ABSTRACT
The Met receptor tyrosine kinase and its ligand hepatocyte growth factor (HGF) play an important role in mediating both tumor progression and tissue regeneration. The N-terminal and first Kringle domains (NK1) of HGF comprise a naturally occurring splice variant that retains the ability to activate the Met receptor. However, NK1 is a weak agonist and is relatively unstable, limiting its therapeutic potential. Here, we engineered NK1 mutants with improved biochemical and biophysical properties that function as Met receptor agonists or antagonists. We first engineered NK1 for increased stability and recombinant expression yield using directed evolution. The NK1 variants isolated from our library screens acted as weak Met receptor antagonists due to a mutation at the NK1 homodimerization interface. We introduced point mutations that restored this NK1 homodimerization interface to create an agonistic ligand, or that further disrupted this interface to create more effective antagonists. The rationally engineered antagonists exhibited melting temperatures up to approximately 64 °C, a 15 °C improvement over antagonists derived from wild-type NK1, and approximately 40-fold improvement in expression yield. Next, we created disulfide-linked NK1 homodimers through introduction of an N-terminal cysteine residue. These covalent dimers exhibited nearly an order of magnitude improved agonistic activity compared to wild-type NK1, approaching the activity of full-length HGF. Moreover, covalent NK1 dimers formed from agonistic or antagonistic monomeric subunits elicited similar activity, further signifying that NK1 dimerization mediates agonistic activity. These engineered NK1 proteins are promising candidates for therapeutic development and will be useful tools for further exploring determinants of Met receptor activation.
Subject(s)
Hepatocyte Growth Factor/metabolism , Peptide Fragments/metabolism , Protein Engineering/methods , Proto-Oncogene Proteins c-met/agonists , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Amino Acid Sequence , Animals , Cell Line , Dogs , Hepatocyte Growth Factor/chemistry , Humans , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Peptide Fragments/chemistry , Peptide Library , Point Mutation/genetics , Protein Binding , Protein Multimerization , Protein StabilityABSTRACT
Phosphotriesterase (PTE) from soil bacteria is known for its ability to catalyze the detoxification of organophosphate pesticides and chemical warfare agents. Most of the organophosphate chemical warfare agents are a mixture of two stereoisomers at the phosphorus center, and the S(P)-enantiomers are significantly more toxic than the R(P)-enantiomers. In previous investigations, PTE variants were created through the manipulation of the substrate binding pockets and these mutants were shown to have greater catalytic activities for the detoxification of the more toxic S(P)-enantiomers of nerve agent analogues for GB, GD, GF, VX, and VR than the less toxic R(P)-enantiomers. In this investigation, alternate strategies were employed to discover additional PTE variants with significant improvements in catalytic activities relative to that of the wild-type enzyme. Screening and selection techniques were utilized to isolate PTE variants from randomized libraries and site specific modifications. The catalytic activities of these newly identified PTE variants toward the S(P)-enantiomers of chromophoric analogues of GB, GD, GF, VX, and VR have been improved up to 15000-fold relative to that of the wild-type enzyme. The X-ray crystal structures of the best PTE variants were determined. Characterization of these mutants with the authentic G-type nerve agents has confirmed the expected improvements in catalytic activity against the most toxic enantiomers of GB, GD, and GF. The values of k(cat)/K(m) for the H257Y/L303T (YT) mutant for the hydrolysis of GB, GD, and GF were determined to be 2 × 10(6), 5 × 10(5), and 8 × 10(5) M(-1) s(-1), respectively. The YT mutant is the most proficient enzyme reported thus far for the detoxification of G-type nerve agents. These results support a combinatorial strategy of rational design and directed evolution as a powerful tool for the discovery of more efficient enzymes for the detoxification of organophosphate nerve agents.
Subject(s)
Bacterial Proteins/chemistry , Chemical Warfare Agents/chemistry , Organophosphorus Compounds/chemistry , Phosphoric Triester Hydrolases/chemistry , Bacterial Proteins/genetics , Catalysis , Catalytic Domain , Crystallography, X-Ray , Enterobacter aerogenes/enzymology , Gene Library , High-Throughput Screening Assays , Hydrolysis , Models, Molecular , Mutagenesis, Site-Directed , Mutation , Phosphoric Diester Hydrolases/chemistry , Phosphoric Diester Hydrolases/genetics , Phosphoric Triester Hydrolases/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Sarin/chemistry , Soman/chemistry , StereoisomerismABSTRACT
Wild-type phosphotriesterase (PTE) preferentially hydrolyzes the R(P) enantiomers of the nerve agents sarin (GB) and cyclosarin (GF) and their chromophoric analogues. The active site of PTE can be subdivided into three binding pockets that have been denoted as the small, large, and leaving group pockets based on high-resolution crystal structures. The sizes and shapes of these pockets dictate the substrate specificity and stereoselectivity for catalysis. Mutants of PTE that exhibit substantial changes in substrate specificity and the ability to differentiate between chiral substrates have been prepared. For example, the G60A mutant is stereoselective for the hydrolysis of the R(P) enantiomer of the chromophoric analogues of sarin and cyclosarin, whereas the H254G/H257W/L303T (GWT) mutant reverses the stereoselectivity for the enantiomers of these two compounds. Molecular dynamics simulations and high-resolution X-ray structures identified the correlations between structural changes in the active site and the experimentally determined kinetic parameters for substrate hydrolysis. New high-resolution structures were determined for the H257Y/L303T (YT), I106G/F132G/H257Y (GGY), and H254Q/H257F (QF) mutants of PTE. Molecular dynamics calculations were conducted using the S(P) and R(P) enantiomers of the analogues for sarin and cyclosarin for the wild-type PTE and the G60A, YT, GGY, QF, and GWT mutants. The experimental stereoselectivity correlated nicely with the difference in the computed angle of attack for the nucleophilic hydroxide relative to the phenolic leaving group of the substrate.
