Substrate specificity of protein tyrosine phosphatases 1B, RPTPα, SHP-1, and SHP-2

We determined the substrate specificities of the protein tyrosine phosphatases (PTPs) PTP1B, RPTPα, SHP-1, and SHP-2 by on-bead screening of combinatorial peptide libraries and solution-phase kinetic analysis of individually synthesized phosphotyrosyl (pY) peptides. These PTPs exhibit different levels of sequence specificity and catalytic efficiency. The catalytic domain of RPTPα has very weak sequence specificity and is approximately 2 orders of magnitude less active than the other three PTPs. The PTP1B catalytic domain has modest preference for acidic residues on both sides of pY, is highly active toward multiply phosphorylated peptides, but disfavors basic residues at any position, a Gly at the pY-1 position, or a Pro at the pY+1 position. By contrast, SHP-1 and SHP-2 share similar but much narrower substrate specificities, with a strong preference for acidic and aromatic hydrophobic amino acids on both sides of the pY residue. An efficient SHP-1/2 substrate generally contains two or more acidic residues on the N-terminal side and one or more acidic residues on the C-terminal side of pY but no basic residues. Subtle differences exist between SHP-1 and SHP-2 in that SHP-1 has a stronger preference for acidic residues at the pY-1 and pY+1 positions and the two SHPs prefer acidic residues at different positions N-terminal to pY. A survey of the known protein substrates of PTP1B, SHP-1, and SHP-2 shows an excellent agreement between the in vivo dephosphorylation pattern and the in vitro specificity profiles derived from library screening. These results suggest that different PTPs have distinct sequence specificity profiles and the intrinsic activity/specificity of the PTP domain is an important determinant of the enzyme's in vivo substrate specificity.     

Screening combinatorial libraries by mass spectrometry. 2. Identification of optimal substrates of protein tyrosine phosphatase SHP-1

Protein tyrosine phosphatases (PTPs) are a large family of enzymes that catalyze the hydrolytic removal of the phosphoryl group from phosphotyrosyl (pY) proteins. In this work, we have developed a novel combinatorial library method, termed "enzyme-catalyzed loss of isotope peak signal enhancement (ECLIPSE)", to determine the substrate specificity of PTPs. This method involves partial labeling of pY at a nonbridging phosphate oxygen atom with 50% (18)O ((16)O/(18)O = 1:1). A 361-member solution-phase peptide library with randomization at the -1 and -2 positions (relative to pY), RNNXXpYA-NH(2) (X = 19 alpha-amino acids except for Cys), was synthesized with the partially (18)O-labeled pY by the split-synthesis method. Each member of the resulting pY peptide library appeared as a doublet peak in the mass spectrum (m/z m and m + 2.0043). Limited treatment of the library with a PTP removed the mass-degenerate phosphoryl group from the most preferred substrates to generate products as singlet peaks, which were readily identified and sequenced by tandem mass spectrometry. Screening of the pY library against the catalytic domain of SHP-1 revealed that SHP-1 prefers an acidic residue at the -2 position, with aspartic acid being slightly better than glutamic acid. At the -1 position, SHP-1 also prefers an acidic residue, although a variety of other amino acids are also tolerated. On the other hand, positively charged residues at these positions render the corresponding peptides very poor substrates of SHP-1. Several selected peptides were individually synthesized and assayed against SHP-1, and the kinetic data confirmed the screening results. These results demonstrate that ECLIPSE is a viable method for studying the substrate specificity of PTPs.     

