Role of Position K+4 in the Phosphorylation and Dephosphorylation Reaction Kinetics of the CheY Response Regulator.

Two-component signaling is a primary method by which microorganisms interact with their environments. A kinase detects stimuli and modulates autophosphorylation activity. The signal propagates by phosphotransfer from the kinase to a response regulator, eliciting a response. Response regulators operate over a range of time scales, corresponding to their related biological processes. Response regulator active site chemistry is highly conserved, but certain variable residues can influence phosphorylation kinetics. An Ala-to-Pro substitution (K+4, residue 113) in the Escherichia coli response regulator CheY triggers a constitutively active phenotype; however, the A113P substitution is too far from the active site to directly affect phosphochemistry. To better understand the activating mechanism(s) of the substitution, we analyzed receiver domain sequences to characterize the evolutionary role of the K+4 position. Although most featured Pro, Leu, Ile, and Val residues, chemotaxis-related proteins exhibited atypical Ala, Gly, Asp, and Glu residues at K+4. Structural and in silico analyses revealed that CheY A113P adopted a partially active configuration. Biochemical data showed that A113P shifted CheY toward a more activated state, enhancing autophosphorylation. By characterizing CheY variants, we determined that this functionality was transmitted through a hydrophobic network bounded by the ß5α5 loop and the α1 helix of CheY. This region also interacts with the phosphodonor CheAP1, suggesting that binding generates an activating perturbation similar to the A113P substitution. Atypical residues like Ala at the K+4 position likely serve two purposes. First, restricting autophosphorylation may minimize background noise generated by intracellular phosphodonors such as acetyl phosphate. Second, optimizing interactions with upstream partners may help prime the receiver domain for phosphorylation.

Exploiting the 1-(4-fluorobenzyl)piperazine fragment for the development of novel tyrosinase inhibitors as anti-melanogenic agents: Design, synthesis, structural insights and biological profile.

The development of Tyrosinase inhibitors (TYRIs) could represent an efficacious strategy for pharmacological intervention on skin pathologies related to aberrant production of melanin. Based on in silico studies we designed and tested a library of twenty-four compounds bearing the 4-(4-fluorobenzyl)piperazin-1-yl]-fragment. As result, we identified several compounds with excellent inhibit effects at low micromolar concentration against TYR from Agaricus bisporus (TyM). Among them, compound 25 (IC50 = 0.96 µM) proved to be ∼20-fold more potent than the reference compound kojic acid (IC50 = 17.76 µM) having wide applications in the cosmetics and pharmaceutical industries. The mode of interaction of active inhibitor 25 was deciphered by means of crystallography as well as molecular docking and these results were consistent with kinetic experiments. Moreover, the identified compound 25 exhibited no considerable cytotoxicity and showed anti-melanogenic effects on B16F10 melanoma cells. Therefore, a combination of computational and biochemical approaches could represent a rational guidelines for further structural modification of this class of compounds as future anti-melanogenic agents.

Filling the Void: Introducing Aromatic Interactions into Solvent Tunnels To Enhance Lipase Stability in Methanol.

An enhanced stability of enzymes in organic solvents is desirable under industrial conditions. The potential of lipases as biocatalysts is mainly limited by their denaturation in polar alcohols. In this study, we focused on selected solvent tunnels in lipase from Geobacillus stearothermophilus T6 to improve its stability in methanol during biodiesel synthesis. Using rational mutagenesis, bulky aromatic residues were incorporated to occupy solvent channels and induce aromatic interactions leading to a better inner core packing. The chemical and structural characteristics of each solvent tunnel were systematically analyzed. Selected residues were replaced with Phe, Tyr, or Trp. Overall, 16 mutants were generated and screened in 60% methanol, from which 3 variants showed an enhanced stability up to 81-fold compared with that of the wild type. All stabilizing mutations were found in the longest tunnel detected in the "closed-lid" X-ray structure. The combination of Phe substitutions in an A187F/L360F double mutant resulted in an increase in unfolding temperature (Tm ) of 7°C in methanol and a 3-fold increase in biodiesel synthesis yield from waste chicken oil. A kinetic analysis with p-nitrophenyl laurate revealed that all mutants displayed lower hydrolysis rates (kcat), though their stability properties mostly determined the transesterification capability. Seven crystal structures of different variants were solved, disclosing new π-π or CH/π intramolecular interactions and emphasizing the significance of aromatic interactions for improved solvent stability. This rational approach could be implemented for the stabilization of other enzymes in organic solvents.IMPORTANCE Enzymatic synthesis in organic solvents holds increasing industrial opportunities in many fields; however, one major obstacle is the limited stability of biocatalysts in such a denaturing environment. Aromatic interactions play a major role in protein folding and stability, and we were inspired by this to redesign enzyme voids. The rational protein engineering of solvent tunnels of lipase from Geobacillus stearothermophilus is presented here, offering a promising approach to introduce new aromatic interactions within the enzyme core. We discovered that longer tunnels leading from the surface to the enzyme active site were more beneficial targets for mutagenesis for improving lipase stability in methanol during biodiesel biosynthesis. A structural analysis of the variants confirmed the generation of new interactions involving aromatic residues. This work provides insights into stability-driven enzyme design by targeting the solvent channel void.

