrates analyzed, Mca-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH(2) displayed the highest specificity constant (30,000/molar second) with MMP-26. This report proposes a working model for the future studies of pro-MMP-26 activation, the design of inhibitors, and the identification of optimal physiological and pathological substrates of MMP-26 in vivo.

The correlation between ovomucoid-derived peptides, human leucocyte antigen class II molecules and T cell receptor-complementarity determining region 3 compositions in patients with egg-white allergy.

Food allergies are more prevalent in children, due to the immature gastrointestinal epithelial membrane barrier allowing more proteins through the barrier and into circulation. Ovomucoid (OM) is one of the major allergens that is found in egg white. The aim of this study was to determine T cell epitopes, antigen-presenting human leucocyte antigen (HLA) class II molecules of the T cell lines (TCLs) and T cell clones (TCCs), and complementarity determining region (CDR) 3 loops of the T cell receptor (TCR) alpha and beta chains of the TCCs specific to OM. We established TCLs and TCCs specific to OM from peripheral blood mononuclear cells (PBMCs) of four atopic patients with egg-white allergy using a mixture of a panel of overlapping synthetic peptides corresponding to the amino acid sequence of the entire OM. We identified the T cell epitopes by antigen-induced proliferative responses, antigen-presenting molecules using allogeneic PBMCs and CDR3 loops of the TCR alpha and beta chains by cloning and sequence analysis. The TCLs and TCCs responded to seven different peptides, and their antigen-presenting molecules were different from each other. Sequence analysis of the TCR alpha and beta gene usage of the TCCs showed marked heterogeneity, and the usage of the CDR3 loop of the TCCs involved heterogenous amino acid residues. Interestingly, TCCs 'IH3.3' and 'YT6.1' recognized the same OM peptides, and had the same TCR Vbeta-Jbeta gene usage. Considering that peptide motifs bind to HLA class II molecules, the electrically charged residue (positive or negative) on the CDR3alpha and the CDR3beta loops of TCR of TCC may form ionic bonds with a charged residue on the HLA class II molecules-peptide complex. TCCs that have the same TCR gene usage were established from patients who had shown similar hypersensitivity-type, indicating that antigen recognition by a specific TCR is closely associated with the characteristics of each patient's symptoms.

The dual role of a loop with low loop contact distance in folding and domain swapping.

Alpha helices, beta strands, and loops are the basic building blocks of protein structure. The folding kinetics of alpha helices and beta strands have been investigated extensively. However, little is known about the formation of loops. Experimental studies show that for some proteins, the formation of a single loop is the rate-determining step for folding, whereas for others, a loop (or turn) can misfold to serve as the hinge loop region for domain-swapped species. Computer simulations of an all-atom model of fragment B of Staphylococcal protein A found that the formation of a single loop initiates the dominant folding pathway. On the other hand, the stability analysis of intermediates suggests that the same loop is a likely candidate to serve as a hinge loop for domain swapping. To interpret the simulation result, we developed a simple structural parameter: the loop contact distance (LCD), or the sequence distance of contacting residues between a loop and the rest of the protein. The parameter is applied to a number of other proteins, including SH3 domains and prion protein. The results suggest that a locally interacting loop (low LCD) can either promote folding or serve as the hinge region for domain swapping. Thus, there is an intimate connection between folding and domain swapping, a possible cause of misfolding and aggregation.

F1-ATPase, the C-terminal End of Subunit γ Is Not Required for ATP Hydrolysis-driven Rotation

ATP hydrolysis by the isolated F(1)-ATPase drives the rotation of the central shaft, subunit gamma, which is located within a hexagon formed by subunits (alphabeta)(3). The C-terminal end of gamma forms an alpha-helix which properly fits into the "hydrophobic bearing" provided by loops of subunits alpha and beta. This "bearing" is expected to be essential for the rotary function. We checked the importance of this contact region by successive C-terminal deletions of 3, 6, 9, 12, 15, and 18 amino acid residues (Escherichia coli F(1)-ATPase). The ATP hydrolysis activity of a load-free ensemble of F(1) with 12 residues deleted decreased to 24% of the control. EF(1) with deletions of 15 or 18 residues was inactive, probably because it failed to assemble. The average torque generated by a single molecule of EF(1) when loaded by a fluorescent actin filament was, however, unaffected by deletions of up to 12 residues, as was their rotational behavior (all samples rotated during 60 +/- 19% of the observation time). Activation energy analysis with the ensemble revealed a moderate decrease from 54 kJ/mol for EF(1) (full-length gamma) to 34 kJ/mol for EF(1)(gamma-12). These observations imply that the intactness of the C terminus of subunit gamma provides structural stability and/or routing during assembly of the enzyme, but that it is not required for the rotary action under load, proper.

