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1.
J Mol Biol ; 314(5): 1147-56, 2001 Dec 14.
Article in English | MEDLINE | ID: mdl-11743730

ABSTRACT

The single mutation L30 K in the Hu-Yap65 WW domain increased the stability of the complex with the peptide GTPPPPYTVG (K(d)=40(+/-5) microM). Here we report the refined solution structure of this complex by NMR spectroscopy and further derived structure-activity relationships by using ligand peptide libraries with truncated sequences and a substitution analysis that yielded acetyl-PPPPY as the smallest high-affinity binding peptide (K(d)=60 microM). The structures of two new complexes with weaker binding ligands chosen based on these results (N-(n-octyl)-GPPPYNH(2) and Ac-PLPPY) comprising the wild-type WW domain of Hu-Yap65 were determined. Comparison of the structures of the three complexes were useful for identifying the molecular basis of high-affinity: hydrophobic and specific interactions between the side-chains of Y28 and W39 and P5' and P4', respectively, and hydrogen bonds between T37 (donnor) and P5' (acceptor) and between W39 (donnor) and T2' (acceptor) stabilize the complex.The structure of the complex L30 K Hu-Yap65 WW domain/GTPPPPYTVG is compared to the published crystal structure of the dystrophin WW domain bound to a segment of the beta-dystroglycan protein and to the solution structure of the first Nedd4 WW domain and its prolin-rich ligand, suggesting that WW sequences bind proline-rich peptides in an evolutionary conserved fashion. The position equivalent to T22 in the Hu-Yap65 WW domain sequence is seen as responsible for differentiation in the binding mode among the WW domains of group I.


Subject(s)
Adaptor Proteins, Signal Transducing , Amino Acid Substitution/genetics , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Peptide Library , Peptides/chemistry , Peptides/metabolism , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Binding Sites , Carrier Proteins/genetics , Humans , Ligands , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Peptides/genetics , Phosphoproteins/genetics , Protein Structure, Tertiary , Sequence Alignment , Solutions , Thermodynamics , Transcription Factors , YAP-Signaling Proteins
2.
EMBO J ; 19(18): 4866-74, 2000 Sep 15.
Article in English | MEDLINE | ID: mdl-10990450

ABSTRACT

We identified evolutionary pathways for the inter- conversion of three sequentially and structurally unrelated peptides, GATPEDLNQKL, GLYEWGGARI and FDKEWNLIEQN, binding to the same site of the hypervariable region of the anti-p24 (HIV-1) monoclonal antibody CB4-1. Conversion of these peptides into each other could be achieved in nine or 10 single amino acid substitution steps without loss of antibody binding. Such pathways were identified by analyzing all 7 620 480 pathways connecting 2560 different peptides, and testing them for CB4-1 binding. The binding modes of intermediate peptides of selected optimal pathways were characterized using complete sets of substitution analogs, revealing that a number of sequential substitutions accumulated without changing the pattern of key interacting residues. At a distinct step, however, one single amino acid exchange induces a sudden change in the binding mode, indicating a flip in specificity and conformation. Our data represent a model of how different specificities, structures and functions might evolve in protein-protein recognition.


Subject(s)
Evolution, Molecular , Peptides/chemistry , Algorithms , Amino Acid Sequence , Amino Acids/chemistry , Antibodies, Monoclonal/chemistry , Biosensing Techniques , Cellulose/chemistry , Crystallography, X-Ray , Enzyme-Linked Immunosorbent Assay , HIV Core Protein p24/chemistry , Ligands , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Peptide Biosynthesis , Phenotype , Protein Binding , Protein Conformation , Structure-Activity Relationship
3.
J Biol Chem ; 274(45): 32001-7, 1999 Nov 05.
Article in English | MEDLINE | ID: mdl-10542231