Structural basis of plasticity in protein tyrosine phosphatase 1B substrate recognition

Protein tyrosine phosphatase 1B (PTP1B) displays a preference for peptides containing acidic as well as aromatic/aliphatic residues immediately NH(2)-terminal to phosphotyrosine. The structure of PTP1B bound with DADEpYL-NH(2) (EGFR(988)(-)(993)) offers a structural explanation for PTP1B's preference for acidic residues [Jia, Z., Barford, D., Flint, A. J., and Tonks, N. K. (1995) Science 268, 1754-1758]. We report here the crystal structures of PTP1B in complex with Ac-ELEFpYMDYE-NH(2) (PTP1B.Con) and Ac-DAD(Bpa)pYLIPQQG (PTP1B.Bpa) determined to 1.8 and 1.9 A resolution, respectively. A structural analysis of PTP1B.Con and PTP1B.Bpa shows how aromatic/aliphatic residues at the -1 and -3 positions of peptide substrates are accommodated by PTP1B. A comparison of the structures of PTP1B.Con and PTP1B.Bpa with that of PTP1B.EGFR(988)(-)(993) reveals the structural basis for the plasticity of PTP1B substrate recognition. PTP1B is able to bind phosphopeptides by utilizing common interactions involving the aromatic ring and phosphate moiety of phosphotyrosine itself, two conserved hydrogen bonds between the Asp48 carboxylate side chain and the main chain nitrogens of the pTyr and residue 1, and a third between the main chain nitrogen of Arg47 and the main chain carbonyl of residue -2. The ability of PTP1B to accommodate both acidic and hydrophobic residues immediately NH(2)-terminal to pTyr appears to be conferred upon PTP1B by a single residue, Arg47. Depending on the nature of the NH(2)-terminal amino acids, the side chain of Arg47 can adopt one of two different conformations, generating two sets of distinct peptide binding surfaces. When an acidic residue is positioned at position -1, a preference for a second acidic residue is also observed at position -2. However, when a large hydrophobic group occupies position -1, Arg47 adopts a new conformation so that it can participate in hydrophobic interactions with both positions -1 and -3.     

Proteolysis of ProPTHrP(1-141) by "prohormone thiol protease" at multibasic residues generates PTHrP-related peptides: implications for PTHrP peptide production in lung cancer cells

The parathyroid hormone-related protein (PTHrP) precursor requires proteolytic processing to generate PTHrP-related peptide products that possess regulatory functions in the control of PTH-like (parathyroid-like) actions and cell growth, calcium transport, and osteoclast activity. Biologically active peptide domains within the PTHrP precursor are typically flanked at their NH2- and COOH-termini by basic residue cleavage sites consisting of multibasic, dibasic, and monobasic residues. These basic residues are predicted to serve as proteolytic cleavage sites for converting the PTHrP precursor into active peptide products. The coexpression of the prohormone processing enzyme PTP ("prohormone thiol protease") in PTHrP-containing lung cancer cells, and the lack of PTP in cell lines that contain little PTHrP, implicate PTP as a candidate processing enzyme for proPTHrP. Therefore, in this study, PTP cleavage of recombinant proPTHrP(1-141) precursor was evaluated by MALDI mass spectrometry to identify peptide products and cleavage sites. PTP cleaved the PTHrP precursor at the predicted basic residue cleavage sites to generate biologically active PTHrP-related peptides that correspond to the NH2-terminal domain (residues 1-37) that possesses PTH-like and growth regulatory activities, the mid-region domain (residues 38-93) that regulates calcium transport, and the COOH-terminal domain (residues 102-141) that modulates osteoclast activity. Lack of cleavage at other types of amino acids demonstrated the specificity of PTP processing at basic residue cleavage sites. Overall, these results demonstrate the ability of PTP to cleave the PTHrP precursor at multibasic, dibasic, and monobasic residue cleavage sites to generate active PTHrP-related peptides. The presence of PTP immunoreactivity in PTHrP-containing lung cancer cells suggests PTP as a candidate processing enzyme for the PTHrP precursor.     

