High-resolution crystal structures of a "half sandwich"-type Ru(II) coordination compound bound to hen egg-white lysozyme and proteinase K.

The high-resolution X-ray crystal structures of the adducts formed between the "half sandwich"-type Ru(II) coordination compound [RuII(1,4,7-trithiacyclononane)(ethane-1,2-diamine)Cl]+ and two proteins, namely hen egg-white lysozyme and proteinase K, are presented. The structures unveil that upon reaction with both enzymes the Ru(II) compound is coordinated by solvent-exposed aspartate residues after releasing the chloride ligand (Asp101 in lysozyme, Asp200 and Asp260 in proteinase K), while retaining the two chelating ligands. The adduct with Asp101 residue at the catalytic cleft of lysozyme is accompanied by residue-specific conformational changes to accommodate the Ru(II) fragment, whereas the complexes bound at the two calcium-binding sites of proteinase K revealed minimal structural perturbation of the enzyme. To the best of our knowledge, proteinase K is used here for the first time as a model system of protein metalation and these are the first X-ray crystal structures of protein adducts of a Ru(II) coordination compound that maintains its coordination sphere almost intact upon binding. Our data demonstrate the role of ligands in stabilizing the protein adducts via hydrophobic/aromatic or hydrogen-bonding interactions, as well as their underlying role in the selection of specific sites on the electrostatic potential surface of the enzymes.

Structural and functional analysis of cyclophilin PpiB mutants supports an in vivo function not limited to prolyl isomerization activity.

Escherichia coli cyclophilin PpiB is a peptidyl-prolyl cis/trans isomerase (PPIase, EC: 5.2.1.8), involved in the negative modulation of various bacterial processes, such as swimming and swarming motility and biofilm formation ability. In this study, we show that PpiB possesses also a chaperone function as it can prevent the thermal denaturation of citrate synthase even with essentially eliminated PPIase activity. We demonstrate, using active site mutations, that the PPIase activity of PpiB is required in all processes, except for the negative effect on swimming, indicating a possible isomerase-independent function. Additionally, we show that the reduced PPIase activity of PpiB does not prevent the association with all prey proteins tested and that the PPIase active site is not involved necessarily in each association. We also used a random mutagenesis approach, to identify amino acid residues apart from the catalytic site, which are necessary for PpiB function. The combination of enzymatic studies concerning the PPIase and chaperone activities of each mutant protein, with structural analyses based on 3D models, provided further insights into the effects of the mutations on the function of PpiB and showed the importance of structural features in addition to the catalytic site, for its in vivo role.

Microbial host selection and periplasmic folding in Escherichia coli affect the biochemical characteristics of a cutinase from Fusarium oxysporum.

A cutinase from the mesophilic fungus Fusarium oxysporum (FoCut5a) was functionally expressed in different hosts and their recombinant products were characterized regarding their activity, thermostability and tolerance in organic solvents. The cutinase gene cut5a was expressed in the BL21 and Origami 2 Escherichia coli strains and the resulting protein was folded either in the cytoplasm or in the periplasmic space, with the aim of correct formation of disulfide bonds. Increase of thermostability occurred when the enzyme was expressed in the oxidative cytoplasm of Origami 2. All expression products showed maximum enzyme activity at 40 °C, while thermostability increased by 73% when expressed in the Origami strain compared to the cytoplasmic expression in BL21 cells. The melting temperature of each protein construct was determined by fluorescence spectroscopy showing an additional transition at about 63 °C for enzymes expressed in Origami cells, indicating the co-presence of a different thermostable species. Kinetic studies performed on three p-nitrophenyl synthetic esters of aliphatic acids (C2, C4, C12) indicated that this cutinase shows higher affinity for the hydrolysis of the butyl ester.

Enzymatic synthesis of model substrates recognized by glucuronoyl esterases from Podospora anserina and Myceliophthora thermophila.