Subject(s)
Phosphoric Triester Hydrolases/chemistry , Phosphoric Triester Hydrolases/metabolism , Catalysis , Chemical Warfare Agents/chemistry , Chemical Warfare Agents/metabolism , Hydrolysis , Molecular Dynamics Simulation , Organophosphorus Compounds , Sarin/metabolism , Stereoisomerism , Substrate Specificity , X-RaysABSTRACT
Organophosphorus compounds include many synthetic, neurotoxic substances that are commonly used as insecticides. The toxicity of these compounds is due to their ability to inhibit the enzyme acetylcholine esterase. Some of the most toxic organophosphates have been adapted for use as chemical warfare agents; the most well-known are GA, GB, GD, GF, VX, and VR. All of these compounds contain a chiral phosphorus center, with the S(P) enantiomers being significantly more toxic than the R(P) enantiomers. Phosphotriesterase (PTE) is an enzyme capable of detoxifying these agents, but the stereochemical preference of the wild-type enzyme is for the R(P) enantiomers. A series of enantiomerically pure chiral nerve agent analogues containing the relevant phosphoryl centers found in GB, GD, GF, VX, and VR has been developed. Wild-type and mutant forms of PTE have been tested for their ability to hydrolyze this series of compounds. Mutant forms of PTE with significantly enhanced, as well as relaxed or reversed, stereoselectivity have been identified. A number of variants exhibited dramatically improved kinetic constants for the catalytic hydrolysis of the more toxic S(P) enantiomers. Improvements of up to 3 orders of magnitude relative to the value of the wild-type enzyme were observed. Some of these mutants were tested against racemic mixtures of GB and GD. The kinetic constants obtained with the chiral nerve agent analogues accurately predict the improved activity and stereoselectivity against the authentic nerve agents used in this study.
Subject(s)
Organophosphorus Compounds/chemistry , Phosphoric Triester Hydrolases/metabolism , Acetylcholinesterase/chemistry , Acetylcholinesterase/metabolism , Catalysis , Chemical Warfare Agents/chemistry , Hydrolysis , Insecticides/chemistry , Organophosphates/chemistry , Phosphoric Triester Hydrolases/chemistry , StereoisomerismABSTRACT
The bacterial phosphotriesterase (PTE) from Pseudomonas diminuta catalyzes the hydrolysis of organophosphate esters at rates close to the diffusion limit. X-ray diffraction studies have shown that a binuclear metal center is positioned in the active site of PTE and that this complex is responsible for the activation of the nucleophilic water from solvent. In this paper, the three-dimensional structure of PTE was determined in the presence of the hydrolysis product, diethyl phosphate (DEP), and a product analogue, cacodylate. In the structure of the PTE-diethyl phosphate complex, the DEP product is found symmetrically bridging the two divalent cations. The DEP displaces the hydroxide from solvent that normally bridges the two divalent cations in structures determined in the presence or absence of substrate analogues. One of the phosphoryl oxygen atoms in the PTE-DEP complex is 2.0 A from the alpha-metal ion, while the other oxygen is 2.2 A from the beta-metal ion. The two metal ions are separated by a distance of 4.0 A. A similar structure is observed in the presence of cacodylate. Analogous complexes have previously been observed for the product complexes of isoaspartyl dipeptidase, d-aminoacylase, and dihydroorotase from the amidohydrolase superfamily of enzymes. The experimentally determined structure of the PTE-diethyl phosphate product complex is inconsistent with a recent proposal based upon quantum mechanical/molecular mechanical simulations which postulated the formation of an asymmetrical product complex bound exclusively to the beta-metal ion with a metal-metal separation of 5.3 A. This structure is also inconsistent with a chemical mechanism for substrate hydrolysis that utilizes the bridging hydroxide as a base to abstract a proton from a water molecule loosely associated with the alpha-metal ion. Density functional theory (DFT) calculations support a reaction mechanism that utilizes the bridging hydroxide as the direct nucleophile in the hydrolysis of organophosphate esters by PTE.
Subject(s)
Bacterial Proteins/chemistry , Metals/chemistry , Models, Molecular , Organophosphates/chemistry , Phosphoric Triester Hydrolases/chemistry , Pseudomonas/enzymology , Binding Sites/physiology , Hydrolysis , Protein Structure, Tertiary/physiology , Structural Homology, ProteinABSTRACT
Hepatocyte growth factor (HGF), through activation of the c-MET receptor, mediates biological processes critical for tissue regeneration; however, its clinical application is limited by protein instability and poor recombinant expression. We previously engineered an HGF fragment (eNK1) that possesses increased stability and expression yield and developed a c-MET agonist by coupling eNK1 through an introduced cysteine residue. Here, we further characterize this eNK1 dimer and show it elicits significantly greater c-MET activation, cell migration, and proliferation than the eNK1 monomer. The efficacy of the eNK1 dimer was similar to HGF, suggesting its promise as a c-MET agonist.