The SPOT technique as a tool for studying protein tyrosine phosphatase substrate specificities

The activity of protein tyrosine phosphatases (PTPs) is restricted by their substrate specificities. The analysis of PTP specificity was greatly helped by the discovery that "substrate-trapping" PTP mutants, such as PTP-1B D181A, stably and specifically bind their substrates. We have set up a PTP substrate specificity assay based on the SPOT technique, which involves the microsynthesis of (phospho)peptides on membranes. To validate this approach, substrate trapping PTP-1B was tested on its cognate ligand, the autophosphorylated insulin receptor (IR). On SPOT membranes, IR peptides with phosphotyrosine 1163 were efficiently bound by PTP1B D181A, and dephosphorylated by PTP-1B. Phosphotyrosine 1163 was preferred over the neighboring 1158 and 1162 phosphotyrosines. PTP-1B also recognized IR-like motifs in Trk autophosphorylation domains, and STAT 5 phosphopeptides. Using a gridded 20-by-20 SPOT library, we show that peptides with the YZM motif (Z: phosphotyrosine) are the strongest ligands for PTP-1B D181A, but not the optimal substrates for dephosphorylation by wild-type PTP1B. In addition we show that PTP-1B and PTP-beta dephosphorylation efficiency is strongly modulated by the introduction of phospho-serine or phospho-threonine in their cognate phospho-tyrosine substrates. Altogether our data illustrate that the SPOT technique is a highly efficient tool for the study of PTP substrate specificity.     

Altered -3 substrate specificity of Escherichia coli signal peptidase 1 mutants as revealed by screening a combinatorial peptide library

Signal peptidase functions to cleave signal peptides from preproteins at the cell membrane. It has a substrate specificity for small uncharged residues at -1 (P1) and aliphatic residues at the -3 (P3) position. Previously, we have reported that certain alterations of the Ile-144 and Ile-86 residues in Escherichia coli signal peptidase I (SPase) can change the specificity such that signal peptidase is able to cleave pro-OmpA nuclease A in vitro after phenylalanine or asparagine residues at the -1 position (Karla, A., Lively, M. O., Paetzel, M. and Dalbey, R. (2005) J. Biol. Chem. 280, 6731-6741). In this study, screening of a fluorescence resonance energy transfer-based peptide library revealed that the I144A, I144C, and I144C/I86T SPase mutants have a more relaxed substrate specificity at the -3 position, in comparison to the wild-type SPase. The double mutant tolerated arginine, glutamine, and tyrosine residues at the -3 position of the substrate. The altered specificity of the I144C/I86T mutant was confirmed by in vivo processing of pre-beta-lactamase containing non-canonical arginine and glutamine residues at the -3 position. This work establishes Ile-144 and Ile-86 as key P3 substrate specificity determinants for signal peptidase I and demonstrates the power of the fluorescence resonance energy transfer-based peptide library approach in defining the substrate specificity of proteases.     

Active-site specificity of digestive aspartic peptidases from the four species of Plasmodium that infect humans using chromogenic combinatorial peptide libraries

Two targeted chromogenic octapeptide combinatorial libraries, comprised of 38 pools each containing 361 different peptides, were used to analyze the enzyme/substrate interactions of five plasmepsins. The first library (P1 library) was based on a good synthetic aspartic peptidase substrate [Westling, J., Cipullo, P., Hung, S. H., Saft, H., Dame, J. B., and Dunn, B. M. (1999) Protein Sci. 8, 2001-2009; Scarborough, P. E., and Dunn, B. M. (1994) Protein Eng. 7, 495-502] and had the sequence Lys-Pro-(Xaa)-Glu-P1*Nph-(Xaa)-Leu. The second library (P1' library) incorporated results with the plasmepsins from the first library and had the sequence Lys-Pro-Ile-(Xaa)-Nph*P1'-Gln-(Xaa). In both cases, P1 and P1' were fixed residues for a given peptide pool, where Nph was a para-nitrophenylalanine chromogenic reporter and Xaa was a mixture of 19 different amino acids. Kinetic assays monitoring the rates of cleavage of these libraries revealed the optimal P1 and P1' residues for the five plasmepsins as hydrophobic substitutions. Extended specificity preferences were obtained utilizing liquid chromatography-mass spectrometry (LC-MS) to analyze the cleavage products produced by enzyme-catalyzed digestion of the best pools of each peptide library. LC-MS analysis of the P1-Phe and P1'-Phe pools revealed the favored amino acids at the P3, P2, P2', and P3' positions. These analyses have provided new insights on the binding preferences of malarial digestive enzymes that were used to design specific methyleneamino peptidomimetic inhibitors of the plasmepsins. Some of these compounds were potent inhibitors of the five plasmepsins, and their possible binding modes were analyzed by computational methods.     

