Diverse recognition of non‐PxxP peptide ligands by the SH3 domains from p67phox, Grb2 and Pex13p

The basic function of the Src homology 3 (SH3) domain is considered to be binding to proline‐rich sequences containing a PxxP motif. Recently, many SH3 domains, including those from Grb2 and Pex13p, were reported to bind sequences lacking a PxxP motif. We report here that the 22 residue peptide lacking a PxxP motif, derived from p47phox, binds to the C‐terminal SH3 domain from p67phox. We applied the NMR cross‐saturation method to locate the interaction sites for the non‐PxxP peptides on their cognate SH3 domains from p67phox, Grb2 and Pex13p. The binding site of the Grb2 SH3 partially overlapped the conventional PxxP‐binding site, whereas those of p67phox and Pex13p SH3s are located in different surface regions. The non‐PxxP peptide from p47phox binds to the p67phox SH3 more tightly when it extends to the N‐terminus to include a typical PxxP motif, which enabled the structure determination of the complex, to reveal that the non‐PxxP peptide segment interacted with the p67phox SH3 in a compact helix–turn–helix structure (PDB entry 1K4U).

[1]  B. Mayer,et al.  SH3 domains: complexity in moderation. , 2001, Journal of cell science.

[2]  S. Grzesiek,et al.  NMRPipe: A multidimensional spectral processing system based on UNIX pipes , 1995, Journal of biomolecular NMR.

[3]  Kenji Ogura,et al.  Novel recognition mode between Vav and Grb2 SH3 domains , 2001, The EMBO journal.

[4]  H. Tabak,et al.  The peroxisomal membrane protein Pex13p shows a novel mode of SH3 interaction , 2000, The EMBO journal.

[5]  S. Grzesiek,et al.  Measurement of homo- and heteronuclear J couplings from quantitative J correlation. , 1994, Methods in enzymology.

[6]  T. Leto,et al.  Role of p67-phox SH3 domains in assembly of the NADPH oxidase system. , 1994, The Journal of biological chemistry.

[7]  Alexander Shekhtman,et al.  A novel, specific interaction involving the Csk SH3 domain and its natural ligand , 2001, Nature Structural Biology.

[8]  B. Chait,et al.  Crystal Structure of the Conserved Core of HIV-1 Nef Complexed with a Src Family SH3 Domain , 1996, Cell.

[9]  Hidekazu Hiroaki,et al.  Solution structure of the PX domain, a target of the SH3 domain , 2001, Nature Structural Biology.

[10]  A. Ullrich,et al.  Solution structure and ligand-binding site of the carboxy-terminal SH3 domain of GRB2. , 1994, Structure.

[11]  G. Wagner,et al.  SH3 domain recognition of a proline‐independent tyrosine‐based RKxxYxxY motif in immune cell adaptor SKAP55 , 2000, The EMBO journal.

[12]  K. Ogura,et al.  An improved double-tuned and isotope-filtered pulse scheme based on a pulsed field gradient and a wide-band inversion shaped pulse , 1996, Journal of biomolecular NMR.

[13]  A. Gronenborn,et al.  Multidimensional heteronuclear nuclear magnetic resonance of proteins. , 1994, Methods in enzymology.

[14]  Kurt Wüthrich,et al.  Protein recognition by NMR , 2000, Nature Structural Biology.

[15]  D. Crane,et al.  Interaction of Pex5p, the Type 1 Peroxisome Targeting Signal Receptor, with the Peroxisomal Membrane Proteins Pex14p and Pex13p* , 2000, The Journal of Biological Chemistry.

[16]  R. Freeman,et al.  Stretched Adiabatic Pulses for Broadband Spin Inversion , 1995 .

[17]  H. Sumimoto,et al.  Roles for proline-rich regions of p47phox and p67phox in the phagocyte NADPH oxidase activation in vitro. , 1997, Biochemical and biophysical research communications.

