Phosphorylation of cMyBP-C Affects Contractile Mechanisms in a Site-specific Manner

Cardiac myosin binding protein-C (cMyBP-C) is a cardiac-specific, thick-filament regulatory protein that is differentially phosphorylated at Ser²⁷³, Ser²⁸², and Ser³⁰² by various kinases and modulates contraction. In this study, phosphorylation-site-specific effects of cMyBP-C on myocardial contractility and cross-bridge kinetics were studied by sinusoidal analysis in papillary and trabecular muscle fibers isolated from t/t (cMyBP-C-null) mice and in their counterparts in which cMyBP-C contains the ADA (Ala²⁷³-Asp²⁸²-Ala³⁰²), DAD (Asp²⁷³-Ala²⁸²-Asp³⁰²), and SAS (Ser²⁷³-Ala²⁸²-Ser³⁰²) mutations; the results were compared to those from mice expressing the wild-type (WT) transgene on the t/t background. Under standard activating conditions, DAD fibers showed significant decreases in tension (∼50%), stiffness, the fast apparent rate constant 2πc, and its magnitude C, as well as its magnitude H, but an increase in the medium rate constant 2πb, with respect to WT. The t/t fibers showed a smaller drop in stiffness and a significant decrease in 2πc that can be explained by isoform shift of myosin heavy chain. In the pCa-tension study using the 8 mM phosphate (Pi) solution, there was hardly any difference in Ca²⁺ sensitivity (pCa₅₀) and cooperativity (n_H) between the mutant and WT samples. However, in the solutions without Pi, DAD showed increased n_H and slightly decreased pCa₅₀. We infer from these observations that the nonphosphorylatable residue 282 combined with phosphomimetic residues Asp²⁷³ and/or Asp³⁰² (in DAD) is detrimental to cardiomyocytes by lowering isometric tension and altering cross-bridge kinetics with decreased 2πc and increased 2πb. In contrast, a single change of residue 282 to nonphosphorylatable Ala (SAS), or to phosphomimetic Asps together with the changes of residues 273 and 302 to nonphosphorylatable Ala (ADA) causes minute changes in fiber mechanics.     

Distinct Sarcomeric Substrates Are Responsible for Protein Kinase D-mediated Regulation of Cardiac Myofilament Ca2+ Sensitivity and Cross-bridge Cycling

Protein kinase D (PKD), a serine/threonine kinase with emerging cardiovascular functions, phosphorylates cardiac troponin I (cTnI) at Ser²²/Ser²³, reduces myofilament Ca²⁺ sensitivity, and accelerates cross-bridge cycle kinetics. Whether PKD regulates cardiac myofilament function entirely through cTnI phosphorylation at Ser²²/Ser²³ remains to be established. To determine the role of cTnI phosphorylation at Ser²²/Ser²³ in PKD-mediated regulation of cardiac myofilament function, we used transgenic mice that express cTnI in which Ser²²/Ser²³ are substituted by nonphosphorylatable Ala (cTnI-Ala₂). In skinned myocardium from wild-type (WT) mice, PKD increased cTnI phosphorylation at Ser²²/Ser²³ and decreased the Ca²⁺ sensitivity of force. In contrast, PKD had no effect on the Ca²⁺ sensitivity of force in myocardium from cTnI-Ala₂ mice, in which Ser²²/Ser²³ were unavailable for phosphorylation. Surprisingly, PKD accelerated cross-bridge cycle kinetics similarly in myocardium from WT and cTnI-Ala₂ mice. Because cardiac myosin-binding protein C (cMyBP-C) phosphorylation underlies cAMP-dependent protein kinase (PKA)-mediated acceleration of cross-bridge cycle kinetics, we explored whether PKD phosphorylates cMyBP-C at its PKA sites, using recombinant C1C2 fragments with or without site-specific Ser/Ala substitutions. Kinase assays confirmed that PKA phosphorylates Ser²⁷³, Ser²⁸², and Ser³⁰², and revealed that PKD phosphorylates only Ser³⁰². Furthermore, PKD phosphorylated Ser³⁰² selectively and to a similar extent in native cMyBP-C of skinned myocardium from WT and cTnI-Ala₂ mice, and this phosphorylation occurred throughout the C-zones of sarcomeric A-bands. In conclusion, PKD reduces myofilament Ca²⁺ sensitivity through cTnI phosphorylation at Ser²²/Ser²³ but accelerates cross-bridge cycle kinetics by a distinct mechanism. PKD phosphorylates cMyBP-C at Ser³⁰², which may mediate the latter effect.     