Mapping of synergistic components of weakly interacting protein-protein motifs using arrays of paired peptides

Protein-protein recognition usually involves multiple interactions among different motifs that are scattered over protein surfaces. To identify such weak interactions, we have developed a novel double peptide synthesis (DS) method. This method allows us to map protein-protein interactions that involve two linear dis- continuous components from a polypeptide by the use of spatially addressable synergistic pairs of synthetic peptides. The DS procedure is based on the "SPOT" membrane-bound peptide synthesis technique, but to synthesize a mixture of two peptides, it uses both Fmoc (N-(9-fluorenyl)methoxycarbonyl))-alanine and Alloc-alanine at the first cycle. This allows their selective deprotection by either piperidine or tributyltin/palladium treatment, respectively. Using SPOT DS, we confirmed as a proof of principle that Elk-1 Ser(383) phosphorylation by ERK-2 kinase is stimulated by the presence of the Elk-1-docking domain. SPOT DS can also be used to dissect protein-protein motifs that define phosphatase substrate affinity. Using this technique, we identified three new regions in the insulin receptor that stimulate the dephosphorylation of the receptor by protein-tyrosine phosphatase (PTP) 1B and presumably increase the selectivity of PTP for this substrate. These data demonstrate that the SPOT DS technique allows the identification of non-linear weakly interacting protein motifs, which are an important determinant of protein kinase and phosphatase substrate specificity and of protein-protein interactions in general.     

Prediction and verification of novel peptide targets of protein tyrosine phosphatase 1B

Phosphotyrosine peptides are useful starting points for inhibitor design and for the search for protein tyrosine phosphatase (PTP) phosphoprotein substrates. To identify novel phosphopeptide substrates of PTP1B, we developed a computational prediction protocol based on a virtual library of protein sequences with known phosphotyrosine sites. To these we applied sequence-based methods, biologically meaningful filters and molecular docking. Five peptides were selected for biochemical testing of their potential as PTP1B substrates. All five peptides were equally good substrates for PTP1B compared to a known peptide substrate whereas appropriate control peptides were not recognized, showing that our protocol can be used to identify novel peptide substrates of PTP1B.     

PTP1B modulates the association of beta-catenin with N-cadherin through binding to an adjacent and partially overlapping target site

The nonreceptor tyrosine phosphatase PTP1B associates with the cytoplasmic domain of N-cadherin and may regulate cadherin function through dephosphorylation of beta-catenin. We have now identified the domain on N-cadherin to which PTP1B binds and characterized the effect of perturbing this domain on cadherin function. Deletion constructs lacking amino acids 872-891 fail to bind PTP1B. This domain partially overlaps with the beta-catenin binding domain. To further define the relationship of these two sites, we used peptides to compete in vitro binding. A peptide representing the most NH(2)-terminal 8 amino acids of the PTP1B binding site, the region of overlap with the beta-catenin target, effectively competes for binding of beta-catenin but is much less effective in competing PTP1B, whereas two peptides representing the remaining 12 amino acids have no effect on beta-catenin binding but effectively compete for PTP1B binding. Introduction into embryonic chick retina cells of a cell-permeable peptide mimicking the 8 most COOH-terminal amino acids in the PTP1B target domain, the region most distant from the beta-catenin target site, prevents binding of PTP1B, increases the pool of free, tyrosine-phosphorylated beta-catenin, and results in loss of N-cadherin function. N-cadherin lacking this same region of the PTP1B target site does not associate with PTP1B or beta-catenin and is not efficiently expressed at the cell surface of transfected L cells. Thus, interaction of PTP1B with N-cadherin is essential for its association with beta-catenin, stable expression at the cell surface, and consequently, cadherin function.     