Targeting Tyrosinase: Development and Structural Insights of Novel Inhibitors Bearing Arylpiperidine and Arylpiperazine Fragments.

The inhibition of tyrosinase (Ty, EC 1.14.18.1) represents an efficient strategy of decreasing melanogenesis and skin hyperpigmentation. A combination of crystallographic and docking studies on two different tyrosinases, that from Bacillus megaterium (TyBm) and that from a mushroom (TyM), has contributed to increasing our knowledge about their structural information and translating that information to the most druggable human Ty (TyH) isozyme. In particular, we designed and synthesized a series of 1-(4-fluorobenzyl)piperazine and 1-(4-fluorobenzyl)piperidine derivatives showing inhibitory activities on TyM at micromolar ranges and more potency than that of the reference compound, kojic acid. The crystal structures of TyBm with inhibitor 3 (IC50 value of 25.11 µM) and 16 (IC50 value of 5.25 µM) were solved, confirming the binding poses hypothesized by in silico studies and revealing the main molecular determinants for the binding recognition of the inhibitors.

Altering 2-Hydroxybiphenyl 3-Monooxygenase Regioselectivity by Protein Engineering for the Production of a New Antioxidant.

2-Hydroxybiphenyl 3-monooxygenase is a flavin-containing NADH-dependent aromatic hydroxylase that oxidizes a broad range of 2-substituted phenols. In order to modulate its activity and selectivity, several residues in the active site pocket were investigated by saturation mutagenesis. Variant M321A demonstrated altered regioselectivity by oxidizing 3-hydroxybiphenyl for the first time, thus enabling the production of a new antioxidant, 3,4-dihydroxybiphenyl, with similar ferric reducing capacity to the well-studied piceatannol. The crystal structure of M321A was determined (2.78 Å), and molecular docking of the 3-substituted phenol provided a rational explanation for the altered regioselectivity. Furthermore, HbpA was found to possess pro-S enantioselectivity towards the production of several chiral sulfoxides, whereas variant M321F exhibited improved enantioselectivity. Based on the biochemical characterization of several mutants, it was suggested that Trp97 stabilized the substrate in the active site, Met223 was involved in NADH entrance or binding to the active site, and Pro320 might facilitate FAD movement.

Matching biochemical reaction kinetics to the timescales of life: structural determinants that influence the autodephosphorylation rate of response regulator proteins.

