In vitro selection and prediction of TIP47 protein-interaction interfaces.
We present a new method for the rapid identification of amino acid residues that contribute to protein-protein interfaces. Tail-interacting protein of 47 kDa (TIP47) binds Rab9 GTPase and the cytoplasmic domains of mannose 6-phosphate receptors and is required for their transport from endosomes to the Golgi apparatus. Cysteine mutations were incorporated randomly into TIP47 by expression in Escherichia coli cells harboring specific misincorporator tRNAs. We made use of the ability of the native TIP47 protein to protect 48 cysteine probes from chemical modification by iodoacetamide as a means to obtain a surface map of TIP47, revealing the identity of surface-localized, hydrophobic residues that are likely to participate in protein-protein interactions. Direct mutation of predicted interface residues confirmed that the protein had altered binding affinity for the mannose 6-phosphate receptor. TIP47 mutants with enhanced or diminished affinities were also selected by affinity chromatography. These methods were validated in comparison with the protein's crystal structure, and provide a powerful means to predict protein-protein interaction interfaces.
Burguete
Alondra Schweizer
AS
Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305-5307, USA.
Harbury
Pehr B
PB
Pfeffer
Suzanne R
SR
D019943:Amino Acid Substitution; D001665:Binding Sites; D003001:Cloning, Molecular / Q000379:methods*; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D000070796:Perilipin-3; D011257:Pregnancy Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D033921:Vesicular Transport Proteins
D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; C038196:PLIN3 protein, human; D000070796:Perilipin-3; D011257:Pregnancy Proteins; D011994:Recombinant Proteins; D033921:Vesicular Transport Proteins; D003545:Cysteine