Glucuronoyl esterases (GEs) are recently discovered enzymes that are suggested to cleave the ester bond between lignin alcohols and xylan-bound 4-O-methyl-D-glucuronic acid. Although their potential use for enhanced enzymatic biomass degradation and synthesis of valuable chemicals renders them attractive research targets for biotechnological applications, the difficulty to purify natural fractions of lignin-carbohydrate complexes hampers the characterization of fungal GEs. In this work, we report the synthesis of three aryl alkyl or alkenyl D-glucuronate esters using lipase B from Candida antarctica (CALB) and their use to determine the kinetic parameters of two GEs, StGE2 from the thermophilic fungus Myceliophthora thermophila (syn. Sporotrichum thermophile) and PaGE1 from the coprophilous fungus Podospora anserina. PaGE1 was functionally expressed in the methylotrophic yeast Pichia pastoris under the transcriptional control of the alcohol oxidase (AOX1) promoter and purified to its homogeneity (63 kDa). The three D-glucuronate esters contain an aromatic UV-absorbing phenol group that facilitates the quantification of their enzymatic hydrolysis by HPLC. Both enzymes were able to hydrolyze the synthetic esters with a pronounced preference towards the cinnamyl-D-glucuronate ester. The experimental results were corroborated by computational docking of the synthesized substrate analogues. We show that the nature of the alcohol portion of the hydrolyzed ester influences the catalytic efficiency of the two GEs.

Structure of a bacterial cytoplasmic cyclophilin A in complex with a tetrapeptide.

Cyclophilins constitute a class of peptidyl-prolyl isomerases which participate in processes related to protein folding, signalling and chaperoning. The crystal structure of the cytoplasmic cyclophilin A (CyPA) from the bacterium Azotobacter vinelandii complexed with a synthetic tetrapeptide was determined by molecular replacement at 2 resolution. The proline in the tetrapeptide is observed to adopt the cis-isomer conformation. Comparisons of this structure with other CyPA structures provide insights into the conformational variability, effects of peptide binding and structure-function relationships of this enzyme.

Discovery of a novel class of non-ATP site DFG-out state p38 inhibitors utilizing computationally assisted virtual fragment-based drug design (vFBDD).

Discovery of a new class of DFG-out p38α kinase inhibitors with no hinge interaction is described. A computationally assisted, virtual fragment-based drug design (vFBDD) platform was utilized to identify novel non-aromatic fragments which make productive hydrogen bond interactions with Arg 70 on the αC-helix. Molecules incorporating these fragments were found to be potent inhibitors of p38 kinase. X-ray co-crystal structures confirmed the predicted binding modes. A lead compound was identified as a potent (p38α IC(50)=22 nM) and highly selective (≥ 150-fold against 150 kinase panel) DFG-out p38 kinase inhibitor.

Analysis of imatinib and sorafenib binding to p38alpha compared with c-Abl and b-Raf provides structural insights for understanding the selectivity of inhibitors targeting the DFG-out form of protein kinases.

Protein kinases c-Abl, b-Raf, and p38alpha are recognized as important targets for therapeutic intervention. c-Abl and b-Raf are major targets of marketed oncology drugs Imatinib (Gleevec) and Sorafenib (Nexavar), respectively, and BIRB-796 is a p38alpha inhibitor that reached Phase II clinical trials. A shared feature of these drugs is the fact that they bind to the DFG-out forms of their kinase targets. Although the discovery of this class of kinase inhibitors has increased the level of emphasis on the design of DFG-out inhibitors, the structural determinants for their binding and stabilization of the DFG-out conformation remain unclear. To improve our understanding of these determinants, we determined cocrystal structures of Imatinib and Sorafenib with p38alpha. We also conducted a detailed analysis of Imatinib and Sorafenib binding to p38alpha in comparison with BIRB-796, including binding kinetics, binding interactions, the solvent accessible surface area (SASA) of the ligands, and stabilization of key structural elements of the protein upon ligand binding. Our results yield an improved understanding of the structural requirements for stabilizing the DFG-out form and a rationale for understanding the genesis of ligand selectivity among DFG-out inhibitors of protein kinases.

