Structural and functional analysis of phosphothreonine-dependent FHA domain interactions.
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.
Division of Molecular Structure, MRC National Institute for Medical Research, The Ridgeway, London NW71AA, UK. spennel@nimr.mrc.ac.uk
Simon
S
Pennell
Sarah
S
Westcott
Miguel
M
Ortiz-Lombardía
Dony
D
Patel
Jiejin
J
Li
Timothy J
TJ
Nott
Duaa
D
Mohammed
Roger S
RS
Buxton
Michael B
MB
Yaffe
Chandra
C
Verma
Stephen J
SJ
Smerdon
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D051858:Forkhead Transcription Factors / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010769:Phosphothreonine / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D054730:Protein Interaction Domains and Motifs / Q000235:genetics / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D051858:Forkhead Transcription Factors; D050505:Mutant Proteins; D010769:Phosphothreonine; D017346:Protein Serine-Threonine Kinases