Cardiac Myosin Binding Protein C Phosphorylation Affects Cross-Bridge Cycle's Elementary Steps in a Site-Specific Manner

Based on our recent finding that cardiac myosin binding protein C (cMyBP-C) phosphorylation affects muscle contractility in a site-specific manner, we further studied the force per cross-bridge and the kinetic constants of the elementary steps in the six-state cross-bridge model in cMyBP-C mutated transgenic mice for better understanding of the influence of cMyBP-C phosphorylation on contractile functions. Papillary muscle fibres were dissected from cMyBP-C mutated mice of ADA (Ala273-Asp282-Ala302), DAD (Asp273-Ala282-Asp302), SAS (Ser273-Ala282-Ser302), and t/t (cMyBP-C null) genotypes, and the results were compared to transgenic mice expressing wide-type (WT) cMyBP-C. Sinusoidal analyses were performed with serial concentrations of ATP, phosphate (Pi), and ADP. Both t/t and DAD mutants significantly reduced active tension, force per cross-bridge, apparent rate constant (2πc), and the rate constant of cross-bridge detachment. In contrast to the weakened ATP binding and enhanced Pi and ADP release steps in t/t mice, DAD mice showed a decreased ADP release without affecting the ATP binding and the Pi release. ADA showed decreased ADP release, and slightly increased ATP binding and cross-bridge detachment steps, whereas SAS diminished the ATP binding step and accelerated the ADP release step. t/t has the broadest effects with changes in most elementary steps of the cross-bridge cycle, DAD mimics t/t to a large extent, and ADA and SAS predominantly affect the nucleotide binding steps. We conclude that the reduced tension production in DAD and t/t is the result of reduced force per cross-bridge, instead of the less number of strongly attached cross-bridges. We further conclude that cMyBP-C is an allosteric activator of myosin to increase cross-bridge force, and its phosphorylation status modulates the force, which is regulated by variety of protein kinases.     

Novel role for p90 ribosomal S6 kinase in the regulation of cardiac myofilament phosphorylation

In myocardium, the 90-kDa ribosomal S6 kinase (RSK) is activated by diverse stimuli and regulates the sarcolemmal Na(+)/H(+) exchanger through direct phosphorylation. Only limited information is available on other cardiac RSK substrates and functions. We evaluated cardiac myosin-binding protein C (cMyBP-C), a sarcomeric regulatory phosphoprotein, as a potential RSK substrate. In rat ventricular myocytes, RSK activation by endothelin 1 (ET1) increased cMyBP-C phosphorylation at Ser(282), which was inhibited by the selective RSK inhibitor D1870. Neither ET1 nor D1870 affected the phosphorylation status of Ser(273) or Ser(302), cMyBP-C residues additionally targeted by cAMP-dependent protein kinase (PKA). Complementary genetic gain- and loss-of-function experiments, through the adenoviral expression of wild-type or kinase-inactive RSK isoforms, confirmed RSK-mediated phosphorylation of cMyBP-C at Ser(282). Kinase assays utilizing as substrate wild-type or mutated (S273A, S282A, S302A) recombinant cMyBP-C fragments revealed direct and selective Ser(282) phosphorylation by RSK. Immunolabeling with a Ser(P)(282) antibody and confocal fluorescence microscopy showed RSK-mediated phosphorylation of cMyBP-C across the C-zones of sarcomeric A-bands. In chemically permeabilized mouse ventricular muscles, active RSK again induced selective Ser(282) phosphorylation in cMyBP-C, accompanied by significant reduction in Ca(2+) sensitivity of force development and significant acceleration of cross-bridge cycle kinetics, independently of troponin I phosphorylation at Ser(22)/Ser(23). The magnitudes of these RSK-induced changes were comparable with those induced by PKA, which phosphorylated cMyBP-C additionally at Ser(273) and Ser(302). We conclude that Ser(282) in cMyBP-C is a novel cardiac RSK substrate and its selective phosphorylation appears to regulate cardiac myofilament function.     

