ABSTRACT
The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high-throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirus-infected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for co-expression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed.
Subject(s)
Eukaryotic Cells/metabolism , Proteomics/methods , Animals , Baculoviridae/genetics , Cells, Cultured , Cloning, Molecular , Gene Expression , Glycosylation , Selenomethionine , Yeasts/metabolismABSTRACT
Protein tyrosine phosphatases PTP-SL and PTPBR7 are isoforms belonging to cytosolic membrane-associated and to receptor-like PTPs (RPTPs), respectively. They represent a new family of PTPs with a major role in activation and translocation of MAP kinases. Specifically, the complex formation between PTP-SL and ERK2 involves an unusual interaction leading to the phosphorylation of PTP-SL by ERK2 at Thr253 and the inactivating dephosphorylation of ERK2 by PTP-SL. This interaction is strictly dependent upon a kinase interaction motif (KIM) (residues 224-239) situated at the N terminus of the PTP-SL catalytic domain. We report the first crystal structure of the catalytic domain for a member of this family (PTP-SL, residues 254-549, identical with residues 361-656 of PTPBR7), providing an example of an RPTP with single cytoplasmic domain, which is monomeric, having an unhindered catalytic site. In addition to the characteristic PTP-core structure, PTP-SL has an N-terminal helix, possibly orienting the KIM motif upon interaction with the target ERK2. An unusual residue in the catalytically important WPD loop promotes formation of a hydrophobically and electrostatically stabilised clamp. This could induce increased rigidity to the WPD loop and therefore reduced catalytic activity, in agreement with our kinetic measurements. A docking model based on the PTP-SL structure suggests that, in the complex with ERK2, the phosphorylation of PTP-SL should be accomplished first. The subsequent dephosphorylation of ERK2 seems to be possible only if a conformational rearrangement of the two interacting partners takes place.
Subject(s)
Catalytic Domain , Mitogen-Activated Protein Kinases/metabolism , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Protein Tyrosine Phosphatases/chemistry , Protein Tyrosine Phosphatases/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Binding Sites , Crystallography, X-Ray , Humans , Intracellular Signaling Peptides and Proteins , Kinetics , Mice , Mitogen-Activated Protein Kinase 1/chemistry , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinases/chemistry , Models, Molecular , Molecular Sequence Data , Phosphorylation , Pliability , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Receptor-Like Protein Tyrosine Phosphatases, Class 7 , Sequence Alignment , Static Electricity , Structure-Activity RelationshipABSTRACT
The receptor-like protein tyrosine phosphatase RPTPmu contains three intracellular domains: the juxtamembrane (JM) and two phosphatase domains (D1 and D2). D1 is catalytically active in vitro. The functional roles of JM and D2 are still unclear. To find out whether and how they modulate the phosphatase activity of D1, we compared the enzymatic characteristics of two constructs, containing a truncated JM and either D1 or both phosphatase domains. p-Nitrophenyl phosphate and two peptide substrates were efficiently dephosphorylated by both constructs. The specificity constant of D1 alone was up to 50% higher. D2 induces (a) decreased K(m) values for peptide substrates, (b) decreased catalytic efficiency for these substrates, (c) shifting of the optimal pH to slightly lower values, and (d) looser binding of competitive inhibitors. These data suggest that the phosphatase activity of D1 is negatively modulated and its ligand binding capacity is sensibly modified by domain D2, having possible functional significance.
Subject(s)
Phosphopeptides/chemistry , Protein Tyrosine Phosphatases/chemistry , Protein Tyrosine Phosphatases/metabolism , Amino Acid Sequence , Catalysis , Catalytic Domain , Cell Membrane/enzymology , Cloning, Molecular , Enzyme Stability , Gastrins/chemistry , Hirudins/chemistry , Humans , Hydrogen-Ion Concentration , Kinetics , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Phosphopeptides/metabolism , Phosphoric Monoester Hydrolases/chemistry , Phosphoric Monoester Hydrolases/metabolism , Phosphotyrosine , Protein Tyrosine Phosphatases/genetics , Receptor-Like Protein Tyrosine Phosphatases, Class 2 , Recombinant Fusion Proteins/chemistry , Restriction Mapping , Substrate SpecificityABSTRACT
The expression of elasticity coefficients for time-dependent enzyme inhibition/activation by an external effector was initially derived. Only a limited number of restrictive assumptions were used, for example, the enzyme was considered to obey Michaelis-Menten kinetics and effectors were taken to be competitive. Then, a simple metabolic system under the control of a time-dependent effector (inhibitor or activator) was analysed and the expressions of the control coefficients were obtained. In addition, two numerical examples were used to represent the control coefficients as functions of time and effector concentration. The results indicate that the control coefficients vary in a relatively limited range of values; however, for certain intervals of time and of effector concentration local minima or major modifications of the coefficients may be recorded. The physiological importance of non-steady state analysis of metabolic systems controlled by external effectors was also discussed. It was stressed that the non-steady state treatment may contribute to creating a more realistic image of the metabolic control processes.
