The ligand specificity of yeast Rad53 FHA domains at the +3 position is determined by nonconserved residues.
On the basis of the results from our laboratory and others, we recently suggested that the ligand specificity of forkhead-associated (FHA) domains is controlled by variations in three major factors: (i) residues interacting with pThr, (ii) residues recognizing the +1 to +3 residues from pThr, and (iii) an extended binding surface. While the first factor has been well established by several solution and crystal structures of FHA-phosphopeptide complexes, the structural bases of the second and third factors are not well understood and are likely to vary greatly between different FHA domains. In this work, we proposed and tested the hypothesis that nonconserved residues G133 and G135 of FHA1 and I681 and D683 of FHA2, located outside of the core FHA region of yeast Rad53 FHA domains, contribute to the specific recognition of the +3 position of different phosphopeptides. By rational mutagenesis of these residues, the specificity of FHA1 has been changed from predominantly pTXXD to be equally acceptable for pTXXD, pTXXL, and pYXL, which are similar to the specificities of the FHA2 domain of Rad53. Conversely, the +3 position specificity of FHA2 has been engineered to be more like FHA1 with the I681A mutation. These results were based on library screening as well as binding analyses of specific phosphopeptides. Furthermore, results of structural analyses by NMR indicate that some of these residues are also important for the structural integrity of the loops.
Yongkiettrakul
Suganya
S
Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, USA.
Byeon
In-Ja L
IJ
Tsai
Ming-Daw
MD
D001120:Arginine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics; D064447:Checkpoint Kinase 2; D017124:Conserved Sequence / Q000235:genetics; D051858:Forkhead Transcription Factors; D005998:Glycine / Q000235:genetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D019151:Peptide Library; D010748:Phosphopeptides / Q000737:chemistry; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D020349:Surface Plasmon Resonance; D013912:Threonine / Q000737:chemistry; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics
D018797:Cell Cycle Proteins; C464906:Fkh1 protein, S cerevisiae; C464958:Fkh2 protein, S cerevisiae; D051858:Forkhead Transcription Factors; D008024:Ligands; D019151:Peptide Library; D010748:Phosphopeptides; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D013912:Threonine; D001224:Aspartic Acid; D001120:Arginine; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; D005998:Glycine