Evidence that serpin architecture intrinsically supports papain-like cysteine protease inhibition: engineering alpha(1)-antitrypsin to inhibit cathepsin proteases.

The closely related serpins squamous cell carcinoma antigen-1 and -2 (SCCA-1 and -2, respectively) are capable of inhibiting cysteine proteases of the papain superfamily. To ascertain whether the ability to inhibit cysteine proteases is an intrinsic property of serpins in general, the reactive center loop (RCL) of the archetypal serine protease inhibitor alpha(1)-antitrypsin was replaced with that of SCCA-1. It was found that this simple substitution could convert alpha(1)-antitrypsin into a cysteine protease inhibitor, albeit an inefficient one. The RCL of SCCA-1 is three residues longer than that of alpha(1)-antitrypsin, and therefore, the effect of loop length on the cysteine protease inhibitory activity was investigated. Mutants in which the RCL was shortened by one, two, or three residues were effective inhibitors with second-order rate constants of 10(5)-10(7) M(-)(1) s(-)(1). In addition to loop length, the identity of the cysteine protease was of considerable importance, since the chimeric molecules inhibited cathepsins L, V, and K efficiently, but not papain or cathepsin B. By testing complexes between an RCL-mimicking peptide and the mutants, it was found that the formation of a stable serpin-cysteine protease complex and the inhibition of a cysteine protease were both critically dependent on RCL insertion. The results strongly indicate that the serpin body is intrinsically capable of supporting cysteine protease inhibition, and that the complex with a papain-like cysteine protease would be expected to be analogous to that seen with serine proteases.

Mechanosensitive induction of apoptosis in fibroblasts is regulated by thrombospondin-1 and integrin associated protein (CD47).

Fibroblasts are cultured in three-dimensional collagen matrices to investigate the effect of mechanical tension on the regulation of apoptosis. Under the influence of mechanical loading, the cells show little apoptosis whereas releasing of tension leads to an increase up to tenfold during the first 24 h and remains constant for further 48 h. An autocrine loop of the integrin alpha(V)beta(3)/CD47 receptor complex and thrombospondin-1 is identified as the molecular coupling device between mechanical loading and apoptosis: The integrin alpha(V)beta(3) is expressed under mechanical loading as well as unloading whereas the CD47 could only be identified after the release of tension. The secreted thrombospondin binds to the active receptor and induces apoptosis. The presented mechanosensitive regulation of apoptosis in fibroblast cultures could be an essential mechanism for the regression of the granulation tissue by apoptosis in the process of wound healing.

The Contribution of Arginine Residues within the P6–P1 Region of α1-Antitrypsin to Its Reaction with Furin

A series of mutants incorporating furin recognition sequences within the P6-P1 region of the reactive site loop of alpha(1)-antitrypsin were constructed. Variants containing different combinations of basic residues in the P1, P2, P4, and P6 positions replacing the wild type (P6)LEAIPM(P1) sequence were evaluated for their capacity to establish SDS-resistant complexes with furin, to affect association rate constants (k(ass) and k'(ass)), or to inhibit furin-dependent proteolysis of a model precursor in vivo. Each variant abolished processing of pro-von Willebrand factor in transfected hEK293 cells. The k(ass) of all variants were found to be similar (1.1-1.7 x 10(6) m(-1) s(-1)) except for one mutant, RERIRR, which had a k(ass) of 3.3 x 10(5) m(-1) s(-1). However, the stoichiometry of inhibition varied with values ranging from 2.9 to >24, indicating rapid formation of the acyl-enzyme intermediate (high k'(ass)). Moreover, those variants having high stoichiometry of inhibition values were accompanied by the rapid formation of cleaved forms of the inhibitors. The data suggest that the rate of conversion of the acyl-enzyme (EI') into the highly stable complex (EI*) was affected by replacement of specific residues within the reactive site loop. Taken together, the results reveal how furin recognition sequences within the context of the biochemical properties of serpins will play a role in the capacity of the protein to follow either the inhibitory or the substrate pathway.