In two-component regulatory systems, covalent phosphorylation typically activates the response regulator signaling protein, and hydrolysis of the phosphoryl group reestablishes the inactive state. Despite highly conserved three-dimensional structures and active-site features, the rates of catalytic autodephosphorylation for different response regulators vary by a factor of almost 10(6). Previous studies identified two variable active-site residues, corresponding to Escherichia coli CheY residues 59 and 89, that modulate response regulator autodephosphorylation rates about 100-fold. Here, a set of five CheY mutants, which match other "model" response regulators (ArcA, CusR, DctD, FixJ, PhoB, or Spo0F) at variable active-site positions corresponding to CheY residues 14, 59, and 89, were characterized functionally and structurally in an attempt to identify mechanisms that modulate autodephosphorylation rate. As expected, the autodephosphorylation rates of the CheY mutants were reduced 6- to 40-fold relative to wild-type CheY, but all still autodephosphorylated 12- to 80-fold faster than their respective model response regulators. Comparison of X-ray crystal structures of the five CheY mutants (complexed with the phosphoryl group analogue BeF(3)(-)) to wild-type CheY or corresponding model response regulator structures gave strong evidence for steric obstruction of the phosphoryl group from the attacking water molecule as one mechanism to enhance phosphoryl group stability. Structural data also suggested that impeding the change of a response regulator from the active to the inactive conformation might retard the autodephosphorylation reaction if the two processes are coupled, and that the residue at position '58' may contribute to rate modulation. A given combination of amino acids at positions '14', '59', and '89' adopted similar conformations regardless of protein context (CheY or model response regulator), suggesting that knowledge of residue identity may be sufficient to predict autodephosphorylation rate, and hence the kinetics of the signaling response, in the response regulator family of proteins.

Single MHC mutation eliminates enthalpy associated with T cell receptor binding.

The keystone of the adaptive immune response is T cell receptor (TCR) recognition of peptide presented by major histocompatibility complex (pMHC) molecules. The crystal structure of AHIII TCR bound to MHC, HLA-A2, showed a large interface with an atypical binding orientation. MHC mutations in the interface of the proteins were tested for changes in TCR recognition. From the range of responses observed, three representative HLA-A2 mutants, T163A, W167A, and K66A, were selected for further study. Binding constants and co-crystal structures of the AHIII TCR and the three mutants were determined. K66 in HLA-A2 makes contacts with both peptide and TCR, and has been identified as a critical residue for recognition by numerous TCR. The K66A mutation resulted in the lowest AHIII T cell response and the lowest binding affinity, which suggests that the T cell response may correlate with affinity. Importantly, the K66A mutation does not affect the conformation of the peptide. The change in affinity appears to be due to a loss in hydrogen bonds in the interface as a result of a conformational change in the TCR complementarity-determining region 3 (CDR3) loop. Isothermal titration calorimetry confirmed the loss of hydrogen bonding by a large loss in enthalpy. Our findings are inconsistent with the notion that the CDR1 and CDR2 loops of the TCR are responsible for MHC restriction, while the CDR3 loops interact solely with the peptide. Instead, we present here an MHC mutation that does not change the conformation of the peptide, yet results in an altered conformation of a CDR3.

Structural elements responsible for conversion of streptavidin to a pseudoenzyme.

Avidin enhances the hydrolysis of biotinyl p-nitrophenyl ester (BNP) under mild alkaline conditions, whereas streptavidin prevents hydrolysis of BNP up to pH 12. Recently, we imposed hydrolytic activity on streptavidin by rational mutagenesis, based on the molecular elements responsible for the hydrolysis by avidin. Three mutants were designed, whereby the desired features, the distinctive L124R point mutation (M1), the L3,4 loop replacement (M2), and the combined mutation (M3), were transferred from avidin to streptavidin. The crystal structures of the mutants, in complex with biotinyl p-nitroanilide (BNA), the stable amide analogue of BNP, were determined. The results demonstrate that the point mutation alone has little effect on hydrolysis, and BNA exhibits a conformation similar to that of streptavidin. Substitution of a lengthier L3,4 loop (from avidin to streptavidin), resulted in an open conformation, thus exposing the ligand to solvent. Moreover, the amide bond of BNA was flipped relative to that of the streptavidin and M1 complexes, thus deflecting the nitro group toward Lys-121. Consequently, the leaving group potential of the nitrophenyl group of BNP is increased, and M2 hydrolyzes BNP at pH values >8.5. To better emulate the hydrolytic potential of avidin, M3 was required. The combination of loop replacement and point mutation served to further increase the leaving group potential by interaction of the nitro group with Arg-124 and Lys-121. The information derived from this study may provide insight into the design of enzymes and transfer of desired properties among homologous proteins.

Dimer-tetramer transition between solution and crystalline states of streptavidin and avidin mutants.

