Structural and functional effects of disease-causing amino acid substitutions affecting residues Ala72 and Glu76 of the protein tyrosine phosphatase SHP-2

Gianfranco Bocchinfuso, Lorenzo Stella, Simone Martinelli, Elisabetta Flex, Claudio Carta, Francesca Pantaleoni, Basilio Pispisa, Mariano Venanzi, Marco Tartaglia, Antonio Palleschi

Research output: Contribution to journalArticle

Abstract

Mutations of the protein tyrosine phosphatase SHP-2 are implicated in human diseases, causing Noonan syndrome (NS) and related developmental disorders or contributing to leukemogenesis depending on the specific amino acid substitution involved. SHP-2 is composed by a catalytic (PTP) and two regulatory (N-SH2 and C-SH2) domains that bind to signaling partners and control the enzymatic activity by limiting the accessibility of the catalytic site. Wild type SHP-2 and four disease-associated mutants recurring in hematologic malignancies (Glu76Lys and Ala72Val) or causing NS (Glu76Asp and Ala72Ser), with affected residues located in the PTP-interacting region of the N-SH2 domain, were analyzed by molecular dynamics simulations and in vitro biochemical assays. Simulations demonstrate that mutations do not affect significantly the conformation of the N-SH2 domain. Rather they destabilize the interaction of this domain with the catalytic site, with more evident effects in the two leukemia associated mutants. Consistent with this structural evidence, mutants exhibit an increased level of basal phosphatase activity in the order Glu76Lys > Ala72Val > Glu76Asp > Ala72Ser > WT. The experimental data also show that the mutants with higher basal activity are more responsive to an activating phosphopeptide. A thermodynamic analysis demonstrates that an increase in the overall phosphopeptide affinity of mutants can be explained by a shift in the equilibrium between the inactive and active SHP-2 structure. These data support the view that an increase in the affinity of SHP-2 for its binding partners, caused by destabilization of the closed, inactive conformation, rather than protein basal activation per se, would represent the molecular mechanism, leading to pathogenesis in these mutants.

Original languageEnglish
Pages (from-to)963-974
Number of pages12
JournalProteins: Structure, Function and Genetics
Volume66
Issue number4
DOIs
Publication statusPublished - Mar 2007

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Non-Receptor Type 11 Protein Tyrosine Phosphatase
Phosphopeptides
src Homology Domains
Amino Acid Substitution
Noonan Syndrome
Conformations
Substitution reactions
Amino Acids
Catalytic Domain
Phosphoric Monoester Hydrolases
Molecular dynamics
Assays
Protein Conformation
Mutation
Chemical activation
Thermodynamics
Hematologic Neoplasms
Molecular Dynamics Simulation
Computer simulation
Leukemia

Keywords

  • Interdomain interaction
  • Leukemia
  • Molecular dynamics simulation
  • Noonan syndrome
  • Phosphatase activity
  • SHP-2 mutants

ASJC Scopus subject areas

  • Genetics
  • Structural Biology
  • Biochemistry

Cite this

Structural and functional effects of disease-causing amino acid substitutions affecting residues Ala72 and Glu76 of the protein tyrosine phosphatase SHP-2. / Bocchinfuso, Gianfranco; Stella, Lorenzo; Martinelli, Simone; Flex, Elisabetta; Carta, Claudio; Pantaleoni, Francesca; Pispisa, Basilio; Venanzi, Mariano; Tartaglia, Marco; Palleschi, Antonio.

In: Proteins: Structure, Function and Genetics, Vol. 66, No. 4, 03.2007, p. 963-974.

