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Giuseppe Inesi - One of the best experts on this subject based on the ideXlab platform.

  • Functional Role of “N” (Nucleotide) and “P” (Phosphorylation) Domain Interactions in the Sarcoplasmic Reticulum (SERCA) ATPase†
    Biochemistry, 2002
    Co-Authors: Suming Hua, David A. Lewis, Giuseppe Inesi, Chikashi Toyoshima
    Abstract:

    Experimental perturbations of the nucleotide site in the N domain of the SR Ca2+ ATPase were produced by chemical derivatization of Lys492 or/and Lys515, mutation of Arg560 to Ala, or addition of inactive nucleotide analogue (TNP-AMP). Selective labeling of either Lys492 or Lys515 produces strong inhibition of ATPase activity and phosphoEnzyme intermediate formation by utilization of ATP, while AcP utilization and reverse ATPase Phosphorylation by Pi are much less affected. Cross-linking of the two residues with DIDS, however, drastically inhibits utilization of both ATP and AcP, as well as of formation of phosphoEnzyme intermediate by utilization of ATP, or reverse Phosphorylation by Pi. Mutation of Arg560 to Ala produces strong inhibition of ATPase activity and Enzyme Phosphorylation by ATP but has a much lower effect on Enzyme Phosphorylation by Pi. TNP-AMP increases the ATPase activity at low concentrations (0.1-0.3 microM), but inhibits ATP, AcP, and Pi utilization at higher concentration (1-10 microM). Cross-linking with DIDS and TNP-AMP binding inhibits formation of the transition state analogue with orthovanadate. It is concluded that in addition to the binding pocket delimited by Lys 492 and Lys515, Arg560 sustains an important and direct role in nucleotide substrate stabilization. Furthermore, the effects of DIDS and TNP-AMP suggest that approximation of N (nucleotide) and P (Phosphorylation) domains is required not only for delivery of nucleotide substrate, but also to favor Enzyme Phosphorylation by nucleotide and nonnucleotide substrates, in the presence and in the absence of Ca2+. Domain separation is then enhanced by secondary nucleotide binding to the phosphoEnzyme, thereby favoring its hydrolytic cleavage.

  • The Role of the M6-M7 Loop (L67) in Stabilization of the Phosphorylation and Ca2+ Binding Domains of the Sarcoplasmic Reticulum Ca2+-ATPase (SERCA)
    The Journal of biological chemistry, 2001
    Co-Authors: Zhongsen Zhang, Giuseppe Inesi, Carlota Sumbilla, David Lewis, Chikashi Toyoshima
    Abstract:

    Abstract The amino acid sequence (L67) intervening between the M6 and M7 transmembrane segments of the Ca2+transport ATPase was subjected to mutational analysis. Mutation of Pro820 to Ala interferes with protein expression even though transcription occurs at normal levels. Single mutations of Lys819 or Arg822 to Ala, Phe, or Glu allow good expression, but produce strong inhibition of ATPase activity. The main defect produced by these mutations is strong interference with Enzyme Phosphorylation by ATP in the presence of Ca2+, and also by Pi in the absence of Ca2+. The Lys819 and Arg822 mutants undergo slight and moderate reduction of Ca2+ binding affinity, respectively. Reduction of overall steady state ATPase velocity is then due to inhibition of phosphorylated intermediate formation. On the other hand, a cluster of conservative mutations of Asp813, Asp815, and Asp818 to Asn interferes strongly with Enzyme activation by Ca2+ binding and formation of phosphorylated Enzyme intermediate by utilization of ATP. Enzyme Phosphorylation by Pi in the absence of Ca2+undergoes slight or no inhibition by the triple aspartate mutation. Therefore, the triple mutation interferes mainly with the calcium-dependent activation of the ATPase. The effect of the triple mutation can be to a large extent reproduced by single mutation of Asp813 (but not of Asp815 or Asp818) to Asn. Functional and structural analysis of the experimental data demonstrates that the L67 loop plays an important role in protein folding and function. This role is sustained by linking the cytosolic catalytic domain and the transmembrane Ca2+binding domain through a network of hydrogen bonds.

