Protein-protein interaction studied by site-directed mutagenesis. Characterization of the annexin II-binding site on p11, a member of the S100 protein family.
p11, a member of the S100 protein family, forms a stable heterotetrameric complex with annexin II. The p11-binding site of annexin II resides in the N-terminal 14 residues, which form an amphiphatic alpha-helix with the hydrophobic face representing the contact site for p11 (Johnsson, N., Marriott, G., and Weber, K. (1988) EMBO J. 7, 2435-2442). We show that a corresponding peptide can be used to purify recombinant p11 by affinity chromatography. To map the annexin II-binding site on p11, we have produced progressively truncated p11 derivatives by site-directed mutagenesis. Our analysis reveals that a highly hydrophobic region between residues 85 and 91 is indispensable for annexin II-binding. It is located in the C-terminal extension, following the second distorted EF-hand. Using a series of single amino acid replacements, we have identified individual hydrophobic residues, which seem to represent contact points for annexin II. Most notably, substitution of tyrosine 85 or phenylalanine 86 by alanine drastically reduces the affinity of p11 for annexin II, whereas replacement of these residues by tryptophan has no or only a marginal effect. Thus, hydrophobic side chains on both annexin II and p11 are involved in complex formation.
Kube
E
E
Department of Biochemistry, Max Planck Institute for Biophysical Chemistry, Goettingen, Federal Republic of Germany.
Becker
T
T
Weber
K
K
Gerke
V
V
D000595:Amino Acid Sequence; D017302:Annexins; D001483:Base Sequence; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides / Q000138:chemical synthesis; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D009418:S100 Proteins / Q000378:metabolism*
D017302:Annexins; D002135:Calcium-Binding Proteins; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides; D011994:Recombinant Proteins; D009418:S100 Proteins; D004247:DNA