ABSTRACT

The proline-rich domain of synaptojanin 1, a synaptic protein with phosphatidylinositol phosphatase activity, binds to amphiphysin and to a family of recently discovered proteins known as the SH3p4/8/13, the SH3-GL, or the endophilin family. These interactions are mediated by SH3 domains and are believed to play a regulatory role in synaptic vesicle recycling. We have precisely mapped the target peptides on human synaptojanin that are recognized by the SH3 domains of endophilins and amphiphysin and proven that they are distinct. By a combination of different approaches, selection of phage displayed peptide libraries, substitution analyses of peptides synthesized on cellulose membranes, and a peptide scan spanning a 252-residue long synaptojanin fragment, we have concluded that amphiphysin binds to two sites, PIRPSR and PTIPPR, whereas endophilin has a distinct preferred binding site, PKRPPPPR. The comparison of the results obtained by phage display and substitution analysis permitted the identification of proline and arginine at positions 4 and 6 in the PIRPSR and PTIPPR target sequence as the major determinants of the recognition specificity mediated by the SH3 domain of amphiphysin 1. More complex is the structural rationalization of the preferred endophilin ligands where SH3 binding cannot be easily interpreted in the framework of the "classical" type I or type II SH3 binding models. Our results suggest that the binding repertoire of SH3 domains may be more complex than originally predicted.


Subject(s)
Adaptor Proteins, Signal Transducing , Carrier Proteins/metabolism , Nerve Tissue Proteins/metabolism , Phosphoric Monoester Hydrolases/metabolism , Proline/metabolism , src Homology Domains , Amino Acid Sequence , Binding Sites , Binding, Competitive , Enzyme-Linked Immunosorbent Assay , Humans , Molecular Sequence Data , Peptide Library
4.
J Pept Res ; 54(4): 319-27, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10532237

ABSTRACT

Positionally addressable syntheses of peptides on continuous cellulose membranes (spot synthesis) have often been reported in detail, but important questions dealing with synthesis quality, reproducibility and subsequent binding assays have largely been under-emphasized. In this report we have investigated some of these problems. The most important results were: (i) the signal intensity of ligate binding to cellulose-bound peptides and the affinity of the corresponding soluble peptides show good correlation, illustrated by three different ligate binding assays; (ii) reducing peptide density on the cellulose avoids the 'ring spot' effect, i.e. where less binding is observed in the spot-center compared to the rim. We recommend a peptide density of 10 nmol/cm2 as a reasonable starting point for further optimization; (iii) statistical analysis of binding assay reproducibility with more than 15000 peptides resulted in a mean standard signal deviation of 0.18; and (iv) optimization of side-chain deprotection revealed that a 30-min pretreatment of the cellulose with 90% trifluoroacetic acid followed by the standard deprotection protocol resulted in higher purity of the synthesized products.


Subject(s)
Peptide Biosynthesis , Peptide Library , Amino Acid Sequence , Binding Sites, Antibody , Cellulose , Chromatography, High Pressure Liquid , Membranes, Artificial , Polyvinyls , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
5.
Nat Struct Biol ; 5(1): 19-24, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9437424

ABSTRACT

Syntrophins are modular proteins belonging to the dystrophin associated glycoprotein complex and are thought to be involved in the regulation of the muscular system. Screening of peptide libraries revealed selectivity of the synotrophin PDZ domain toward the motif R/K/Q-E-S/T-X-V-COO- found to be highly conserved in the alpha-subunit C-terminus of vertebrate voltage gated sodium channels (VGSCs). The solution structure of the domain in complex with the peptide G-V-K-E-S-L-V shows specific interactions between the conserved residues in the peptide and syntrophin-characteristic residues in the domain. We propose that syntrophins localize VGSCs to the dystrophin network through its PDZ domain.


Subject(s)
Membrane Proteins/chemistry , Muscle Proteins/chemistry , Sodium Channels/chemistry , Amino Acid Sequence , Animals , Binding Sites , Calcium-Binding Proteins , Hydrogen Bonding , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Peptide Library , Protein Binding , Protein Structure, Tertiary , Rabbits , Sequence Alignment , Sequence Homology, Amino Acid
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