A Distinct Meta-active Conformation in the 1.1-Å Resolution Structure of Wild-type ApoCheY

CheY is the best characterized member of the response regulator superfamily, and as such it has become the principal model for understanding the initial molecular mechanisms of signaling in two-component systems. Normal signaling by response regulators requires phosphorylation, in combination with an activation mechanism whose conformational effects are not completely understood. CheY activation involves three events, phosphorylation, a conformational change in the beta(4)--alpha(4) loop, and a rotational restriction of the side chain of tyrosine 106. An outstanding question concerns the nature of an active conformation in the apoCheY population. The details of this 1.08-A resolution crystal structure of wild-type apoCheY shows the beta(4)--alpha(4) loop in two distinctly different conformations that sterically correlate with the two rotameric positions of the tyrosine 106 side chain. One of these conformational states of CheY is the inactive form, and we propose that the other is a meta-active form, responsible for the active properties seen in apoCheY.

Cytoplasmic surface structure of bacteriorhodopsin consisting of interhelical loops and C-terminal alpha helix, modified by a variety of environmental factors as studied by (13)C-NMR.

We have examined the (13)C-NMR spectra of [3-(13)C] Ala-labeled bacteriorhodopsin and its mutants by varying a variety of environmental or intrinsic factors such as ionic strength, temperature, pH, truncation of the C-terminal alpha helix, and site-directed mutation at cytoplasmic loops, in order to gain insight into a plausible surface structure arising from the C-terminal alpha helix and loops. It is found that the surface structure can be characterized as a complex stabilized by salt bridges or metal-mediated linkages among charged side chains. The surface complex in bacteriorhodopsin is most pronounced under the conditions of 10 mM NaCl at neutral pH but is destabilized to yield relaxed states when environmental factors are changed to high ionic strength, low pH and higher temperature. These two states were readily distinguished by associated spectral changes, including suppressed (cross polarization-magic angle spinning NMR) or displaced (upfield) (13)C signals from the C-terminal alpha helix, or modified spectral features in the loop region. It is also noteworthy that such spectral changes, when going from the complexed to relaxed states, occur either when the C-terminal alpha helix is deleted or site-directed mutations were introduced at a cytoplasmic loop. These observations clearly emphasize that organization of the cytoplasmic surface complex is important in the stabilization of the three-dimensional structure at ambient temperature, and subsequently plays an essential role in biological functions.

Molecular determinants of the functional interaction between syntaxin and N-type Ca2+ channel gating.

Syntaxin is a key presynaptic protein that binds to N- and P/Q-type Ca(2+) channels in biochemical studies and affects gating of these Ca(2+) channels in expression systems and in synaptosomes. The present study was aimed at understanding the molecular basis of syntaxin modulation of N-type channel gating. Mutagenesis of either syntaxin 1A or the pore-forming alpha(1B) subunit of N-type Ca(2+) channels was combined with functional assays of N-type channel gating in a Xenopus oocyte coexpression system and in biochemical binding experiments in vitro. Our analysis showed that the transmembrane region of syntaxin and a short region within the H3 helical cytoplasmic domain of syntaxin, containing residues Ala-240 and Val-244, appeared critical for the channel modulation but not for biochemical association with the "synprint site" in the II/III loop of alpha(1B). These results suggest that syntaxin and the alpha(1B) subunit engage in two kinds of interactions: an anchoring interaction via the II/III loop synprint site and a modulatory interaction via another site located elsewhere in the channel sequence. The segment of syntaxin H3 found to be involved in the modulatory interaction would lie hidden within the four-helix structure of the SNARE complex, supporting the hypothesis that syntaxin's ability to regulate N-type Ca(2+) channels would be enabled after SNARE complex disassembly after synaptic vesicle exocytosis.