Insights into binding modes of tumstatin peptide T7 with the active site of αvβ3 integrin

Through interaction with the active site of α_vβ₃ integrin, tumstatin T7 peptide inhibits both the angiogenesis and the proliferation of tumour cells. In this work, docking in conjunction with molecular dynamics simulation was used to explore the binding mode of T7 peptide and α_vβ₃ integrin. The binding mode analysis revealed that the residues Ser⁹⁰, Arg⁹¹, Asp⁹³ and Tyr⁹⁴ in T7 peptide, and (α)-Asp¹⁵⁰, (β)-Arg²¹⁴, (α)-Asp¹⁴⁸ (α)-Gln²¹⁴ and (α)-Glu¹²³ in the active site of α_vβ₃ integrin were most likely the key interaction sites. The hydroxyl of Tyr⁹⁴ coordinates α_vβ₃ via a Mn²⁺ ion, revealing that Mn²⁺ is also an important factor for the interaction. The insight into these key interaction sites not only suggests that the active site of α_vβ₃ integrin can bind to molecules through multiple binding mechanisms, but also provides some useful information for structure-based drug design.     

Phosphorylation of actopaxin regulates cell spreading and migration

Actopaxin is an actin and paxillin binding protein that localizes to focal adhesions. It regulates cell spreading and is phosphorylated during mitosis. Herein, we identify a role for actopaxin phosphorylation in cell spreading and migration. Stable clones of U2OS cells expressing actopaxin wild-type (WT), nonphosphorylatable, and phosphomimetic mutants were developed to evaluate actopaxin function. All proteins targeted to focal adhesions, however the nonphosphorylatable mutant inhibited spreading whereas the phosphomimetic mutant cells spread more efficiently than WT cells. Endogenous and WT actopaxin, but not the nonphosphorylatable mutant, were phosphorylated in vivo during cell adhesion/spreading. Expression of the nonphosphorylatable actopaxin mutant significantly reduced cell migration, whereas expression of the phosphomimetic increased cell migration in scrape wound and Boyden chamber migration assays. In vitro kinase assays demonstrate that extracellular signal-regulated protein kinase phosphorylates actopaxin, and treatment of U2OS cells with the MEK1 inhibitor UO126 inhibited adhesion-induced phosphorylation of actopaxin and also inhibited cell migration.     

PKA Phosphorylation of Cardiac Troponin I Modulates Activation and?Relaxation Kinetics of Ventricular Myofibrils

Protein kinase A (PKA) phosphorylation of myofibril proteins constitutes an important pathway for β-adrenergic modulation of cardiac contractility and relaxation. PKA targets the N-terminus (Ser-23/24) of cardiac troponin I (cTnI), cardiac myosin-binding protein C (cMyBP-C) and titin. The effect of PKA-mediated phosphorylation on the magnitude of contraction has been studied in some detail, but little is known about how it modulates the kinetics of thin filament activation and myofibril relaxation as Ca²⁺ levels vary. Troponin C (cTnC) interaction with cTnI (C-I interaction) is a critical step in contractile activation that can be modulated by cTnI phosphorylation. We tested the hypothesis that altering C-I interactions by PKA, or by cTnI phosphomimetic mutations (S23D/S24D-cTnI), directly affects thin filament activation and myofilament relaxation kinetics. Rat ventricular myofibrils were isolated and endogenous cTn was exchanged with either wild-type cTnI, or S23D/S24D-cTnI recombinant cTn. Contractile mechanics were monitored at maximum and submaximal Ca²⁺ concentrations. PKA treatment of wild-type cTn or exchange of cTn containing S23D/S24D-cTnI resulted in an increase in the rate of early, slow phase of relaxation (k_REL,slow) and a decrease in its duration (t_REL,slow). These effects were greater for submaximal Ca²⁺ activated contractions. PKA treatment also reduced the rate of contractile activation (k_ACT) at maximal, but not submaximal Ca²⁺, and reduced the Ca²⁺ sensitivity of contraction. Using a fluorescent probe coupled to cTnC (C35S-IANBD), the Ca²⁺-cTn binding affinity and C-I interaction were monitored. Ca²⁺ binding to cTn (pCa₅₀) was significantly decreased when cTnI was phosphorylated by PKA (ΔpCa₅₀ = 0.31). PKA phosphorylation of cTnI also weakened C-I interaction in the presence of Ca²⁺. These data suggest that weakened C-I interaction, via PKA phosphorylation of cTnI, may slow thin filament activation and result in increased myofilament relaxation kinetics, the latter of which could enhance early phase diastolic relaxation during β-adrenergic stimulation.     