Conformational dynamics of the EGFR kinase domain reveals structural features involved in activation.

The epidermal growth factor receptor (EGFR) has been the focus of intensive studies because of its importance in cancer research. Thus, a broader understanding of the molecular mechanism of activation of the EGFR kinase will have profound significance for the development of novel therapeutics. Numerous crystal structures of EGFR kinase, including the structure of the activating-kinase dimer, have provided snapshots of the specific pathway. Herein, we performed unrestrained-, as well as targeted-molecular dynamics simulations based on these data, to gain further insight into the conformational changes responsible for activation. Comparison of the monomer- versus activating-EGFR-dimer simulations indicates that the dimerization is stabilizing structural elements associated with the activated state and predicts new salt-bridge interactions involving activation-loop residues that may also be associated with that state. Targeted molecular dynamics simulations of the inactive-to-active EGFR transition, as well as the reverse pathway, confirm the formation of conserved structural features of functional importance for the activity or stabilization of either conformation. Interestingly, simulations of the L834R mutant, which is associated with cancer, suggest that the structural basis of the activation induced by that mutation might be the ability of the mutated R834 residue to consecutively form salt bridges with neighboring acidic residues and cause destabilization of a hydrophobic cluster in the inactive state.

Structure-Function Analysis of the Periplasmic Escherichia coli Cyclophilin PpiA in Relation to Biofilm Formation.

The presence of peptidyl-prolyl cis/trans isomerases (PPIases, EC: 5.2.1.8) in all domains of life indicates their biological importance. Cyclophilin PpiA, present in the periplasm of gram-negative bacteria, possesses PPIase activity but its physiological functions are still not clearly defined. Here, we demonstrate that the ΔppiA deletion strain from Escherichia coli exhibits an increased ability for biofilm formation and enhanced swimming motility compared to the wild-type strain. To identify structural features of PpiA which are necessary for the negative modulation of biofilm formation, we constructed a series of mutant PpiA proteins using a combination of error-prone and site-directed mutagenesis approaches. We show that the negative effect of PpiA on biofilm formation is not dependent on its PPIase activity, since PpiA mutants with a reduced PPIase activity are able to complement the ΔppiA strain during biofilm growth.

Crystal structure of the alpha1beta1 integrin I domain in complex with an antibody Fab fragment.

The alpha1beta1 (VLA-1) integrin is a cell-surface receptor for collagen and laminin and has been implicated in biological pathways involved in several pathological processes. These processes may be inhibited by the monoclonal antibody AQC2, which binds with high affinity to human alpha1beta1 integrin. To understand the structural basis of the inhibition we determined the crystal structure of the complex of a chimeric rat/human I domain of the alpha1beta1 integrin and the Fab fragment of humanized AQC2 antibody. The structure of the complex shows that the antibody blocks the collagen binding site of the I domain. An aspartate residue, from the CDR3 loop of the antibody heavy chain, coordinates the MIDAS metal ion in a manner similar to that of a glutamate residue from collagen. Substitution of the aspartate residue by alanine or arginine results in significant reduction of antibody binding affinity. Interestingly, although the mode of metal ion coordination resembles that of the open conformation, the I domain maintains an overall closed conformation previously observed only for unliganded I domains.

Crystal structure of extracellular human BAFF, a TNF family member that stimulates B lymphocytes.

B cell activating factor (BAFF), a ligand belonging to the tumor necrosis factor (TNF) family, plays a critical role in regulating survival and activation of peripheral B cell populations and has been associated with autoimmune disease. BAFF is known to interact with three receptors, BCMA, TACI and BAFF-R, that have distant similarities with other receptors of the TNF family. We have determined the crystal structure of the TNF-homologous domain of BAFF at 2.8 A resolution. The structure reveals significant differences when compared to other TNF family members, including an unusually long D-E loop that participates in the formation of a deep, concave and negatively charged region in the putative receptor binding site. The BAFF structure was further used to generate a homology model of APRIL, a closely related TNF family ligand that also binds to BCMA and TACI, but not BAFF-R. Analysis of the putative receptor binding sites of BAFF and APRIL suggests that differences in the D-E loop structure and electrostatic surface potentials may be important for determining binding specificities for BCMA, TACI and BAFF-R.