Excitation-Contraction Coupling Is Not Affected by Scrambled Sequence in Residues 681–690 of the Dihydropyridine Receptor II-III Loop

A peptide corresponding to residues 681-690 of the II-III loop of the skeletal muscle dihydropyridine receptor alpha(1) subunit (DHPR, alpha(1S)) has been reported to activate the skeletal muscle ryanodine receptor (RyR1) in vitro. Within this region of alpha(1S), a cluster of basic residues, Arg(681)-Lys(685), was previously reported to be indispensable for the activation of RyR1 in microsomal preparations and lipid bilayers. We have used an intact alpha(1S) subunit with scrambled sequence in this region of the II-III loop (alpha(1S)-scr) to test the importance of residues 681-690 and the basic motif for skeletal-type excitation-contraction (EC) coupling and retrograde signaling in vivo. When expressed in dysgenic myotubes (which lack endogenous alpha(1S)), alpha(1S)-scr restored calcium currents that were indistinguishable, in current density and voltage dependence, from those restored by wild-type alpha(1S). The scrambled DHPR also rescued skeletal-type EC coupling, as indicated by electrically evoked contractions in the presence of 0.5 mm Cd(2+) and 0.1 mm La(3+). Furthermore, the release of intracellular Ca(2+), as assayed by the indicator dye, Fluo-3, had similar kinetics and voltage dependence for alpha(1S) and alpha(1S)-scr. These data suggest that residues 681-690 of the alpha(1S) II-III loop are not essential in muscle cells for normal functioning of the DHPR, including skeletal-type EC coupling and retrograde signaling.

Pairwise Interactions between Neuronal α7Acetylcholine Receptors and α-Conotoxin PnIB

This work uses alpha-conotoxin PnIB to probe the agonist binding site of neuronal alpha(7) acetylcholine receptors. We mutated the 13 non-cysteine residues in CTx PnIB, expressed alpha(7)/5-hydroxytryptamine-3 homomeric receptors in 293 HEK cells, and measured binding of each mutant toxin to the expressed receptors by competition against the initial rate of (125)I-alpha-bungarotoxin binding. The results reveal that residues Ser-4, Leu-5, Pro-6, Pro-7, Ala-9, and Leu-10 endow CTx PnIB with affinity for alpha(7)/5-hydroxytryptamine-3 receptors; side chains of these residues cluster in a localized region within the three-dimensional structure of CTx PnIB. We next mutated key residues in the seven loops of alpha(7) that converge at subunit interfaces to form the agonist binding site. The results reveal predominant contributions by residues Trp-149 and Tyr-93 in alpha(7) and smaller contributions by Ser-34, Arg-186, Tyr-188, and Tyr-195. To identify pairwise interactions that stabilize the receptor-conotoxin complex, we measured binding of receptor and toxin mutations and analyzed the results by double mutant cycles. The results reveal a single dominant interaction between Leu-10 of CTx PnIB and Trp-149 of alpha(7) that anchors the toxin to the binding site. We also find weaker interactions between Pro-6 of CTx PnIB and Trp-149 and between both Pro-6 and Pro-7 and Tyr-93 of alpha(7). The overall results demonstrate that a localized hydrophobic region in CTx PnIB interacts with conserved aromatic residues on one of the two faces of the alpha(7) binding site.

Alpha 3 beta 1 adhesion to laminin-5 and invasin: critical and differential role of integrin residues clustered at the boundary between alpha 3 N-terminal repeats 2 and 3.

Integrin/ligand interaction is a therapeutic target for many diseases. We previously reported that residues critical for ligand binding are clustered in N-terminal repeat 3 (in the predicted 2-3 loop) of alpha 4, alpha 5 and alpha IIb. Here we have localized residues critical for ligand binding in the alpha 3 subunit of integrin alpha 3 beta 1 with distinct ligand specificity (laminin-5). We identified an alpha 3 epitope common to several function-blocking anti-alpha 3 antibodies at the boundary between repeats 1 and 2 (residues 75-80). We found that swapping the predicted 4-1 loop (residues 153-165) at the boundary between repeats 2 and 3 with the corresponding alpha 4 sequence and mutating Thr-162 and Gly-163 residues in this predicted loop block laminin-5 binding. Thr-162 and Gly-163 and the antibody epitope are separated in the primary structure; however, they are close to each other in the proposed beta-propeller model. Mutating residues recently reported to block (Tyr-186 and Trp-188) or enhance (Asp-122) laminin-5 binding to alpha 3 beta 1 [Krukonis, E. S., Dersch, P., Eble, J. A., and Isberg, R. R.(1998) J. Biol. Chem. 273, 31837-31843] did not affect laminin-5 binding under the assay conditions used. Thr-162 and Gly-163 are not critical for adhesion to invasin, indicating that laminin-5 and invasin may use different recognition mechanisms, and that mutation of Thr-162 and Gly-163 does not drastically affect the integrity of alpha 3 beta 1. These results suggest that residues critical for ligand binding may be similarly (but not identically) located in repeat 3 of the alpha subunit regardless of ligand specificity.