  • ATPase Gene Transfer and Mutational Analysis of the Cation Translocation Mechanism
    Annals of the New York Academy of Sciences, 1997
    Co-Authors: Giuseppe Inesi, Dorothy E. Lewis, Carlota Sumbilla, A. Nandi, Mary E. Kirtley, C. P. Ordahl
    Abstract:

    The peptide segment interposed between cation binding and Phosphorylation domains retains a high degree of homology in all cation transport ATPases. Mutational analysis and chimeric replacements of Ca2+ ATPase components with corresponding Na+,K(+)-ATPase components indicate that this segment is utilized by various cation ATPases as a common structural device for a long-range functional linkage of Enzyme Phosphorylation and cation transport. Vectorial displacement of bound cation is rendered possible by a transmembrane channel formed by four clustered helices (M4, M5, M6, and M8). Originating from the four helices, the oxygen functions of Glu309, Glu771, Thr799, Asp800, and Glu908 form a duplex Ca2+ binding site in the middle of the channel, while Lys297 seals the luminal end of the channel with its positively charged side chain. The perturbation triggered by Enzyme Phosphorylation is apparently transmitted through the linkage segment to produce rotational displacement of the M4 helix with minimal change of secondary structure. The cation binding site is thereby disrupted and the Lys297 side chain removed, permitting Ca2+ to dissociate in exchange for H+ and to flow through the luminal end of the channel.

  • Ca2+ binding and translocation by the sarcoplasmic reticulum ATPase: functional and structural considerations.
    Bioscience reports, 1995
    Co-Authors: Giuseppe Inesi, David A. Lewis, Carlota Sumbilla, Li Chen, Mary E. Kirtley
    Abstract:

    Three experimental systems are described including sarcoplasmic reticulum (SR) vesicles, reconstituted proteoliposomes, and recombinant protein obtained by gene transfer and expression in foreign cells. It is shown that the Ca(2+) ATPase of sarcoplasmic reticulum (SR) includes an extramembranous globular head which is connected through a stalk to a membrane bound region. Cooperative binding of two calcium ions occurs sequentially, within a channel formed by four clustered helices within the membrane bound region. Destabilization of the helical cluster is produced following Enzyme Phosphorylation by ATP at the catalytic site in the extramembranous region. The affinity and orientation of the Ca2+ binding site are thereby changed, permitting vectorial dissociation of bound Ca2+ against a concentration gradient. A long range linkage between Phosphorylation and Ca2+ binding sites is provided by an intervening peptide segment that retains high homology in cation transport ATPases, and whose function is highly sensitive to mutational perturbations.

Chikashi Toyoshima - One of the best experts on this subject based on the ideXlab platform.

  • Functional Role of “N” (Nucleotide) and “P” (Phosphorylation) Domain Interactions in the Sarcoplasmic Reticulum (SERCA) ATPase†
    Biochemistry, 2002
    Co-Authors: Suming Hua, David A. Lewis, Giuseppe Inesi, Chikashi Toyoshima
    Abstract:

    Experimental perturbations of the nucleotide site in the N domain of the SR Ca2+ ATPase were produced by chemical derivatization of Lys492 or/and Lys515, mutation of Arg560 to Ala, or addition of inactive nucleotide analogue (TNP-AMP). Selective labeling of either Lys492 or Lys515 produces strong inhibition of ATPase activity and phosphoEnzyme intermediate formation by utilization of ATP, while AcP utilization and reverse ATPase Phosphorylation by Pi are much less affected. Cross-linking of the two residues with DIDS, however, drastically inhibits utilization of both ATP and AcP, as well as of formation of phosphoEnzyme intermediate by utilization of ATP, or reverse Phosphorylation by Pi. Mutation of Arg560 to Ala produces strong inhibition of ATPase activity and Enzyme Phosphorylation by ATP but has a much lower effect on Enzyme Phosphorylation by Pi. TNP-AMP increases the ATPase activity at low concentrations (0.1-0.3 microM), but inhibits ATP, AcP, and Pi utilization at higher concentration (1-10 microM). Cross-linking with DIDS and TNP-AMP binding inhibits formation of the transition state analogue with orthovanadate. It is concluded that in addition to the binding pocket delimited by Lys 492 and Lys515, Arg560 sustains an important and direct role in nucleotide substrate stabilization. Furthermore, the effects of DIDS and TNP-AMP suggest that approximation of N (nucleotide) and P (Phosphorylation) domains is required not only for delivery of nucleotide substrate, but also to favor Enzyme Phosphorylation by nucleotide and nonnucleotide substrates, in the presence and in the absence of Ca2+. Domain separation is then enhanced by secondary nucleotide binding to the phosphoEnzyme, thereby favoring its hydrolytic cleavage.