[18]  S. Schreiber,et al.  Two binding orientations for peptides to the Src SH3 domain: development of a general model for SH3-ligand interactions. , 1995, Science.

[19]  Hideo Takahashi,et al.  A novel NMR method for determining the interfaces of large protein–protein complexes , 2000, Nature Structural Biology.

[20]  T Nose,et al.  Role of Src homology 3 domains in assembly and activation of the phagocyte NADPH oxidase. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[21]  S. Grzesiek,et al.  Measurement of HN-Hα J couplings in calcium-free calmodulin using new 2D and 3D water-flip-back methods , 1994, Journal of biomolecular NMR.

[22]  J. Thornton,et al.  AQUA and PROCHECK-NMR: Programs for checking the quality of protein structures solved by NMR , 1996, Journal of biomolecular NMR.

[23]  K. Miyazawa,et al.  A Deubiquitinating Enzyme UBPY Interacts with the Src Homology 3 Domain of Hrs-binding Protein via a Novel Binding Motif PX(V/I)(D/N)RXXKP* , 2000, The Journal of Biological Chemistry.

[24]  W. Birchmeier,et al.  The C-terminal SH3 domain of the adapter protein Grb2 binds with high affinity to sequences in Gab1 and SLP-76 which lack the SH3-typical P-x-x-P core motif , 2001, Oncogene.

[25]  J. Cloutier,et al.  Sequence Requirements for Association of Protein-tyrosine Phosphatase PEP with the Src Homology 3 Domain of Inhibitory Tyrosine Protein Kinase p50 csk * , 1998, The Journal of Biological Chemistry.

[26]  A. Bax,et al.  An alternative 3D NMR technique for correlating backbone 15N with side chain Hβ resonances in larger proteins , 1991 .

[27]  F E Cohen,et al.  Exploiting the basis of proline recognition by SH3 and WW domains: design of N-substituted inhibitors. , 1998, Science.

[28]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[29]  M C Peitsch,et al.  Protein modelling for all. , 1999, Trends in biochemical sciences.

[30]  Andrea Musacchio,et al.  A novel peptide–SH3 interaction , 1999, The EMBO journal.

[31]  K. Wüthrich,et al.  Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional 13C labeling. , 1989, Biochemistry.

[32]  M. Billeter,et al.  MOLMOL: a program for display and analysis of macromolecular structures. , 1996, Journal of molecular graphics.

[33]  P. Finan,et al.  An SH3 domain and proline-rich sequence mediate an interaction between two components of the phagocyte NADPH oxidase complex. , 1994, The Journal of biological chemistry.

[34]  S. Grzesiek,et al.  Two-Dimensional NMR Methods for Determining χ1 Angles of Aromatic Residues in Proteins from Three-Bond JC‘Cγ and JNCγ Couplings , 1997 .

[35]  Tetsuro Ago,et al.  Novel modular domain PB1 recognizes PC motif to mediate functional protein–protein interactions , 2001, The EMBO journal.

[36]  Wendell A. Lim,et al.  Structural determinants of peptide-binding orientation and of sequence specificity in SH3 domains , 1995, Nature.

[37]  M. Sudol,et al.  The importance of being proline: the interaction of proline‐rich motifs in signaling proteins with their cognate domains , 2000, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[38]  J. Ladbury,et al.  Searching for specificity in SH domains. , 2000, Chemistry & biology.

[39]  R. Levy,et al.  Estimation of interatomic distances in proteins from NOE spectra at longer mixing times using an empirical two-spin equation , 1993 .

[40]  A. Shiose,et al.  Arachidonic Acid and Phosphorylation Synergistically Induce a Conformational Change of p47 phox to Activate the Phagocyte NADPH Oxidase* , 2000, The Journal of Biological Chemistry.

[41]  S. Schreiber,et al.  Specific interactions outside the proline-rich core of two classes of Src homology 3 ligands. , 1995, Proceedings of the National Academy of Sciences of the United States of America.