Structure and function of the phosphotyrosine binding (PTB) domain.
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Amino Acid Sequence↗
Biomedical subjects
M M Zhou
Publications and source records attributed to M M Zhou.
Solution structure of a low molecular weight protein tyrosine phosphatase.
Protein tyrosine phosphatases (PTPs) are important enzymes involved in signal transduction, cell cycle regulation, and the control of differentiation. Despite the importance of this class of enzymes in the control of critical cell processes, very little structural information is available for this family of proteins. In this paper, we present the first solution structure of a protein tyrosine phosphatase. This protein is a low molecular weight cytosolic PTP that was initially isolated from bovine heart. The structure that was determined from 1747 NMR-derived restraints consists of a central four-stranded parallel beta-sheet surrounded by four alpha-helices and a short 3(10) helix. The phosphate binding site, identified by chemical shift changes upon the addition of the competitive inhibitors phosphate and vanadate, is in a loop region connecting the C-terminal end of the first beta-strand with the first alpha-helix. Residues in the second, fourth, and fifth alpha-helices and in some of the loop regions connecting the elements of regular secondary structure also contribute to the binding site. The structure determined here is consistent with previous mutagenesis and chemical modification studies conducted on this protein.
Animals↗
Backbone 1H, 13C, and 15N assignments and secondary structure of bovine low molecular weight phosphotyrosyl protein phosphatase.
Phosphotyrosyl protein phosphatases play an important role in mediating cellular signal transduction; yet three-dimensional structures of this important class of proteins have not been reported. We present the sequence-specific 1H, 13C, and 15N backbone assignments for the low molecular weight bovine heart phosphotyrosyl protein phosphatase (BHPTPase) (157 residues, 17,900). The assignments were obtained from a combination of double- and triple-resonance multidimensional NMR experiments. From these assignments, the secondary structure of BHPTPase was determined from an analysis of NOE patterns, 3JHNH alpha coupling constants, 13C alpha and 13CO chemical shifts, and amide 1H exchange rates. BHPTPase was found to consist of a four-stranded parallel beta-sheet (residues K6-C12, W39-A45, Y87-M91, and K112-L116), four alpha-helices (residues I21-D32, R58-G67, S94-N104, and D135-R157), and one stretch of beta 10-helix (residues K79-F85). The secondary structure is characteristic of the beta alpha beta structural motif. The secondary structure elements identified in this study are consistent with previous chemical and mutagenesis studies of BHPTPase structure.
Amino Acid Sequence↗
Kinetic and site-directed mutagenesis studies of the cysteine residues of bovine low molecular weight phosphotyrosyl protein phosphatase.
The roles of the 8 conserved cysteines and 1 arginine in the low molecular weight phosphotyrosyl protein phosphatases were investigated using site-directed mutagenesis of the recombinant bovine heart enzyme. Single mutants of cysteine to serine were studied for each cysteine; alanine replacements were also made for Cys-12, Cys-17, and Arg-18. The CD spectra of the purified proteins were effectively superimposable, consistent with the conclusion that no major structural alterations had occurred, but 1H NMR spectroscopy did reveal some spectral shifts in the aromatic region. Kinetic analysis of the mutant proteins demonstrated that only Cys-12, Cys-17, and Arg-18 had significantly altered catalytic activity toward the substrate p-nitrophenyl phosphate at pH 5. The Cys-12 and Arg-18 mutants were effectively inactive. Thus, it is concluded that Cys-12 is the catalytic nucleophile, and Arg-18 presumably serves an essential function in substrate binding. The C17S mutant had 6% residual activity compared with wild type protein, whereas the C17A mutant had 37% activity. Consistent with the observed activity of the Cys-17 mutant, a covalent phosphocysteine intermediate was trapped and identified by 31P NMR. Further kinetic analysis of C17A using several aryl phosphate monoester substrates with different leaving group pK alpha values indicated that no change in the rate-determining step of the catalytic mechanism had occurred, that is, dephosphorylation of the covalent phosphoenzyme intermediate remains rate-limiting. The C17A mutant had a 4-fold higher phosphate Ki and slightly higher Km values for p-nitrophenyl phosphate suggesting that Cys-17 may be important for optimal positioning of the substrate phosphate moiety.
Amino Acid Sequence↗
Spectroscopic and kinetic studies of the histidine residues of bovine low-molecular-weight phosphotyrosyl protein phosphatase.
The role of the two conserved histidine residues in the low-molecular-weight phosphotyrosyl protein phosphatases was investigated using site-directed mutagenesis of the recombinant bovine heart enzyme. His-66 and His-72 were individually mutated to alanine and to asparagine. A double mutant, containing only alanines in place of the histidines, was also created. The 1H NMR spectra of the purified proteins revealed no apparent tertiary structure alterations. Microscopic pKas for the two histidines were determined from a pH titration of the wild-type enzyme using 1H NMR spectroscopy and an MLEV-17 spectral editing scheme to more readily follow shifts in the specific histidine resonance peaks. His-66 titrates with an apparent pKa of 8.4 while for His-72 the value is 9.2. Since earlier chemical modification experiments indicated that the wild-type enzyme was inactivated by the histidine-selective modification reagent diethyl pyrocarbonate (DEP), the histidine mutants were tested for sensitivity to DEP. Both of the histidine single mutants were inactivated by DEP, and surprisingly, the double mutant containing no histidines was also readily inactivated by DEP. Thus, for this protein, modification by DEP is not specific for histidine residues. Kinetic studies of the mutant proteins reveal that neither histidine is essential in the catalytic mechanism. His-66 mutants showed virtually identical catalytic properties compared to wild-type enzyme, whereas His-72 mutants had reduced specific activity and higher phosphate Ki and lower Km values at pH5 and higher. It is proposed that His-72, although not essential for catalysis, may serve a significant structural role at the enzyme active site.