Determination of the Substrate Specificity of Protein-tyrosine Phosphatase TULA-2 and Identification of Syk as a TULA-2 Substrate

TULA-1 (UBASH3A/STS-2) and TULA-2 (p70/STS-1) represent a novel class of protein-tyrosine phosphatases. Previous studies suggest that TULA-2 is sequence-selective toward phosphotyrosyl (Tyr(P)) peptides. In this work the substrate specificity of TULA-1 and -2 was systematically evaluated by screening a combinatorial Tyr(P) peptide library. Although TULA-1 showed no detectable activity toward any of the Tyr(P) peptides in the library, TULA-2 recognizes two distinct classes of Tyr(P) substrates. On the N-terminal side of Tyr(P), the class I substrates contain a proline at the Tyr(P)−1 position, a hydrophilic residue at the Tyr(P)−2 position, and aromatic hydrophobic residues at positions Tyr(P)−3 and beyond. The class II substrates typically contain two or more acidic residues, especially at Tyr(P)−1 to Tyr(P)−3 positions, and aromatic hydrophobic residues at other positions. At the C-terminal side of Tyr(P), TULA-2 generally prefers acidic and aromatic residues. The library screening results were confirmed by kinetic analysis of representative peptides selected from the library as well as Tyr(P) peptides derived from various Tyr(P) proteins. TULA-2 is highly active toward peptides corresponding to the Tyr(P)-323 and Tyr(P)-352 sites of Syk, and the Tyr(P)-397 site of focal adhesion kinase and has lower activity toward other Tyr(P) sites in these proteins. In glycoprotein VI-stimulated platelets, knock-out of the TULA-2 gene significantly increased the phosphorylation level of Syk at Tyr-323 and Tyr-352 sites and to a lesser degree at the Tyr-525/526 sites. These results suggest that Syk is a bona fide TULA-2 substrate in platelets.     

Steric hindrance as a basis for structure-based design of selective inhibitors of protein-tyrosine phosphatases.

Utilizing structure-based design, we have previously demonstrated that it is possible to obtain selective inhibitors of protein-tyrosine phosphatase 1B (PTP1B). A basic nitrogen was introduced into a general PTP inhibitor to form a salt bridge to Asp48 in PTP1B and simultaneously cause repulsion in PTPs containing an asparagine in the equivalent position [Iversen, L. F., et al. (2000) J. Biol. Chem. 275, 10300-10307]. Further, we have recently demonstrated that Gly259 in PTP1B forms the bottom of a gateway that allows easy access to the active site for a broad range of substrates, while bulky residues in the same position in other PTPs cause steric hindrance and reduced substrate recognition capacity [Peters, G. H., et al. (2000) J. Biol. Chem. 275, 18201-18209]. The current study was undertaken to investigate the feasibility of structure-based design, utilizing these differences in accessibility to the active site among various PTPs. We show that a general, low-molecular weight PTP inhibitor can be developed into a highly selective inhibitor for PTP1B and TC-PTP by introducing a substituent, which is designed to address the region around residues 258 and 259. Detailed enzyme kinetic analysis with a set of wild-type and mutant PTPs, X-ray protein crystallography, and molecular modeling studies confirmed that selectivity for PTP1B and TC-PTP was achieved due to steric hindrance imposed by bulky position 259 residues in other PTPs.     

Intact LIM 3 and LIM 4 domains of paxillin are required for the association to a novel polyproline region (Pro 2) of protein-tyrosine phosphatase-PEST.