Variation in the ordered structure of complexes between CD154 and anti-CD154 monoclonal antibodies.

The cell surface co-stimulatory protein CD154 (CD40L) is a target for monoclonal antibody (mAb) inhibitors of T-cell mediated immune diseases. This protein, like most other members of the TNF ligand family, forms homotrimeric complexes on the cell surface and in solution, with a three-fold axis of symmetry. We find that several different anti-CD154 monoclonal antibodies form distinctive complexes with soluble CD154. These soluble complexes have been analyzed using size exclusion chromatography, static and dynamic light scattering, and electron microscopy and shown to consist of caged structures of various geometries. The cell surface complexes have been analyzed by confocal microscopy and, depending on the mAb, remain as small, separate complexes or form large aggregates. The formation of these complexes in solution is likely to have an impact on measures of affinity, while the cell surface complexes could affect binding potency and provoke other biological effects.

The interaction of the chemotherapeutic drug chlorambucil with human glutathione transferase A1-1: kinetic and structural analysis.

Glutathione transferases (GSTs) are enzymes that contribute to cellular detoxification by catalysing the nucleophilic attack of glutathione (GSH) on the electrophilic centre of a number of xenobiotic compounds, including several chemotherapeutic drugs. In the present work we investigated the interaction of the chemotherapeutic drug chlorambucil (CBL) with human GSTA1-1 (hGSTA1-1) using kinetic analysis, protein crystallography and molecular dynamics. In the presence of GSH, CBL behaves as an efficient substrate for hGSTA1-1. The rate-limiting step of the catalytic reaction between CBL and GSH is viscosity-dependent and kinetic data suggest that product release is rate-limiting. The crystal structure of the hGSTA1-1/CBL-GSH complex was solved at 2.1 Å resolution by molecular replacement. CBL is bound at the H-site attached to the thiol group of GSH, is partially ordered and exposed to the solvent, making specific interactions with the enzyme. Molecular dynamics simulations based on the crystal structure indicated high mobility of the CBL moiety and stabilization of the C-terminal helix due to the presence of the adduct. In the absence of GSH, CBL is shown to be an alkylating irreversible inhibitor for hGSTA1-1. Inactivation of the enzyme by CBL followed a biphasic pseudo-first-order saturation kinetics with approximately 1 mol of CBL per mol of dimeric enzyme being incorporated. Structural analysis suggested that the modifying residue is Cys112 which is located at the entrance of the H-site. The results are indicative of a structural communication between the subunits on the basis of mutually exclusive modification of Cys112, indicating that the two enzyme active sites are presumably coordinated.

Study of the inclusion of the (R)- and (S)-camphor enantiomers in alpha-cyclodextrin by X-ray crystallography and molecular dynamics.

The inclusion of (R)- and (S)-camphor compounds in alpha-cyclodextrin has been studied by X-ray crystallography. The crystal structures of the complexes reveal that one guest molecule is accommodated inside the cavity formed by a head-to-head cyclodextrin dimer. In the crystal lattice, the dimers form layers which are successively shifted by half a dimer. In both (R)- and (S)-cases, the camphor molecule exhibits disorder and occupies three major sites with orientations that can be described as either 'polar' or 'equatorial'. Molecular dynamics simulations performed for the observed complexes indicate that although the carbonyl oxygen of both (R)- and (S)-camphor switches between different hydrogen bonding partners, it maintains the observed mode of 'polar' or 'equatorial' alignment.

Discovery of potent vascular endothelial growth factor receptor-2 inhibitors.