Animals↗
Identification and pKa determination of the histidine residues of human low-molecular-weight phosphotyrosyl protein phosphatases: a convenient approach using an MLEV-17 spectral editing scheme.
A useful approach using an MLEV-17 pulse sequence was developed to identify histidine C epsilon 1H magnetic resonances of proteins. This technique can be readily applied to proteins dissolved directly in deuterium oxide solution and eliminates the necessity for an exhaustive exchange of NH to ND. Because of its sensitivity, this technique makes it possible to significantly extend the limitations on protein size. The utility of this spin-lock sequence is demonstrated using ribonuclease, subtilisin, and human prostatic acid phosphatase, with molecular weights ranging from 12K to 100K. With this technique, all three or four of the histidine 1H NMR signals of two human low-molecular-weight phosphotyrosyl protein phosphatases (HCPTP-A or -B, respectively) were readily detected. Histidine peak assignments were accomplished through the use of histidine to alanine mutants of HCPTP-A and -B and a homologous bovine enzyme. Analysis of the pH titration curves of these signals provided microscopic pKa's for the histidines in the human enzymes. A comparison of corresponding histidine pKa values of the two isoenzymes, together with an examination of the 1H NMR spectra of the proteins, provided evidence of significant differences in secondary structure. Titration of HCPTP-A and -B with vanadate, a strongly bound competitive inhibitor, caused the His-72 peak to appear as two signals at nearly equimolar concentrations of protein and vanadate, while the other histidine peaks were not affected. This is interpreted to mean that His-72 is at the enzyme active site.
Amino Acid Sequence↗
Overexpression, site-directed mutagenesis, and mechanism of Escherichia coli acid phosphatase.
Site-directed mutagenesis was used to examine the catalytic importance of 2 histidine and 4 arginine residues in Escherichia coli periplasmic acid phosphatase (EcAP). The residues that were selected as targets for mutagenesis were those that were also conserved in a number of high molecular weight acid phosphatases from eukaryotic organisms, including human prostatic and lysosomal acid phosphatases. Both wild type EcAP and mutant proteins were overproduced in E. coli using an expression system based on the T7 RNA polymerase promoter, and the proteins were purified to homogeneity. Examination of the purified mutant proteins by circular dichroism and proton NMR spectroscopy revealed no significant conformational changes. The replacement of Arg16 and His17 residues that were localized in a conserved N-terminal RHGXRXP motif resulted in the complete elimination of EcAP enzymatic activity. Critical roles for Arg20, Arg92, and His303 were also established because the corresponding mutant proteins exhibited residual activities that were not higher than 0.4% of that of wild type enzyme. In contrast, the replacement of Arg63 did not cause a significant alteration of the kinetic parameters. The results are in agreement with a previously postulated distant relationship between acid phosphatases, phosphoglycerate mutases, and fructose-2,6-bisphosphatase. These and earlier results are also consistent with the conclusion that 2 histidine residues participate in the catalytic mechanism of acid phosphatases, with His17 playing the role of a nucleophilic acceptor of the phospho group, whereas His303 may act as a proton donor to the alcohol or phenol.
Acid Phosphatase↗
Cloning, expression, and catalytic mechanism of the low molecular weight phosphotyrosyl protein phosphatase from bovine heart.
The first representative of a group of mammalian, low molecular weight phosphotyrosyl protein phosphatases was cloned, sequenced and expressed in Escherichia coli. Using a 61-mer oligonucleotide probe based on the amino acid sequence of the purified enzyme, several overlapping cDNA clones were isolated from a bovine heart cDNA library. A full-length clone was obtained consisting of a 27-bp 5' noncoding region, an open reading frame encoding the expected 157 amino acid protein, and an extensive 3' nontranslated sequence. The identification of the clone as full length was consistent with results obtained in mRNA blotting experiments using poly(A)+ mRNA from bovine heart. The coding sequence was placed downstream of a bacteriophage T7 promoter, and protein was expressed in E. coli. The expressed enzyme was soluble, and catalytically active and was readily isolated and purified. The recombinant protein had the expected Mr of 18,000 (estimated by SDS-PAGE), and it showed cross-reactivity with antisera that had been raised against both the bovine heart and the human placenta enzymes. The amino acid sequence of the N-terminal region of the expressed protein showed that methionine had been removed, resulting in a sequence identical to that of the enzyme isolated from the bovine tissue, with the exception that the N-terminal alanine of the protein from tissue is acetylated. A kinetically competent phosphoenzyme intermediate was trapped from a phosphatase-catalyzed reaction. Using 31P NMR, the covalent intermediate was identified as a cysteinyl phosphate. By analogy with the nomenclature used for serine esterases, these enzymes may be called cysteine phosphatases.
Amino Acid Sequence↗