Regulation of conventional protein kinase C isozymes by phosphoinositide-dependent kinase 1 (PDK-1).

Phosphorylation critically regulates the catalytic function of most members of the protein kinase superfamily. One such member, protein kinase C (PKC), contains two phosphorylation switches: a site on the activation loop that is phosphorylated by another kinase, and two autophosphorylation sites in the carboxyl terminus. For conventional PKC isozymes, the mature enzyme, which is present in the detergent-soluble fraction of cells, is quantitatively phosphorylated at the carboxy-terminal sites but only partially phosphorylated on the activation loop. This study identifies the recently discovered phosphoinositide-dependent kinase 1, PDK-1, as a regulator of the activation loop of conventional PKC isozymes. First, studies in vivo revealed that PDK-1 controls the amount of mature (carboxy-terminally phosphorylated) conventional PKC. More specifically, co-expression of the conventional PKC isoform PKC betaII with a catalytically inactive form of PDK-1 in COS-7 cells resulted in both the accumulation of non-phosphorylated PKC and a corresponding decrease in PKC activity. Second, studies in vitro using purified proteins established that PDK-1 specifically phosphorylates the activation loop of PKC alpha and betaII. The phosphorylation of the mature PKC enzyme did not modulate its basal activity or its maximal cofactor-dependent activity. Rather, the phosphorylation of non-phosphorylated enzyme by PDK-1 triggered carboxy-terminal phosphorylation of PKC, thus providing the first step in the generation of catalytically competent (mature) enzyme. We have shown that PDK-1 controls the phosphorylation of conventional PKC isozymes in vivo. Studies performed in vitro establish that PDK-1 directly phosphorylates PKC on the activation loop, thereby allowing carboxy-terminal phosphorylation of PKC. These data suggest that phosphorylation of the activation loop by PDK-1 provides the first step in the processing of conventional PKC isozymes by phosphorylation.

The long internal loop of the alpha 3 subunit targets nAChRs to subdomains within individual synapses on neurons in vivo.

Different types of neurotransmitter receptors coexist within single neurons and must be targeted to discrete synaptic regions for proper function. In chick ciliary ganglion neurons, nicotinic acetylcholine receptors (nAChRs) containing alpha 3 and alpha 5 subunits are concentrated in the postsynaptic membrane, whereas alpha-bungarotoxin receptors composed of alpha 7 subunits are localized perisynaptically and excluded from the synapse. Using retroviral vector-mediated gene transfer in vivo, we show that the long cytoplasmic loop of alpha 3 targets chimeric alpha 7 subunits to the synapse and reduces endogenous nAChR surface levels, whereas the alpha 5 loop does neither. These results show that a particular domain of one subunit targets specific receptor subtypes to the interneuronal synapse in vivo. Moreover, our findings suggest a difference in the mechanisms that govern assembly of interneuronal synapses as compared to the neuromuscular junction in vertebrates.

Identification of the minimum essential region in the II-III loop of the dihydropyridine receptor alpha 1 subunit required for activation of skeletal muscle-type excitation-contraction coupling.