PKA Phosphorylation of Cardiac Troponin I Modulates Activation and Relaxation Kinetics of Ventricular Myofibrils

Protein kinase A (PKA) phosphorylation of myofibril proteins constitutes an important pathway for β-adrenergic modulation of cardiac contractility and relaxation. PKA targets the N-terminus (Ser-23/24) of cardiac troponin I (cTnI), cardiac myosin-binding protein C (cMyBP-C) and titin. The effect of PKA-mediated phosphorylation on the magnitude of contraction has been studied in some detail, but little is known about how it modulates the kinetics of thin filament activation and myofibril relaxation as Ca²⁺ levels vary. Troponin C (cTnC) interaction with cTnI (C-I interaction) is a critical step in contractile activation that can be modulated by cTnI phosphorylation. We tested the hypothesis that altering C-I interactions by PKA, or by cTnI phosphomimetic mutations (S23D/S24D-cTnI), directly affects thin filament activation and myofilament relaxation kinetics. Rat ventricular myofibrils were isolated and endogenous cTn was exchanged with either wild-type cTnI, or S23D/S24D-cTnI recombinant cTn. Contractile mechanics were monitored at maximum and submaximal Ca²⁺ concentrations. PKA treatment of wild-type cTn or exchange of cTn containing S23D/S24D-cTnI resulted in an increase in the rate of early, slow phase of relaxation (k_REL,slow) and a decrease in its duration (t_REL,slow). These effects were greater for submaximal Ca²⁺ activated contractions. PKA treatment also reduced the rate of contractile activation (k_ACT) at maximal, but not submaximal Ca²⁺, and reduced the Ca²⁺ sensitivity of contraction. Using a fluorescent probe coupled to cTnC (C35S-IANBD), the Ca²⁺-cTn binding affinity and C-I interaction were monitored. Ca²⁺ binding to cTn (pCa₅₀) was significantly decreased when cTnI was phosphorylated by PKA (ΔpCa₅₀ = 0.31). PKA phosphorylation of cTnI also weakened C-I interaction in the presence of Ca²⁺. These data suggest that weakened C-I interaction, via PKA phosphorylation of cTnI, may slow thin filament activation and result in increased myofilament relaxation kinetics, the latter of which could enhance early phase diastolic relaxation during β-adrenergic stimulation.     

Two Salt Bridges Differentially Contribute to the Maintenance of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Function

Previous studies have identified two salt bridges in human CFTR chloride ion channels, Arg³⁵²-Asp⁹⁹³ and Arg³⁴⁷-Asp⁹²⁴, that are required for normal channel function. In the present study, we determined how the two salt bridges cooperate to maintain the open pore architecture of CFTR. Our data suggest that Arg³⁴⁷ not only interacts with Asp⁹²⁴ but also interacts with Asp⁹⁹³. The tripartite interaction Arg³⁴⁷-Asp⁹²⁴-Asp⁹⁹³ mainly contributes to maintaining a stable s2 open subconductance state. The Arg³⁵²-Asp⁹⁹³ salt bridge, in contrast, is involved in stabilizing both the s2 and full (f) open conductance states, with the main contribution being to the f state. The s1 subconductance state does not require either salt bridge. In confirmation of the role of Arg³⁵² and Asp⁹⁹³, channels bearing cysteines at these sites could be latched into a full open state using the bifunctional cross-linker 1,2-ethanediyl bismethanethiosulfonate, but only when applied in the open state. Channels remained latched open even after washout of ATP. The results suggest that these interacting residues contribute differently to stabilizing the open pore in different phases of the gating cycle.     