Substantial evidence over the last decades has implicated uncontrolled angiogenesis with various pathological states, including cancer. Vascular endothelial growth factor (VEGF) plays a critical role in its regulation. Because the tyrosine kinase VEGF receptor-2 (VEGFR-2) is the major mediator of the mitogenic, angiogenic, and permeability-enhancing effects of VEGF, it has become one of the most profound anti-angiogenesis targets. Inspired by the anthranilamide class of VEGFR-2 inhibitors, we performed a computational analysis of some potent representative members, using docking and molecular dynamics calculations. Based on the observations drawn from introducing the effect of the receptor's flexibility in implicit aqueous environment, we designed, synthesized, and characterized several new analogues of related scaffolds with modifications in their steric and electronic characteristics. In vitro evaluation of these compounds revealed several novel VEGFR-2 inhibitors that are less cytotoxic and more potent than the parent compounds.

Prednisolone exerts late mitogenic and biphasic effects on resistant acute lymphoblastic leukemia cells: Relation to early gene expression.

Resistance or sensitivity to glucocorticoids is considered to be of crucial importance for disease prognosis in childhood acute lymphoblastic leukemia. Prednisolone exerted a delayed biphasic effect on the resistant CCRF-CEM leukemic cell line, necrotic at low doses and apoptotic at higher doses. At low doses, prednisolone exerted a pre-dominant mitogenic effect despite its induction on total cell death, while at higher doses, prednisolone's mitogenic and cell death effects were counterbalanced. Early gene microarray analysis revealed notable differences in 40 genes. The mitogenic/biphasic effects of prednisolone are of clinical importance in the case of resistant leukemic cells. This approach might lead to the identification of gene candidates for future molecular drug targets in combination therapy with glucocorticoids, along with early markers for glucocorticoid resistance.

Mutagenesis of p38alpha MAP kinase establishes key roles of Phe169 in function and structural dynamics and reveals a novel DFG-OUT state.

In order to study the role of Phe169 in p38alpha MAP kinase structure and function, wild-type p38alpha and five p38alpha DFG motif mutants were examined in vitro for phosphorylation by MKK6, kinase activity toward ATF2 substrate, thermal stability, and X-ray crystal structure. All six p38alpha variants were efficiently phosphorylated by MKK6. However, only one activated p38alpha mutant (F169Y) possessed measurable kinase activity (1% compared to wild-type). The loss of kinase activity among the DFG mutants may result from an inability to correctly position Asp168 in the activated form of p38alpha. Two mutations significantly increased the thermal stability of p38alpha (F169A DeltaTm = 1.3 degrees C and D168G DeltaTm = 3.8 degrees C), and two mutations significantly decreased the stability of p38alpha (F169R DeltaTm = -3.2 degrees C and F169G DeltaTm = -4.7 degrees C). Interestingly, X-ray crystal structures of two thermally destabilized p38alpha-F169R and p38alpha-F169G mutants revealed a DFG-OUT conformation in the absence of an inhibitor molecule. This DFG-OUT conformation, termed alpha-DFG-OUT, is different from the ones previously identified in p38alpha crystal structures with bound inhibitors and postulated from high-temperature molecular dynamics simulations. Taken together, these results indicate that Phe169 is optimized for p38alpha functional activity and structural dynamics, rather than for structural stability. The alpha-DFG-OUT conformation observed for p38alpha-F169R and p38alpha-F169G may represent a naturally occurring intermediate state of p38alpha that provides access for binding of allosteric inhibitors. A model of the local forces driving the DFG IN-OUT transition in p38alpha is proposed.

Two classes of p38alpha MAP kinase inhibitors having a common diphenylether core but exhibiting divergent binding modes.

