title: The SH3 domain of HS1 protein recognizes lysine-rich polyproline motifs.
abstract: HS1 is a protein involved in erythroid proliferation and apoptotic cell death, containing several structurally significant motifs including a C-terminal SH3 domain. HPK1 is a member of the Ste20-related kinase family, which contains four proline-rich sequences and is constitutively associated with HS1 in hematopoietic cells. Recombinant fusion protein GST-SH3(HS1) was expressed to assess the binding properties of 16 peptides derived from the HPK1 proline-rich regions. The binding affinities were determined by non-immobilized ligand interaction assay by circular dichroism. Our results revealed that the classical PxxPxK class II binding motif is not sufficient to induce the interaction with the GST-SH3(HS1) domain, an event dependent on the presence of additional basic residue(s) located at the C-terminus of the PxxPxK motif: Lys(-5) in P2 peptide and Lys(-8) in P4c peptide. Lys replacement by Arg residues decreases the ligand binding affinity. The finding that both SH3(HS1) domain and full-length HS1 protein bind to P2 peptide with similar affinity demonstrates that the whole protein sequence does not affect the interaction properties of the domain. In silico models of SH3(HS1) as a complex with P2 or P4c highlight the domain residues that interact with the recognition determinants of the peptide ligand and that cooperate in the complex stabilization.
authors.affiliation: Diamond Light Source Ltd., Rutherford Appleton Laboratory, Chilton, Didcot, Oxfordshire, OX11 0QX, UK.
authors.forename: Giuliano
authors.initials: G
authors.lastname: Siligardi
authors.forename: Paolo
authors.initials: P
authors.lastname: Ruzza
authors.forename: Rohanah
authors.initials: R
authors.lastname: Hussain
authors.forename: Luca
authors.initials: L
authors.lastname: Cesaro
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Brunati
authors.forename: Lorenzo A
authors.initials: LA
authors.lastname: Pinna
authors.forename: Arianna
authors.initials: A
authors.lastname: Donella-Deana
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D018909:src Homology Domains
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D001798:Blood Proteins; C061909:HCLS1 protein, human; D010455:Peptides; C011083:polyproline; D008239:Lysine