Cardiac MyBP-C phosphorylation regulates the Frank–Starling relationship in murine hearts

The Frank–Starling relationship establishes that elevated end-diastolic volume progressively increases ventricular pressure and stroke volume in healthy hearts. The relationship is modulated by a number of physiological inputs and is often depressed in human heart failure. Emerging evidence suggests that cardiac myosin-binding protein-C (cMyBP-C) contributes to the Frank–Starling relationship. We measured contractile properties at multiple levels of structural organization to determine the role of cMyBP-C and its phosphorylation in regulating (1) the sarcomere length dependence of power in cardiac myofilaments and (2) the Frank–Starling relationship in vivo. We compared transgenic mice expressing wild-type cMyBP-C on the null background, which have ∼50% phosphorylated cMyBP-C (Controls), to transgenic mice lacking cMyBP-C (KO) and to mice expressing cMyBP-C that have serine-273, -282, and -302 mutated to aspartate (cMyBP-C t3SD) or alanine (cMyBP-C t3SA) on the null background to mimic either constitutive PKA phosphorylation or nonphosphorylated cMyBP-C, respectively. We observed a continuum of length dependence of power output in myocyte preparations. Sarcomere length dependence of power progressively increased with a rank ordering of cMyBP-C KO = cMyBP-C t3SA < Control < cMyBP-C t3SD. Length dependence of myofilament power translated, at least in part, to hearts, whereby Frank–Starling relationships were steepest in cMyBP-C t3SD mice. The results support the hypothesis that cMyBP-C and its phosphorylation state tune sarcomere length dependence of myofibrillar power, and these regulatory processes translate across spatial levels of myocardial organization to control beat-to-beat ventricular performance.     

Differential Regulation of Kainate Receptor Trafficking by Phosphorylation of Distinct Sites on GluR6

Kainate receptors are widely expressed in the brain, and are present at pre- and postsynaptic sites where they play a prominent role in synaptic plasticity and the regulation of network activity. Within individual neurons, kainate receptors of different subunit compositions are targeted to various locations where they serve distinct functional roles. Despite this complex targeting, relatively little is known about the molecular mechanisms regulating kainate receptor subunit trafficking. Here we investigate the role of phosphorylation in the trafficking of the GluR6 kainate receptor subunit. We identify two specific residues on the GluR6 C terminus, Ser⁸⁴⁶ and Ser⁸⁶⁸, which are phosphorylated by protein kinase C (PKC) and dramatically regulate GluR6 surface expression. By using GluR6 containing phosphomimetic and nonphosphorylatable mutations for these sites expressed in heterologous cells or in neurons lacking endogenous GluR6, we show that phosphorylation of Ser⁸⁴⁶ or Ser⁸⁶⁸ regulates receptor trafficking through the biosynthetic pathway. Additionally, Ser⁸⁴⁶ phosphorylation dynamically regulates endocytosis of GluR6 at the plasma membrane. Our findings thus demonstrate that phosphorylation of PKC sites on GluR6 regulates surface expression of GluR6 at distinct intracellular trafficking pathways, providing potential molecular mechanisms for the PKC-dependent regulation of synaptic kainate receptor function observed during various forms of synaptic plasticity.     

Ser123 Is Essential for the Water Channel Activity of McPIP2;1 from Mesembryanthemum crystallinum

The increased expression of McPIP2;1 (MipC), a root-specific aquaporin (AQP) from Mesembryanthemum crystallinum, under salt stress has suggested a role for this AQP in the salt tolerance of the plant. However, whether McPIP2;1 transports water or another solute and how its activity is regulated are so far unknown. Therefore, wild type (wt) or mutated McPIP2;1 protein was expressed in Xenopus laevis oocytes. Then, the osmotic water permeability (P_f) of the oocytes membrane was assessed by hypotonic challenges. Selectivity of McPIP2;1 to water was determined by radiolabeled glycerol or urea uptake assays. Moreover, swelling and in vitro phosphorylation assays revealed that both water permeation and phosphorylation status of McPIP2;1 were significantly increased by the phosphorylation agonists okadaic acid (OA), phorbol myristate acetate (PMA), and 8-Br-cAMP, and markedly decreased by the inhibitory peptides PKI 14-22 and PKC 20-28, inhibitors of protein kinases A (PKA) and C (PKC), respectively. Substitution of Ser¹²³ or both, Ser¹²³ and Ser²⁸², abolished the water channel activity of McPIP2;1 while substitution of Ser²⁸² only partially inhibited it (51.9% inhibition). Despite lacking Ser¹²³ and/or Ser²⁸², the McPIP2;1 mutant forms were still phosphorylated in vitro, which suggests that phosphorylation may have a dual role on this AQP. Our results indicate that McPIP2;1 water permeability depends completely on Ser¹²³ and is positively regulated by PKA- and PKC-mediated phosphorylation. Regulation of the phosphorylation status of McPIP2;1 may contribute to control water transport through root cells when the plant is subjected to high salinity conditions.     