  • The Role of the M6-M7 Loop (L67) in Stabilization of the Phosphorylation and Ca2+ Binding Domains of the Sarcoplasmic Reticulum Ca2+-ATPase (SERCA)
    The Journal of biological chemistry, 2001
    Co-Authors: Zhongsen Zhang, Giuseppe Inesi, Carlota Sumbilla, David Lewis, Chikashi Toyoshima
    Abstract:

    Abstract The amino acid sequence (L67) intervening between the M6 and M7 transmembrane segments of the Ca2+transport ATPase was subjected to mutational analysis. Mutation of Pro820 to Ala interferes with protein expression even though transcription occurs at normal levels. Single mutations of Lys819 or Arg822 to Ala, Phe, or Glu allow good expression, but produce strong inhibition of ATPase activity. The main defect produced by these mutations is strong interference with Enzyme Phosphorylation by ATP in the presence of Ca2+, and also by Pi in the absence of Ca2+. The Lys819 and Arg822 mutants undergo slight and moderate reduction of Ca2+ binding affinity, respectively. Reduction of overall steady state ATPase velocity is then due to inhibition of phosphorylated intermediate formation. On the other hand, a cluster of conservative mutations of Asp813, Asp815, and Asp818 to Asn interferes strongly with Enzyme activation by Ca2+ binding and formation of phosphorylated Enzyme intermediate by utilization of ATP. Enzyme Phosphorylation by Pi in the absence of Ca2+undergoes slight or no inhibition by the triple aspartate mutation. Therefore, the triple mutation interferes mainly with the calcium-dependent activation of the ATPase. The effect of the triple mutation can be to a large extent reproduced by single mutation of Asp813 (but not of Asp815 or Asp818) to Asn. Functional and structural analysis of the experimental data demonstrates that the L67 loop plays an important role in protein folding and function. This role is sustained by linking the cytosolic catalytic domain and the transmembrane Ca2+binding domain through a network of hydrogen bonds.

Mary E. Kirtley - One of the best experts on this subject based on the ideXlab platform.

  • ATPase Gene Transfer and Mutational Analysis of the Cation Translocation Mechanism
    Annals of the New York Academy of Sciences, 1997
    Co-Authors: Giuseppe Inesi, Dorothy E. Lewis, Carlota Sumbilla, A. Nandi, Mary E. Kirtley, C. P. Ordahl
    Abstract:

    The peptide segment interposed between cation binding and Phosphorylation domains retains a high degree of homology in all cation transport ATPases. Mutational analysis and chimeric replacements of Ca2+ ATPase components with corresponding Na+,K(+)-ATPase components indicate that this segment is utilized by various cation ATPases as a common structural device for a long-range functional linkage of Enzyme Phosphorylation and cation transport. Vectorial displacement of bound cation is rendered possible by a transmembrane channel formed by four clustered helices (M4, M5, M6, and M8). Originating from the four helices, the oxygen functions of Glu309, Glu771, Thr799, Asp800, and Glu908 form a duplex Ca2+ binding site in the middle of the channel, while Lys297 seals the luminal end of the channel with its positively charged side chain. The perturbation triggered by Enzyme Phosphorylation is apparently transmitted through the linkage segment to produce rotational displacement of the M4 helix with minimal change of secondary structure. The cation binding site is thereby disrupted and the Lys297 side chain removed, permitting Ca2+ to dissociate in exchange for H+ and to flow through the luminal end of the channel.

