Structural and functional analysis of phosphothreonine-dependent FHA domain interactions

Structure. 2010 Dec 8;18(12):1587-95. doi: 10.1016/j.str.2010.09.014.

Abstract

FHA domains are well established as phospho-dependent binding modules mediating signal transduction in Ser/Thr kinase signaling networks in both eukaryotic and prokaryotic species. Although they are unique in binding exclusively to phosphothreonine, the basis for this discrimination over phosphoserine has remained elusive. Here, we attempt to dissect overall binding specificity at the molecular level. We first determined the optimal peptide sequence for Rv0020c FHA domain binding by oriented peptide library screening. This served as a basis for systematic mutagenic and binding analyses, allowing us to derive relative thermodynamic contributions of conserved protein and peptide residues to binding and specificity. Structures of phosphopeptide-bound and uncomplexed Rv0020c FHA domain then directed molecular dynamics simulations which show how the extraordinary discrimination in favor of phosphothreonine occurs through formation of additional hydrogen-bonding networks that are ultimately stabilized by van der Waals interactions of the phosphothreonine γ-methyl group with a conserved pocket on the FHA domain surface.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Crystallography, X-Ray
  • Forkhead Transcription Factors / metabolism
  • Humans
  • Hydrogen Bonding
  • Models, Molecular
  • Molecular Dynamics Simulation
  • Mutagenesis, Site-Directed
  • Mutant Proteins / chemistry
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism
  • Phosphothreonine / chemistry
  • Phosphothreonine / metabolism*
  • Phosphothreonine / pharmacology*
  • Protein Binding / genetics
  • Protein Binding / physiology
  • Protein Interaction Domains and Motifs* / genetics
  • Protein Interaction Domains and Motifs* / physiology
  • Protein-Serine-Threonine Kinases / chemistry*
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*
  • Structure-Activity Relationship
  • Substrate Specificity

Substances

  • Forkhead Transcription Factors
  • Mutant Proteins
  • Phosphothreonine
  • Protein-Serine-Threonine Kinases

Associated data

  • PDB/3PO8
  • PDB/3POA