Determinants of Versican-V1 Proteoglycan Processing by the Metalloproteinase ADAMTS5

Proteolysis of the Glu⁴⁴¹-Ala⁴⁴² bond in the glycosaminoglycan (GAG) β domain of the versican-V1 variant by a disintegrin-like and metalloproteinase domain with thrombospondin type 1 motif (ADAMTS) proteases is required for proper embryo morphogenesis. However, the processing mechanism and the possibility of additional ADAMTS-cleaved processing sites are unknown. We demonstrate here that if Glu⁴⁴¹ is mutated, ADAMTS5 cleaves inefficiently at a proximate upstream site but normally does not cleave elsewhere within the GAGβ domain. Chondroitin sulfate (CS) modification of versican is a prerequisite for cleavage at the Glu⁴⁴¹-Ala⁴⁴² site, as demonstrated by reduced processing of CS-deficient or chondroitinase ABC-treated versican-V1. Site-directed mutagenesis identified the N-terminal CS attachment sites Ser⁵⁰⁷ and Ser⁵²⁵ as essential for processing of the Glu⁴⁴¹-Ala⁴⁴² bond by ADAMTS5. A construct including only these two GAG chains, but not downstream GAG attachment sites, was cleaved efficiently. Therefore, CS chain attachment to Ser⁵⁰⁷ and Ser⁵²⁵ is necessary and sufficient for versican proteolysis by ADAMTS5. Mutagenesis of Glu⁴⁴¹ and an antibody to a peptide spanning Thr⁴³²-Gly⁴⁴⁵ (i.e. containing the scissile bond) reduced versican-V1 processing. ADAMTS5 lacking the C-terminal ancillary domain did not cleave versican, and an ADAMTS5 ancillary domain construct bound versican-V1 via the CS chains. We conclude that docking of ADAMTS5 with two N-terminal GAG chains of versican-V1 via its ancillary domain is required for versican processing at Glu⁴⁴¹-Ala⁴⁴². V1 proteolysis by ADAMTS1 demonstrated a similar requirement for the N-terminal GAG chains and Glu⁴⁴¹. Therefore, versican cleavage can be inhibited substantially by mutation of Glu⁴⁴¹, Ser⁵⁰⁷, and Ser⁵²⁵ or by an antibody to the region of the scissile bond.     

cMyBPC phosphorylation modulates the effect of omecamtiv mecarbil on myocardial force generation

Omecamtiv mecarbil (OM), a direct myosin motor activator, is currently being tested as a therapeutic replacement for conventional inotropes in heart failure (HF) patients. It is known that HF patients exhibit dysregulated β-adrenergic signaling and decreased cardiac myosin-binding protein C (cMyBPC) phosphorylation, a critical modulator of myocardial force generation. However, the functional effects of OM in conditions of altered cMyBPC phosphorylation have not been established. Here, we tested the effects of OM on force generation and cross-bridge (XB) kinetics using murine myocardial preparations isolated from wild-type (WT) hearts and from hearts expressing S273A, S282A, and S302A substitutions (SA) in the M domain, between the C1 and C2 domains of cMyBPC, which cannot be phosphorylated. At submaximal Ca²⁺ activations, OM-mediated force enhancements were less pronounced in SA than in WT myocardial preparations. Additionally, SA myocardial preparations lacked the dose-dependent increases in force that were observed in WT myocardial preparations. Following OM incubation, the basal differences in the rate of XB detachment (k_rel) between WT and SA myocardial preparations were abolished, suggesting that OM differentially affects the XB behavior when cMyBPC phosphorylation is reduced. Similarly, in myocardial preparations pretreated with protein kinase A to phosphorylate cMyBPC, incubation with OM significantly slowed k_rel in both the WT and SA myocardial preparations. Collectively, our data suggest there is a strong interplay between the effects of OM and XB behavior, such that it effectively uncouples the sarcomere from cMyBPC phosphorylation levels. Our findings imply that OM may significantly alter the in vivo cardiac response to β-adrenergic stimulation.     