Research output: Contribution to journalArticle

Bocchinfuso, Gianfranco ; Stella, Lorenzo ; Martinelli, Simone ; Flex, Elisabetta ; Carta, Claudio ; Pantaleoni, Francesca ; Pispisa, Basilio ; Venanzi, Mariano ; Tartaglia, Marco ; Palleschi, Antonio. / Structural and functional effects of disease-causing amino acid substitutions affecting residues Ala72 and Glu76 of the protein tyrosine phosphatase SHP-2. In: Proteins: Structure, Function and Genetics. 2007 ; Vol. 66, No. 4. pp. 963-974.
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abstract = "Mutations of the protein tyrosine phosphatase SHP-2 are implicated in human diseases, causing Noonan syndrome (NS) and related developmental disorders or contributing to leukemogenesis depending on the specific amino acid substitution involved. SHP-2 is composed by a catalytic (PTP) and two regulatory (N-SH2 and C-SH2) domains that bind to signaling partners and control the enzymatic activity by limiting the accessibility of the catalytic site. Wild type SHP-2 and four disease-associated mutants recurring in hematologic malignancies (Glu76Lys and Ala72Val) or causing NS (Glu76Asp and Ala72Ser), with affected residues located in the PTP-interacting region of the N-SH2 domain, were analyzed by molecular dynamics simulations and in vitro biochemical assays. Simulations demonstrate that mutations do not affect significantly the conformation of the N-SH2 domain. Rather they destabilize the interaction of this domain with the catalytic site, with more evident effects in the two leukemia associated mutants. Consistent with this structural evidence, mutants exhibit an increased level of basal phosphatase activity in the order Glu76Lys > Ala72Val > Glu76Asp > Ala72Ser > WT. The experimental data also show that the mutants with higher basal activity are more responsive to an activating phosphopeptide. A thermodynamic analysis demonstrates that an increase in the overall phosphopeptide affinity of mutants can be explained by a shift in the equilibrium between the inactive and active SHP-2 structure. These data support the view that an increase in the affinity of SHP-2 for its binding partners, caused by destabilization of the closed, inactive conformation, rather than protein basal activation per se, would represent the molecular mechanism, leading to pathogenesis in these mutants.",
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T1 - Structural and functional effects of disease-causing amino acid substitutions affecting residues Ala72 and Glu76 of the protein tyrosine phosphatase SHP-2

AU - Bocchinfuso, Gianfranco

AU - Stella, Lorenzo

AU - Martinelli, Simone

AU - Flex, Elisabetta

AU - Carta, Claudio

AU - Pantaleoni, Francesca

AU - Pispisa, Basilio

AU - Venanzi, Mariano

AU - Tartaglia, Marco

AU - Palleschi, Antonio

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N2 - Mutations of the protein tyrosine phosphatase SHP-2 are implicated in human diseases, causing Noonan syndrome (NS) and related developmental disorders or contributing to leukemogenesis depending on the specific amino acid substitution involved. SHP-2 is composed by a catalytic (PTP) and two regulatory (N-SH2 and C-SH2) domains that bind to signaling partners and control the enzymatic activity by limiting the accessibility of the catalytic site. Wild type SHP-2 and four disease-associated mutants recurring in hematologic malignancies (Glu76Lys and Ala72Val) or causing NS (Glu76Asp and Ala72Ser), with affected residues located in the PTP-interacting region of the N-SH2 domain, were analyzed by molecular dynamics simulations and in vitro biochemical assays. Simulations demonstrate that mutations do not affect significantly the conformation of the N-SH2 domain. Rather they destabilize the interaction of this domain with the catalytic site, with more evident effects in the two leukemia associated mutants. Consistent with this structural evidence, mutants exhibit an increased level of basal phosphatase activity in the order Glu76Lys > Ala72Val > Glu76Asp > Ala72Ser > WT. The experimental data also show that the mutants with higher basal activity are more responsive to an activating phosphopeptide. A thermodynamic analysis demonstrates that an increase in the overall phosphopeptide affinity of mutants can be explained by a shift in the equilibrium between the inactive and active SHP-2 structure. These data support the view that an increase in the affinity of SHP-2 for its binding partners, caused by destabilization of the closed, inactive conformation, rather than protein basal activation per se, would represent the molecular mechanism, leading to pathogenesis in these mutants.

AB - Mutations of the protein tyrosine phosphatase SHP-2 are implicated in human diseases, causing Noonan syndrome (NS) and related developmental disorders or contributing to leukemogenesis depending on the specific amino acid substitution involved. SHP-2 is composed by a catalytic (PTP) and two regulatory (N-SH2 and C-SH2) domains that bind to signaling partners and control the enzymatic activity by limiting the accessibility of the catalytic site. Wild type SHP-2 and four disease-associated mutants recurring in hematologic malignancies (Glu76Lys and Ala72Val) or causing NS (Glu76Asp and Ala72Ser), with affected residues located in the PTP-interacting region of the N-SH2 domain, were analyzed by molecular dynamics simulations and in vitro biochemical assays. Simulations demonstrate that mutations do not affect significantly the conformation of the N-SH2 domain. Rather they destabilize the interaction of this domain with the catalytic site, with more evident effects in the two leukemia associated mutants. Consistent with this structural evidence, mutants exhibit an increased level of basal phosphatase activity in the order Glu76Lys > Ala72Val > Glu76Asp > Ala72Ser > WT. The experimental data also show that the mutants with higher basal activity are more responsive to an activating phosphopeptide. A thermodynamic analysis demonstrates that an increase in the overall phosphopeptide affinity of mutants can be explained by a shift in the equilibrium between the inactive and active SHP-2 structure. These data support the view that an increase in the affinity of SHP-2 for its binding partners, caused by destabilization of the closed, inactive conformation, rather than protein basal activation per se, would represent the molecular mechanism, leading to pathogenesis in these mutants.

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