The focal adhesion protein p130(Cas) was identified as a substrate for the protein-tyrosine phosphatase (PTP)-PEST, and the specificity of this interaction is mediated by a dual mechanism involving a Src homology 3 domain-mediated binding and PTP domain recognition. Recently, paxillin was also demonstrated to interact with PTP-PEST (Shen, Y., Schneider, G., Cloutier, J. F., Veillette, A., and Schaller, M. D. (1998) J. Biol. Chem. 273, 6474-6481). In the present study, we show that amino acids 344-397 of PTP-PEST are sufficient for the binding to paxillin. We demonstrate that a proline-rich segment of PTP-PEST (Pro 2), 355PPEPHPVPPILTPSPPSAFP374, is essential for this interaction in vivo. Furthermore, mutation of proline residues within the Pro 2 motif reveal that proline 362 is critical for the binding of paxillin. Conversely, using deletion and point mutants of paxillin, LIM 3 and 4 domains were both found to be necessary for binding of PTP-PEST. Finally, using a "substrate trapping" approach, we demonstrate that, unlike p130(Cas), paxillin is not a substrate for PTP-PEST. In conclusion, we show that a novel proline-rich motif found in PTP-PEST serves as a ligand for the LIM domains of paxillin. Interestingly, the focal adhesion targeting of paxillin is mediated by LIM 3. Thus, we propose that PTP-PEST, by a competition with the ligand of paxillin in the focal adhesion complex, could contribute to the removal of paxillin from the adhesion sites and consequently promote focal adhesion turnover.     

Biochemical properties of a putative signal peptide peptidase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1

We have performed the first biochemical characterization of a putative archaeal signal peptide peptidase (SppA(Tk)) from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. SppA(Tk), comprised of 334 residues, was much smaller than its counterpart from Escherichia coli (618 residues) and harbored a single predicted transmembrane domain near its N terminus. A truncated mutant protein without the N-terminal 54 amino acid residues (deltaN54SppA(Tk)) was found to be stable against autoproteolysis and was examined further. DeltaN54SppA(Tk) exhibited peptidase activity towards fluorogenic peptide substrates and was found to be highly thermostable. Moreover, the enzyme displayed a remarkable stability and preference for alkaline pH, with optimal activity detected at pH 10. DeltaN54SppA(Tk) displayed a K(m) of 240 +/- 18 microM and a V(max) of 27.8 +/- 0.7 micromol min(-1) mg(-1) towards Ala-Ala-Phe-4-methyl-coumaryl-7-amide at 80 degrees C and pH 10. The substrate specificity of the enzyme was examined in detail with a FRETS peptide library. By analyzing the cleavage products with liquid chromatography-mass spectrometry, deltaN54SppA(Tk) was found to efficiently cleave peptides with a relatively small side chain at the P-1 position and a hydrophobic or aromatic residue at the P-3 position. The positively charged Arg residue was preferred at the P-4 position, while substrates with negatively charged residues at the P-2, P-3, or P-4 position were not cleaved. When predicted signal sequences from the T. kodakaraensis genome sequence were examined, we found that the substrate specificity of deltaN54SppA(Tk) was in good agreement with its presumed role as a signal peptide peptidase in this archaeon.     

SUMO assay with peptide arrays on solid support: insights into SUMO target sites

The modification of proteins by SUMO (small ubiquitin-like modifier) regulates various cellular processes. Sumoylation often occurs on a specific lysine residue within the consensus motif psiKxE/D. However, little is known about the specificity and selectivity of SUMO target sites. We describe here a SUMO assay with peptide array on solid support for the simultaneous characterization of hundreds of different SUMO target sites. This approach was used to characterize known SUMO substrates. The position of the motif within the peptide and the amino acids flanking the acceptor site affected the efficiency of SUMO modification. Interestingly, a sequence of only four amino acids, corresponding to the SUMO consensus motif without flanking amino acids, was a bona fide target site. Analysis of a peptide library for all variants of the psiKxE/D consensus motif revealed that the first and third positions in the tetrapeptide preferably contain aromatic amino acid residues. Furthermore, by adding the SUMO E3 ligase PIAS1 to the reaction mixture, we show specific enhancement of the modification of a PIAS1-dependent SUMO substrate in this system. Overall, our results demonstrate that the sumoylation assay with peptide array on solid support can be used for the high-throughput characterization of SUMO target sites, and provide new insights into the composition, selectivity and specificity of SUMO target sites.     