Critical Roles of Interdomain Interactions for Modulatory ATP Binding to Sarcoplasmic Reticulum Ca2+-ATPase

ATP has dual roles in the reaction cycle of sarcoplasmic reticulum Ca²⁺-ATPase. Upon binding to the Ca₂E1 state, ATP phosphorylates the enzyme, and by binding to other conformational states in a non-phosphorylating modulatory mode ATP stimulates the dephosphorylation and other partial reaction steps of the cycle, thereby ensuring a high rate of Ca²⁺ transport under physiological conditions. The present study elucidates the mechanism underlying the modulatory effect on dephosphorylation. In the intermediate states of dephosphorylation the A-domain residues Ser¹⁸⁶ and Asp²⁰³ interact with Glu⁴³⁹ (N-domain) and Arg⁶⁷⁸ (P-domain), respectively. Single mutations to these residues abolish the stimulation of dephosphorylation by ATP. The double mutation swapping Asp²⁰³ and Arg⁶⁷⁸ rescues ATP stimulation, whereas this is not the case for the double mutation swapping Ser¹⁸⁶ and Glu⁴³⁹. By taking advantage of the ability of wild type and mutant Ca²⁺-ATPases to form stable complexes with aluminum fluoride (E2·AlF) and beryllium fluoride (E2·BeF) as analogs of the E2·P phosphoryl transition state and E2P ground state, respectively, of the dephosphorylation reaction, the mutational effects on ATP binding to these intermediates are demonstrated. In the wild type Ca²⁺-ATPase, the ATP affinity of the E2·P phosphoryl transition state is higher than that of the E2P ground state, thus explaining the stimulation of dephosphorylation by nucleotide-induced transition state stabilization. We find that the Asp²⁰³-Arg⁶⁷⁸ and Ser¹⁸⁶-Glu⁴³⁹ interdomain bonds are critical, because they tighten the interaction with ATP in the E2·P phosphoryl transition state. Moreover, ATP binding and the Ser¹⁸⁶-Glu⁴³⁹ bond are mutually exclusive in the E2P ground state.     

Ser422 phosphorylation blocks human Tau cleavage by caspase-3: Biochemical implications to Alzheimer’s Disease

Proteolytic truncation of microtubule associated human (h) Tau protein by caspase-3 at the carboxy (C) terminus has been linked to the pathogenesis of Alzheimer’s Disease (AD). This cleavage likely occurs between Asp⁴²¹↓Ser⁴²² leading to the formation of 421-mer truncated Tau protein which has been found to be present as aggregate in high level after phosphorylation in mortal AD brain tissue compared to normal. At least 50 phosphorylation sites involving Ser, Thr and Tyr residues have been identified or proposed in hTau and a selected number of them have been implicated in hTau aggregation following latter’s proteolytic truncation. Interestingly, it is further noted that Ser⁴²² residue present in the P1′ position of hTau caspase-3 cleavage region is a potential phosphorylation site. So we became interested to examine in vitro the effect of phospho-Ser⁴²² residue on hTau cleavage by caspase-3 which is a crucial upstream event associated with hTau self-assembly leading to AD pathogenesis. The goal of this project is to study in vitro the caspase-3 cleavage site of hTau protein and to examine the kinetics of this cleavage following Ser⁴²² phosphorylation and treatment with caspase-3 inhibitors. This is achieved by designing peptides from the sequence of hTau protein containing the proposed caspase-3 cleavage region. Peptides were designed from 441-mer major human Tau protein sequence that encompasses the proposed caspase-3 cleavage site [Asp⁴²¹↓Ser⁴²²]. Corresponding phospho-, dextro-Ser⁴²² and dextro-Asp⁴²¹ analogs were also designed. Peptides were synthesized by solid phase chemistry, purified and fully characterized by mass spectrometry. These were then incubated with recombinant caspase-3 enzyme under identical condition for digestion and analyzed for cleavage by mass spectrometry and RP-HPLC chromatograms. Our results indicated that while the control peptide is efficiently cleaved by caspase-3 at Asp⁴²¹↓Ser⁴²² site producing the expected N- and C-terminal fragment peptides, the corresponding phospho-Ser⁴²² peptide remained completely resistant to the cleavage. Substitution of Asp⁴²¹ by its dextro isoform also blocks peptide cleavage by caspase-3. However substitution of Ser⁴²² by its dextro isoform in the peptide did not affect the cleavage significantly. The above results were further confirmed by caspase-3 digestion experiment in the presence of varying amounts of caspase-3 inhibitor (Ac-DQVD-aldehyde) which was found to block this cleavage in a highly effective manner. Our results highlighted the crucial significance of Ser⁴²² phosphorylation and suggest that the kinase associated with this Ser-phosphorylation may protect Tau from aggregation. Thus specific promoters/activators of this kinase may find useful therapeutic benefits in arresting Tau truncation by caspase-3 and the progression of AD. In addition our data demonstrated that Tau-peptides where Ser⁴²² or Asp⁴²¹ are substituted by their respective dextro isomers, exhibit different cleavage kinetics by caspase-3 and this may have important implications in therapeutic intervention of Tau aggregation and associated AD.     

