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 journalArticlepeer-review

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

Keywords

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

ASJC Scopus subject areas

  • Genetics
  • Structural Biology
  • Biochemistry

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