RESUMO
Src-homology (SH3) domain belongs to a class of ubiquitous modular protein domains found in nature. SH3 domains have a conserved surface that recognises proline-rich peptides in ligand proteins, but additional contacts also contribute to binding. Using the SH3 domain of hematopoietic cell kinase as a test case, we show that SH3 binding properties can be profoundly altered by modifications within a hexapeptide sequence in the RT-loop region that is not involved in recognition of currently known consensus SH3 target peptides. These results highlight the role of non-conserved regions in SH3 target selection, and introduce a strategy that may be generally feasible for generating artificial SH3 domains with desired ligand binding properties.
Assuntos
Engenharia de Proteínas/métodos , Domínios de Homologia de src , Proteínas ADAM/química , Proteínas ADAM/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Complexo CD3/química , Complexo CD3/metabolismo , Sequência Conservada , Produtos do Gene nef/química , Produtos do Gene nef/metabolismo , Imunoensaio/métodos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Biológicos , Modelos Moleculares , Proteínas Oncogênicas v-mos/química , Proteínas Oncogênicas v-mos/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Secundária de Proteína , Transporte Proteico , Proteína SOS1/química , Proteína SOS1/metabolismo , Quinases Ativadas por p21RESUMO
We have determined the human genome to contain 296 different Src homology-3 (SH3) domains and cloned them into a phage-display vector. This provided a powerful and unbiased system for simultaneous assaying of the complete human SH3 proteome for the strongest binding to target proteins of interest, without the limitations posed by short linear peptide ligands or confounding variables of more indirect methods for protein interaction screening. Studies involving three ligand proteins, human immunodeficiency virus-1 Nef, p21-activated kinase (PAK)2 and ADAM15, showed previously reported as well as novel SH3 partners with nanomolar affinities specific for them. This argues that SH3 domains may have a more dominant role in directing cellular protein interactions than has been assumed. Besides showing potentially important new SH3-directed interactions, these studies also led to the discovery of novel signalling proteins, such as the PAK2-binding adaptor protein POSH2 and the ADAM15-binding sorting nexin family member SNX30.
Assuntos
Biblioteca de Peptídeos , Proteínas/metabolismo , Proteoma , Domínios de Homologia de src/fisiologia , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Produtos do Gene nef/genética , Produtos do Gene nef/metabolismo , Vetores Genéticos , Glutationa Transferase/metabolismo , Humanos , Ligantes , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/genética , Proteínas Recombinantes de Fusão/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Quinases Ativadas por p21 , Domínios de Homologia de src/genéticaRESUMO
The simian immunodeficiency virus (SIV) Nef protein contains a consensus Src-homology 3 (SH3) binding motif. However, no SH3-domain proteins showing strong binding to SIV Nef have yet been found, and its potential capacity for high-affinity SH3 binding has therefore remained unproven. Here we have used phage-display-assisted protein engineering to develop artificial SH3 domains that bind tightly to SIV strain mac (SIVmac) Nef. Substitution of six amino acids in the RT loop region of Hck-SH3 with the sequence E/DGWWG resulted in SH3 domains that bound in vitro to SIVmac Nef much better than the natural Hck- or Fyn-SH3 domains. These novel SH3 domains also efficiently associated with SIVmac Nef when co-expressed in 293T cells and displayed a strikingly differential specificity when compared with SH3 domains similarly targeted for binding to human immunodeficiency virus type 1 (HIV-1) Nef. Thus, SIVmac Nef is competent for high-affinity SH3 binding, but its natural SH3 protein partners are likely to be different from those of HIV-1 Nef.