We have previously shown that among several peptides encompassing various regions of the II-III loop of the dihydropyridine receptor alpha 1 subunit, only one peptide corresponding to the Thr671-Leu690 region (designated as peptide A) activated ryanodine binding to and induced calcium release from the sarcoplasmic reticulum [El-Hayek et al. (1995) J. Biol. Chem. 270, 22116-22118]. To further localize within peptide A the minimum unit essential for activating the sarcoplasmic reticulum calcium release channel, we synthesized variously truncated forms of peptide A and examined their ability to activate ryanodine binding. We found that the carboxy-terminal 10-residue region of peptide A encompassing Arg681-Leu690 (peptide As-10; s, skeletal muscle-type sequence) activated ryanodine binding in a RyR1-specific manner and induced calcium release even more efficiently than the 20-residue peptide A. Further truncation of one or more residue(s) of peptide As-10 virtually abolished both functions of activating ryanodine binding and inducing Ca2+ release. The activating ability of As-10 seems to be determined by at least two factors: (1) the distribution of the positively charged residues, and (2) the skeletal muscle-type amino acid sequence, as deduced from the comparison of various peptides with modified structures. These results provide evidence that the minimum essential unit for the in situ trigger of skeletal muscle excitation-contraction coupling is localized in the Arg681-Leu690 region of the II-III loop of the alpha 1 subunit of the dihydropyridine receptor.

Signal transmission and transduction in excitation-contraction coupling.

For the better understanding of the molecular mechanism of E-C coupling, two key questions remain to be resolved: (a) how the excitation signal elicited in the T-tubule membrane is transmitted to the ryanodine receptor, RyR (signal transmission), and (b) how the signal transmitted from the T-tubule to the RyR is translated into the action of opening the sarcoplasmic reticulum Ca2+ channel to induce Ca2+ release and muscle contraction (signal transduction). Our recent studies on E-C coupling with the use of the isolated triads and synthetic peptides have provided several pieces of new information. It appears that the signal transmission is mediated by the voltage-controlled binding of the Thr671-Leu690 region (Trigger) of the cytoplasmic II-III loop of the dihydropyridine receptor alpha 1 a subunit to the putative activator site on the RyR. The transmitted signal is translated to the action of channel opening by mediation of rapid conformational changes occurring in the RyR. Upon T-tubule polarization the Glu724-Pro760 region of the loop (Blocker) replaces the RyR-bound Trigger. This reprimes the RyR to the original conformational state.

Closed structure of phosphoglycerate kinase from Thermotoga maritima reveals the catalytic mechanism and determinants of thermal stability.

Phosphoglycerate kinase (PGK) is essential in most living cells both for ATP generation in the glycolytic pathway of aerobes and for fermentation in anaerobes. In addition, in many plants the enzyme is involved in carbon fixation. Like other kinases, PGK folds into two distinct domains, which undergo a large hinge-bending motion upon catalysis. The monomeric 45 kDa enzyme catalyzes the transfer of the C1-phosphoryl group from 1, 3-bisphosphoglycerate to ADP to form 1,3-bisphosphoglycerate to ADP to form 3-phosphoglycerate and ATP. For decades, the conformation of the enzyme during catalysis has been enigmatic. The crystal structure of PGK from the hyperthermophilic organism Thermotoga maritima (TmPGK) represents the first structure of an extremely thermostable PGK. It adds to a series of four known crystal structures of PGKs from mesophilic via moderately thermophilic to a hyperthermophilic organism, allowing a detailed analysis of possible structural determinants of thermostability. The crystal structure of TmPGK was determined to 2.0 A resolution, as a ternary complex with the product 3-phosphoglycerate and the product analogue AMP-PNP (adenylyl-imido diphosphate). The complex crystallizes in a closed conformation with a drastically reduced inter-domain angle and a distance between the two bound ligands of 4.4 A, presumably representing the active conformation of the enzyme. The structure provides new details of the catalytic mechanism. An inter-domain salt bridge between residues Arg62 and Asp200 forms a strap to hold the two domains in the closed state. We identify Lys197 as a residue involved in stabilization of the transition state phosphoryl group, and so term it the 'phosphoryl gripper'. The hinge-bending motion of the two domains upon closure of the structure, as seen in the Trypanosoma PGK structure, is confirmed. This closed conformation obviously occurs after binding of both substrates and is locked by the Arg62-Asp200 salt bridge. Re-orientations in the conserved active-site loop region around Thr374 also bring both domains into direct contact in the core region of the former inter-domain cleft, to form the complete catalytic site. Comparison of extremely thermostable TmPGK with less thermostable homologues reveals that its increased rigidity is achieved by a raised number of intramolecular interactions, such as an increased number of ion pairs and additional stabilization of alpha helix and loop regions. The covalent fusion with triosephosphate isomerase might represent an additional stabilization strategy.