Two new classes of diphenylether inhibitors of p38alpha MAP kinase are described. Both chemical classes are based on a common diphenylether core that is identified by simulated fragment annealing as one of the most favored chemotypes within a prominent hydrophobic pocket of the p38alpha ATP-binding site. In the fully elaborated molecules, the diphenylether moiety acts as an anchor occupying the deep pocket, while polar extensions make specific interactions with either the adenine binding site or the phosphate binding site of ATP. The synthesis, crystallographic analysis, and biological activity of these p38alpha inhibitors are discussed.

Substitution of Ser for Arg-443 in the regulatory domain of human housekeeping (GLUD1) glutamate dehydrogenase virtually abolishes basal activity and markedly alters the activation of the enzyme by ADP and L-leucine.

Human glutamate dehydrogenase (GDH) exists in GLUD1 (housekeeping) and in GLUD2-specified (brain-specific) isoforms, which differ markedly in their basal activity and allosteric regulation. To determine the structural basis of these functional differences, we mutagenized the GLUD1 GDH at four residues that differ from those of the GLUD2 isoenzyme. Functional analyses revealed that substitution of Ser for Arg-443 (but not substitution of Thr for Ser-331, Leu for Met-370, or Leu for Met-415) virtually abolished basal activity and totally abrogated the activation of the enzyme by l-leucine (1-10 mm) in the absence of other effectors. However, when ADP (0.025-0.1 mm) was present in the reaction mixture, l-leucine (0.3-6.0 mm) activated the mutant enzyme up to >2,000%. The R443S mutant was much less sensitive to ADP (SC(50) = 383.9 +/- 14.6 microm) than the GLUD1 GDH (SC(50) = 31.7 +/- 4.2 microm; p < 0.001); however, at 1 mm ADP the V(max) for the mutant (136.67 micromol min(-1) mg(-1)) was comparable with that of the GLUD1 GDH (152.95 micromol min(-1) mg(-1)). Varying the composition and the pH of the reaction buffer differentially affected the mutant and the wild-type GDH. Arg-443 lies in the "antenna" structure, in a helix that undergoes major conformational changes during catalysis and is involved in intersubunit communication. Its replacement by Ser is sufficient to impair both the catalytic and the allosteric function of human GDH.

Improved expression, purification, and crystallization of p38alpha MAP kinase.

p38alpha mitogen-activated protein (MAP) kinase is widely expressed in many mammalian tissues and is activated as a part of signal transduction cascades that respond to inflammatory stimuli. The activation of p38 is known to trigger various biological effects, including cell death, differentiation, and proliferation. The central role played by p38alpha in cellular signaling events, including those that control a wide range of inflammatory and autoimmune diseases, makes it an attractive drug target. To develop optimized small molecule therapeutics targeting p38alpha, different techniques must be employed for the detailed biochemical, biophysical, and structural characterization of the interactions of p38alpha with lead compounds. These methods typically require large quantities of highly purified p38alpha protein. We describe here an improved expression and purification method for recombinant p38alpha production that reproducibly yields over 70 mg of highly purified protein per liter of shake flask bacterial culture. This yield is significantly higher than that previously reported for p38alpha production in Escherichia coli. We achieved a significant increase in soluble p38alpha protein expression by using the genetically modified E. coli strain BL21 DE3 Rosetta, which is optimized for expression of eukaryotic proteins with codons rarely used in E. coli. The p38alpha protein was purified to near homogeneity using a simple two-step procedure including nickel-chelating Sepharose chromatography followed by anion-exchange chromatography using MonoQ resin. Purified p38alpha was characterized using the standard commercially available small molecule inhibitor SB-203580. The binding association and dissociation rate constants determined by Biacore are in excellent agreement with previously reported values. The purified p38alpha protein was efficiently activated by MKK6 kinase to yield phosphorylated p38alpha. Purified p38alpha protein was also successfully crystallized, producing crystals diffracting to 1.9 angstroms, exceeding the highest resolution for p38alpha reported in the Protein DataBank. The simplicity and efficiency of this approach should prove useful for many laboratories that are interested in production of p38alpha for biochemical and biophysical studies and structure-based drug design.