  • Ca2+ binding and translocation by the sarcoplasmic reticulum ATPase: functional and structural considerations.
    Bioscience reports, 1995
    Co-Authors: Giuseppe Inesi, David A. Lewis, Carlota Sumbilla, Li Chen, Mary E. Kirtley
    Abstract:

    Three experimental systems are described including sarcoplasmic reticulum (SR) vesicles, reconstituted proteoliposomes, and recombinant protein obtained by gene transfer and expression in foreign cells. It is shown that the Ca(2+) ATPase of sarcoplasmic reticulum (SR) includes an extramembranous globular head which is connected through a stalk to a membrane bound region. Cooperative binding of two calcium ions occurs sequentially, within a channel formed by four clustered helices within the membrane bound region. Destabilization of the helical cluster is produced following Enzyme Phosphorylation by ATP at the catalytic site in the extramembranous region. The affinity and orientation of the Ca2+ binding site are thereby changed, permitting vectorial dissociation of bound Ca2+ against a concentration gradient. A long range linkage between Phosphorylation and Ca2+ binding sites is provided by an intervening peptide segment that retains high homology in cation transport ATPases, and whose function is highly sensitive to mutational perturbations.

Carlota Sumbilla - One of the best experts on this subject based on the ideXlab platform.

  • The Role of the M6-M7 Loop (L67) in Stabilization of the Phosphorylation and Ca2+ Binding Domains of the Sarcoplasmic Reticulum Ca2+-ATPase (SERCA)
    The Journal of biological chemistry, 2001
    Co-Authors: Zhongsen Zhang, Giuseppe Inesi, Carlota Sumbilla, David Lewis, Chikashi Toyoshima
    Abstract:

    Abstract The amino acid sequence (L67) intervening between the M6 and M7 transmembrane segments of the Ca2+transport ATPase was subjected to mutational analysis. Mutation of Pro820 to Ala interferes with protein expression even though transcription occurs at normal levels. Single mutations of Lys819 or Arg822 to Ala, Phe, or Glu allow good expression, but produce strong inhibition of ATPase activity. The main defect produced by these mutations is strong interference with Enzyme Phosphorylation by ATP in the presence of Ca2+, and also by Pi in the absence of Ca2+. The Lys819 and Arg822 mutants undergo slight and moderate reduction of Ca2+ binding affinity, respectively. Reduction of overall steady state ATPase velocity is then due to inhibition of phosphorylated intermediate formation. On the other hand, a cluster of conservative mutations of Asp813, Asp815, and Asp818 to Asn interferes strongly with Enzyme activation by Ca2+ binding and formation of phosphorylated Enzyme intermediate by utilization of ATP. Enzyme Phosphorylation by Pi in the absence of Ca2+undergoes slight or no inhibition by the triple aspartate mutation. Therefore, the triple mutation interferes mainly with the calcium-dependent activation of the ATPase. The effect of the triple mutation can be to a large extent reproduced by single mutation of Asp813 (but not of Asp815 or Asp818) to Asn. Functional and structural analysis of the experimental data demonstrates that the L67 loop plays an important role in protein folding and function. This role is sustained by linking the cytosolic catalytic domain and the transmembrane Ca2+binding domain through a network of hydrogen bonds.

  • ATPase Gene Transfer and Mutational Analysis of the Cation Translocation Mechanism
    Annals of the New York Academy of Sciences, 1997
    Co-Authors: Giuseppe Inesi, Dorothy E. Lewis, Carlota Sumbilla, A. Nandi, Mary E. Kirtley, C. P. Ordahl
    Abstract:

    The peptide segment interposed between cation binding and Phosphorylation domains retains a high degree of homology in all cation transport ATPases. Mutational analysis and chimeric replacements of Ca2+ ATPase components with corresponding Na+,K(+)-ATPase components indicate that this segment is utilized by various cation ATPases as a common structural device for a long-range functional linkage of Enzyme Phosphorylation and cation transport. Vectorial displacement of bound cation is rendered possible by a transmembrane channel formed by four clustered helices (M4, M5, M6, and M8). Originating from the four helices, the oxygen functions of Glu309, Glu771, Thr799, Asp800, and Glu908 form a duplex Ca2+ binding site in the middle of the channel, while Lys297 seals the luminal end of the channel with its positively charged side chain. The perturbation triggered by Enzyme Phosphorylation is apparently transmitted through the linkage segment to produce rotational displacement of the M4 helix with minimal change of secondary structure. The cation binding site is thereby disrupted and the Lys297 side chain removed, permitting Ca2+ to dissociate in exchange for H+ and to flow through the luminal end of the channel.