Residue 259 is a key determinant of substrate specificity of protein-tyrosine phosphatases 1B and alpha

The aim of this study was to define the structural elements that determine the differences in substrate recognition capacity of two protein-tyrosine phosphatases (PTPs), PTP1B and PTPalpha, both suggested to be negative regulators of insulin signaling. Since the Ac-DADE(pY)L-NH(2) peptide is well recognized by PTP1B, but less efficiently by PTPalpha, it was chosen as a tool for these analyses. Calpha regiovariation analyses and primary sequence alignments indicate that residues 47, 48, 258, and 259 (PTP1B numbering) define a selectivity-determining region. By analyzing a set of DADE(pY)L analogs with a series of PTP mutants in which these four residues were exchanged between PTP1B and PTPalpha, either in combination or alone, we here demonstrate that the key selectivity-determining residue is 259. In PTPalpha, this residue is a glutamine causing steric hindrance and in PTP1B a glycine allowing broad substrate recognition. Significantly, replacing Gln(259) with a glycine almost turns PTPalpha into a PTP1B-like enzyme. By using a novel set of PTP inhibitors and x-ray crystallography, we further provide evidence that Gln(259) in PTPalpha plays a dual role leading to restricted substrate recognition (directly via steric hindrance) and reduced catalytic activity (indirectly via Gln(262)). Both effects may indicate that PTPalpha regulates highly selective signal transduction processes.     

The YRD motif is a major determinant of substrate and inhibitor specificity in T-cell protein-tyrosine phosphatase

We have studied T-cell protein-tyrosine phosphatase (TCPTP) as a model phosphatase in an attempt to unravel amino acid residues that may influence the design of specific inhibitors. Residues 48--50, termed the YRD motif, a region that is found in protein-tyrosine phosphatases, but absent in dual-specificity phosphatases was targeted. YRD derivatives of TCPTP were characterized by steady-state kinetics and by inhibition studies with BzN-EJJ-amide, a potent inhibitor of TCPTP. Substitution of Asp(50) to alanine or Arg(49) to lysine, methionine, or alanine significantly affected substrate hydrolysis and led to a substantial decrease in affinity for BzN-EJJ-amide. The influence of residue 49 on substrate/inhibitor selectivity was further investigated by comparing subsite amino acid preferences of TCPTP and its R49K derivative by affinity selection coupled with mass spectrometry. The greatest effect on selectivity was observed on the residue that precedes the phosphorylated tyrosine. Unlike wild-type TCPTP, the R49K derivative preferred tyrosine to aspartic or glutamic acid. BzN-EJJ-amide which retains the preferred specificity requirements of TCPTP and PTP1B was equipotent on both enzymes but greater than 30-fold selective over other phosphatases. These results suggest that Arg(49) and Asp(50) may be targeted for the design of potent and selective inhibitors of TCPTP and PTP1B.     

Thermodynamic study of ligand binding to protein-tyrosine phosphatase 1B and its substrate-trapping mutants