The biotin-binding tetrameric proteins, streptavidin from Streptomyces avidinii and chicken egg white avidin, are excellent models for the study of subunit-subunit interactions of a multimeric protein. Efforts are thus being made to prepare mutated forms of streptavidin and avidin, which would form monomers or dimers, in order to examine their effect on quaternary structure and assembly. In the present communication, we compared the crystal structures of binding site W-->K mutations in streptavidin and avidin. In solution, both mutant proteins are known to form dimers, but upon crystallization, both formed tetramers with the same parameters as the native proteins. All of the intersubunit bonds were conserved, except for the hydrophobic interaction between biotin and the tryptophan that was replaced by lysine. In the crystal structure, the binding site of the mutated apo-avidin contains 3 molecules of structured water instead of the 5 contained in the native protein. The lysine side chain extends in a direction opposite that of the native tryptophan, the void being partially filled by an adjacent lysine residue. Nevertheless, the binding-site conformation observed for the mutant tetramer is an artificial consequence of crystal packing that would not be maintained in the solution-phase dimer. It appears that the dimer-tetramer transition may be concentration dependent, and the interaction among subunits obeys the law of mass action.

Structure-based rational design of streptavidin mutants with pseudo-catalytic activity.

Introduction of enzymatic activity into proteins or other types of polymers by rational design is a major objective in the life sciences. To date, relatively low levels of enzymatic activity could be introduced into antibodies by using transition-state analogues of haptens. In the present study, we identify the structural elements that contribute to the observed hydrolytic activity in egg white avidin, which promote the cleavage of active biotin esters (notably biotinyl p-nitrophenyl ester). The latter elements were then incorporated into bacterial streptavidin via genetic engineering. The streptavidin molecule was thus converted from a protector to an enhancer of hydrolysis of biotin esters. The conversion was accomplished by the combined replacement of a "lid-like loop" (L3,4) and a leucine-to-arginine point mutation in streptavidin. Interestingly, neither of these elements play a direct role in the hydrolytic reaction. The latter features were thus shown to be responsible for enhanced substrate hydrolysis. This work indicates that structural and non-catalytic elements of a protein can be modified to promote the induced fit of a substrate for subsequent interaction with either a catalytic residue or water molecules. This approach complements the conventional design of active sites that involves direct modifications of catalytic residues.

Ligand exchange between proteins. Exchange of biotin and biotin derivatives between avidin and streptavidin.

We have studied the structural elements that affect ligand exchange between the two high affinity biotin-binding proteins, egg white avidin and its bacterial analogue, streptavidin. For this purpose, we have developed a simple assay based on the antipodal behavior of the two proteins toward hydrolysis of biotinyl p-nitrophenyl ester (BNP). The assay provided the experimental basis for these studies. It was found that biotin migrates unidirectionally from streptavidin to avidin. Conversely, the biotin derivative, BNP, is transferred in the opposite direction, from avidin to streptavidin. A previous crystallographic study (Huberman, T., Eisenberg-Domovich, Y., Gitlin, G., Kulik, T., Bayer, E. A., Wilchek, M., and Livnah, O. (2001) J. Biol. Chem. 276, 32031-32039) provided insight into a plausible explanation for these results. These data revealed that the non-hydrolyzable BNP analogue, biotinyl p-nitroanilide, was almost completely sheltered in streptavidin as opposed to avidin in which the disordered conformation of a critical loop resulted in the loss of several hydrogen bonds and concomitant exposure of the analogue to the solvent. In order to determine the minimal modification of the biotin molecule required to cause the disordered loop conformation, the structures of avidin and streptavidin were determined with norbiotin, homobiotin, and a common long-chain biotin derivative, biotinyl epsilon-aminocaproic acid. Six new crystal structures of the avidin and streptavidin complexes with the latter biotin analogues and derivatives were thus elucidated. It was found that extending the biotin side chain by a single CH(2) group (i.e. homobiotin) is sufficient to result in this remarkable conformational change in the loop of avidin. These results bear significant biotechnological importance, suggesting that complexes containing biotinylated probes with streptavidin would be more stable than those with avidin. These findings should be heeded when developing new drugs based on lead compounds because it is difficult to predict the structural and conformational consequences on the resultant protein-ligand interactions.