The structure/function relationship of a dual-substrate (betaalpha)8-isomerase

Two structures of phosphoribosyl isomerase A (PriA) from Streptomyces coelicolor, involved in both histidine and tryptophan biosynthesis, were solved at 1.8 Å resolution. A closed conformer was obtained, which represents the first complete structure of PriA, revealing hitherto unnoticed molecular interactions and the occurrence of conformational changes. Inspection of these conformers, including ligand-docking simulations, allowed identification of residues involved in substrate recognition, chemical catalysis and conformational changes. These predictions were validated by mutagenesis and functional analysis. Arg¹⁹ and Ser⁸¹ were shown to play critical roles within the carboxyl and amino phosphate-binding sites, respectively; the catalytic residues Asp¹¹ and Asp¹³⁰ are responsible for both activities; and Thr¹⁶⁶ and Asp¹⁷¹, which make an unusual contact, are likely to elicit the conformational changes needed for adopting the active site architectures. This represents the first report of the structure/function relationship of this (βα)₈-isomerase.     

Novel dextranase catalyzing cycloisomaltooligosaccharide formation and identification of catalytic amino acids and their functions using chemical rescue approach

A novel endodextranase from Paenibacillus sp. (Paenibacillus sp. dextranase; PsDex) was found to mainly produce isomaltotetraose and small amounts of cycloisomaltooligosaccharides (CIs) with a degree of polymerization of 7–14 from dextran. The 1,696-amino acid sequence belonging to the glycosyl hydrolase family 66 (GH-66) has a long insertion (632 residues; Thr⁴⁵¹–Val¹⁰⁸²), a portion of which shares identity (35% at Ala³⁹–Ser¹³⁰⁴ of PsDex) with Pro³²–Ala⁷⁵⁵ of CI glucanotransferase (CITase), a GH-66 enzyme that catalyzes the formation of CIs from dextran. This homologous sequence (Val⁸³⁷–Met⁹³² for PsDex and Tyr⁴⁰⁴–Tyr⁴⁹² for CITase), similar to carbohydrate-binding module 35, was not found in other endodextranases (Dexs) devoid of CITase activity. These results support the classification of GH-66 enzymes into three types: (i) Dex showing only dextranolytic activity, (ii) Dex catalyzing hydrolysis with low cyclization activity, and (iii) CITase showing CI-forming activity with low dextranolytic activity. The fact that a C-terminal truncated enzyme (having Ala³⁹–Ser¹³⁰⁴) has 50% wild-type PsDex activity indicates that the C-terminal 392 residues are not involved in hydrolysis. GH-66 enzymes possess four conserved acidic residues (Asp¹⁸⁹, Asp³⁴⁰, Glu⁴¹², and Asp¹²⁵⁴ of PsDex) of catalytic candidates. Their amide mutants decreased activity (1/1, 500 to 1/40, 000 times), and D1254N had 36% activity. A chemical rescue approach was applied to D189A, D340G, and E412Q using α-isomaltotetraosyl fluoride with NaN₃. D340G or E412Q formed a β- or α-isomaltotetraosyl azide, respectively, strongly indicating Asp³⁴⁰ and Glu⁴¹² as a nucleophile and acid/base catalyst, respectively. Interestingly, D189A synthesized small sized dextran from α-isomaltotetraosyl fluoride in the presence of NaN₃.