The binding of several phosphonodifluoromethyl phenylalanine (F(2)Pmp)-containing peptides to protein-tyrosine phosphatase 1B (PTP1B) and its substrate-trapping mutants (C215S and D181A) has been studied using isothermal titration calorimetry. The binding of a high affinity ligand, Ac-Asp-Ala-Asp-Glu-F(2)Pmp-Leu-NH(2), to PTP1B (K(d) = 0.24 microm) is favored by both enthalpic and entropic contributions. Disruption of ionic interactions between the side chain of Arg-47 and the N-terminal acidic residues reduces the binding affinity primarily through the reduction of the TDeltaS term. The role of Arg-47 may be to maximize surface contact between PTP1B and the peptide, which contributes to high affinity binding. The active site Cys-215 --> Ser mutant PTP1B binds ligands with the same affinity as the wild-type enzyme. However, unlike wild-type PTP1B, peptide binding to C215S is predominantly driven by enthalpy change, which likely results from the elimination of the electrostatic repulsion between the thiolate anion and the phosphonate group. The increased enthalpic contribution is offset by reduction in the binding entropy, which may be the result of increased entropy of the unbound protein caused by this mutation. The general acid-deficient mutant D181A binds the peptide 5-fold tighter than the C215S mutant, consistent with the observation that the Asp to Ala mutant is a better "substrate-trapping" reagent than C215S. The increased binding affinity for D181A as compared with the wild-type PTP1B results primarily from an increase in the DeltaH of binding in the mutant, which may be related to decreased electrostatic repulsion between the phosphate moiety and PTP1B. These results have important implications for the design of high affinity PTP1B inhibitors.     

A broad-spectrum fluorescence-based peptide library for the rapid identification of protease substrates

Identification of peptide substrates for proteases can be a major undertaking. To overcome issues such as feasibility and deconvolution, associated with large peptide libraries, a 'small but smart' generic fluorescence resonance energy transfer rapid endopeptidase profiling library (REPLi) was synthesised as a tool for rapidly identifying protease substrates. Within a tripeptide core, flanked by Gly residues, similar amino acids were paired giving rise to a relatively small library of 3375 peptides divided into 512 distinct pools each containing only 8 peptides. The REPLi was validated with trypsin, pepsin, the matrix metalloprotease (MMP)-12 and MMP-13 and calpains-1 and -2. In the case of calpain-2, a single iteration step involving LC-MS, provided the definitive residue specificity from which a highly sensitive fluorogenic substrate, (FAM)-Gly-Gly-Gly-Gln-Leu-Tyr-Gly-Gly-DPA-Arg-Arg-Lys-(TAMRA), was then designed. The thorough validation of this 'small but smart' peptide library with representatives from each of the four mechanistic protease classes indicates that the REPLi will be useful for the rapid identification of substrates for multiple proteases.     

Determination of peptide substrate specificity for mu-calpain by a peptide library-based approach: the importance of primed side interactions

Calpains are proteases that catalyze the limited cleavage of target proteins in response to Ca(2+) signaling. Because of their involvement in pathological conditions such as post-ischemic injury and Alzheimer and Parkinson disease, calpains form a class of pharmacologically significant targets for inhibition. We have determined the sequence preference for the hydrolysis of peptide substrates of the ubiquitous mu-calpain isoform by a peptide library-based approach using the proteolytic core of mu-calpain (muI-II). The approach, first described by Turk et al. (Turk, B. E., Huang, L. L., Piro, E. T., and Cantley, L. C. (2001) Nat. Biotechnol. 19, 661-667), involved the digestion of an N-terminally acetylated degenerate peptide library in conjunction with Edman sequencing to determine the specificity for residues found at primed positions. The cleavage consensus for these positions was then used to design a second, partially degenerate library, to determine specificity at unprimed positions. We have improved upon the original methodology by using a degenerate peptide dendrimer for determination of specificity at unprimed positions. By using this modified approach, the complete cleavage specificity profile for muI-II was determined for all positions flanking the cleaved peptide. A previously known preference of calpains for hydrophobic amino acids at unprimed positions was confirmed. In addition, a novel residue specificity for primed positions was revealed to highlight the importance of these sites for substrate recognition. The optimal primed site motif (MER) was shown to be capable of directing cleavage to a specific peptide bond. Accordingly, we designed a fluorescent resonance energy transfer-based substrate with optimal cleavage motifs on the primed and non-primed sides (PLFAER). The mu-calpain core shows a far greater turnover rate for our substrate than for those based on the cleavage site of alpha-spectrin or the proteolytic sequence consensus compiled from substrate alignments.