  • Ca2+ binding and translocation by the sarcoplasmic reticulum ATPase: functional and structural considerations.
    Bioscience reports, 1995
    Co-Authors: Giuseppe Inesi, David A. Lewis, Carlota Sumbilla, Li Chen, Mary E. Kirtley
    Abstract:

    Three experimental systems are described including sarcoplasmic reticulum (SR) vesicles, reconstituted proteoliposomes, and recombinant protein obtained by gene transfer and expression in foreign cells. It is shown that the Ca(2+) ATPase of sarcoplasmic reticulum (SR) includes an extramembranous globular head which is connected through a stalk to a membrane bound region. Cooperative binding of two calcium ions occurs sequentially, within a channel formed by four clustered helices within the membrane bound region. Destabilization of the helical cluster is produced following Enzyme Phosphorylation by ATP at the catalytic site in the extramembranous region. The affinity and orientation of the Ca2+ binding site are thereby changed, permitting vectorial dissociation of bound Ca2+ against a concentration gradient. A long range linkage between Phosphorylation and Ca2+ binding sites is provided by an intervening peptide segment that retains high homology in cation transport ATPases, and whose function is highly sensitive to mutational perturbations.

Antonio-carlos G. De Almeida - One of the best experts on this subject based on the ideXlab platform.

  • Palytoxin and the sodium/potassium pump--Phosphorylation and potassium interaction.
    Physical biology, 2009
    Co-Authors: Antônio M. Rodrigues, Antonio Fernando Catelli Infantosi, Antonio-carlos G. De Almeida
    Abstract:

    We proposed a reaction model for investigating interactions between K+ and the palytoxin-sodium-potassium (PTX-Na+/K+) pump complex under conditions where Enzyme Phosphorylation may occur. The model is composed of (i) the Albers-Post model for Na+/K+-ATPase, describing Na+ and K+ pumping; (ii) the reaction model proposed for Na+/K+-ATPase interactions with its ligands (Na+, K+, ATP, ADP and P) and with PTX. A mathematical model derived for representing the reactions was used to simulate experimental studies of the PTX-induced current, in different concentrations for the pump ligands. The simulations allow interpretation of the simultaneous action of Na+/K+-ATPase Phosphorylation and K+ on the PTX-induced channels. The results suggest that(i) Phosphorylation increases the PTX toxic effect, increasing its affinity and reducing the K+occlusion rate, and (ii) K+ causes channel blockage, increases the toxin dissociation rate and impedes the induced channel Phosphorylation, implying reduction of the PTX toxic effect.

  • palytoxin and the sodium potassium pump Phosphorylation and potassium interaction
    Physical Biology, 2009
    Co-Authors: Antônio M. Rodrigues, Antonio Fernando Catelli Infantosi, Antonio-carlos G. De Almeida
    Abstract:

    We proposed a reaction model for investigating interactions between K+ and the palytoxin-sodium-potassium (PTX-Na+/K+) pump complex under conditions where Enzyme Phosphorylation may occur. The model is composed of (i) the Albers-Post model for Na+/K+-ATPase, describing Na+ and K+ pumping; (ii) the reaction model proposed for Na+/K+-ATPase interactions with its ligands (Na+, K+, ATP, ADP and P) and with PTX. A mathematical model derived for representing the reactions was used to simulate experimental studies of the PTX-induced current, in different concentrations for the pump ligands. The simulations allow interpretation of the simultaneous action of Na+/K+-ATPase Phosphorylation and K+ on the PTX-induced channels. The results suggest that(i) Phosphorylation increases the PTX toxic effect, increasing its affinity and reducing the K+occlusion rate, and (ii) K+ causes channel blockage, increases the toxin dissociation rate and impedes the induced channel Phosphorylation, implying reduction of the PTX toxic effect.