Biomedical subjects

K D Beebe

Publications and source records attributed to K D Beebe.

4 recordsLinked to original sources

PubMed2000

Determination of the binding specificity of the SH2 domains of protein tyrosine phosphatase SHP-1 through the screening of a combinatorial phosphotyrosyl peptide library.

A method for the rapid identification of high-affinity ligands to Src homology-2 (SH2) domains is reported. A phosphotyrosyl (pY) peptide library containing completely randomized residues at positions -2 to +3 relative to the pY was synthesized on TentaGel resin, with a unique peptide sequence on each resin bead (total 2.5 x 10(6) different sequences). The library was screened against the biotinylated N- and C-terminal SH2 domains of protein tyrosine phosphatase SHP-1, and the beads that carry high-affinity ligands of the SH2 domains were identified using an enzyme-linked assay involving a streptavidin-alkaline phosphatase conjugate. Peptide ladder sequencing of the selected beads using matrix-assisted laser desorption ionization mass spectrometry revealed consensus sequences for both SH2 domains. The N-terminal SH2 domain strongly selects for peptides with a leucine at the -2 position; at the C-terminal side of the pY residue, it can recognize two distinct classes of peptides with consensus sequences of LXpY(M/F)X(F/M) and LXpYAXL (X = any amino acid), respectively. The C-terminal SH2 domain exhibits almost exclusive selectivity for peptides of the consensus sequence, (V/I/L)XpYAX(L/V). Several representative sequences selected from the library were individually synthesized and tested for binding to the SH2 domains by surface plasmon resonance and for their ability to stimulate the catalytic activity of SHP-1. Both experiments have demonstrated that the selected peptides are capable of binding to the SH2 domains with dissociation constants (K(D)) in the low micromolar range.

Amino Acid Sequence↗

PubMed2000

II. Structure and specificity of the interaction between the FHA2 domain of Rad53 and phosphotyrosyl peptides.

The forkhead-associated (FHA) domain is a protein module found in many proteins involved in cell signaling in response to DNA damage. It has been suggested to bind to pThr sites of its target protein. Recently we have determined the first structure of an FHA domain, FHA2 from the yeast protein Rad53, and demonstrated that FHA2 binds to a pTyr-containing peptide (826)EDI(pY)YLD(832) from Rad9, with a moderate affinity (K(d) ca. 100 microM). We now report the solution structure of the complex of FHA2 bound with this pTyr peptide. The structure shows that the phosphate group of pTyr interacts directly with three arginine residues (605, 617, and 620), and that the leucine residue at the +2 position from the pTyr interacts with a hydrophobic surface on FHA2. The sequence specificity of FHA2 was determined by screening a combinatorial pTyr library. The results clearly show that FHA2 recognizes specific sequences C-terminal to pTyr with the following consensus: XX(pY)N(1)N(2)N(3), where N(1)=Leu, Met, Phe, or Ile, N(2)=Tyr, Phe, Leu, or Met, and N(3)=Phe, Leu, or Met. Two of the selected peptides, GF(pY)LYFIR and DV(pY)FYMIR, bind FHA2 with K(d) values of 1.1 and 5.0 microM, respectively. The results, along with other recent reports, demonstrate that the FHA domain is a new class of phosphoprotein-binding domain, capable of binding both pTyr and pThr sequences.

Amino Acid Sequence↗

PubMed2000

Substrate recognition through a PDZ domain in tail-specific protease.

Tail-specific protease (Tsp) is a periplasmic enzyme that selectively degrades proteins bearing a nonpolar C-terminus. Its substrate specificity suggests that Tsp may contain a substrate recognition domain, which selectively binds to the nonpolar C-termini of substrate proteins, separate from its catalytic site. In this work, we show that substrate recognition of Tsp is mediated by a PDZ domain, a small protein module that promotes protein-protein interactions by binding to internal or C-terminal sequences of their partner proteins. Partial proteolysis by V8 protease at a single peptide bond immediately N-terminal to the PDZ domain resulted in two distinct and relatively stable fragments and complete loss of catalytic activity. Photoaffinity labeling with a fluorescent nonpolar peptide caused the covalent attachment of the peptide to a single site on the Tsp protein. Systematic deletion mutagenesis of Tsp localized the binding site to amino acids 206-307, a region that completely encompasses the putative PDZ domain (217-301). The isolated PDZ domain (amino acids 206-334) is capable of folding into a well-behaved structure and binds to a nonpolar peptide with a dissociation constant (K(D)) of 1.9 microM, similar to that of the intact Tsp protein. Site-directed mutagenesis of a surface residue at the peptide binding site of the PDZ domain, valine 229, to Glu or Gln resulted in an increase in the K(M) value but had no effect on the k(cat) value. The use of a separate substrate recognition domain such as a PDZ domain may be a general mechanism for achieving selective protein degradation.

Binding Sites↗

PubMed1998

A continuous fluorimetric assay for tail-specific protease.

A continuous fluorimetric assay for tail-specific protease (Tsp) has been developed using a fluorescence donor/quencher system, in which 5-[(2-aminoethyl) amino]naphthalene-1-sulfonic acid (EDANS) and 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL) are attached to the N-terminus and the lysyl side chain of peptide AARAAK-(6-aminocaproyl)2-ENYALAA, respectively. Tsp-mediated cleavage of the Ala-Arg peptide bond separates the quencher, DABCYL, from the donor, EDANS, and results in a large increase in the fluorescent yield of EDANS (>50-fold). Using this sensitive assay, Escherichia coli tail-specific protease was shown to exhibit typical Michaelis-Menten kinetics with a kcat of 0.086 +/- 0.002 s-1, KM of 4.0 +/- 0.3 microM, and kcat/KM of 2.2 x 10(4) M-1 s-1. A control substrate, which only differs from the above substrate by having a charged residue (glutamate) at the C-terminus, showed drastically reduced activity to Tsp (kcat/KM = 58 M-1 s-1). A peptide containing the C-terminal sequence of the substrate, GRGYALAA, was shown to be a competitive inhibitor of Tsp with a KI value of 31 microM. These results demonstrate the utility of this assay for the rapid assessment of Tsp activity.

Binding, Competitive↗