Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies of a 12R-LOX-chaperone complex.

Lipoxygenases are a family of nonheme iron-containing dioxygenases. An Escherichia coli expression system producing the bacterial chaperones GroES and GroEL was engineered and successfully used to produce large quantities of recombinant human 12R-LOX (LOXR; MW 80.34 kDa; 701 amino-acid residues). The co-overproduction of the two chaperones with 12R-LOX resulted in increased solubility of 12R-LOX and allowed the purification of milligram amounts of active enzyme for structural studies by X-ray diffraction. The lipoxygenase protein was purified on an affinity column and a gel-filtration column with chaperone protein (MW 57.16 kDa). The LOXR-chaperone complex was crystallized with ligand by the hanging-drop vapor-diffusion method using 1.5 M ammonium hydrogen phosphate as precipitant. The crystals belonged to the monoclinic system, space group P2(1), with unit-cell parameters a = 138.97, b = 266.11, c = 152.26 Å, ß = 101.07°. Based on the calculated Matthews coefficient (3.1 Å(3) Da(-1)), it is estimated that one molecule of LOXR complexed with two molecules of chaperone is present in the asymmetric unit of the crystal lattice. X-ray diffraction data were collected to 4 Šresolution using synchrotron radiation.

Chemotactic activity of S100A7 (Psoriasin) is mediated by the receptor for advanced glycation end products and potentiates inflammation with highly homologous but functionally distinct S100A15.

Human S100A7 (psoriasin) is overexpressed in inflammatory diseases. The recently discovered, co-evolved hS100A15 is almost identical in sequence and up-regulated with hS100A7 during cutaneous inflammation. The functional role of these closely related proteins for inflammation remains undefined. By generating specific Abs, we demonstrate that hS100A7 and hS100A15 proteins are differentially expressed by specific cell types in the skin. Although highly homologous, both proteins are chemoattractants with distinct chemotactic activity for leukocyte subsets. We define RAGE (receptor for advanced glycation end products) as the hS100A7 receptor, whereas hS100A15 functions through a Gi protein-coupled receptor. hS100A7-RAGE binding, signaling, and chemotaxis are zinc-dependent in vitro, reflecting the previously reported zinc-mediated changes in the hS100A7 dimer structure. When combined, hS100A7 and hS100A15 potentiate inflammation in vivo. Thus, proinflammatory synergism in disease may be driven by the diverse biology of these almost identical proteins that have just recently evolved. The identified S100A7 interaction with RAGE may provide a novel therapeutic target for inflammation.

A BAR domain in the N terminus of the Arf GAP ASAP1 affects membrane structure and trafficking of epidermal growth factor receptor.

BACKGROUND: Arf GAPs are multidomain proteins that function in membrane traffic by inactivating the GTP binding protein Arf1. Numerous Arf GAPs contain a BAR domain, a protein structural element that contributes to membrane traffic by either inducing or sensing membrane curvature. We have examined the role of a putative BAR domain in the function of the Arf GAP ASAP1. RESULTS: ASAP1's N terminus, containing the putative BAR domain together with a PH domain, dimerized to form an extended structure that bound to large unilamellar vesicles containing acidic phospholipids, properties that define a BAR domain. A recombinant protein containing the BAR domain of ASAP1, together with the PH and Arf GAP domains, efficiently bent the surface of large unilamellar vesicles, resulting in the formation of tubular structures. This activity was regulated by Arf1*GTP binding to the Arf GAP domain. In vivo, the tubular structures induced by ASAP1 mutants contained epidermal growth factor receptor (EGFR) and Rab11, and ASAP1 colocalized in tubular structures with EGFR during recycling of receptor. Expression of ASAP1 accelerated EGFR trafficking and slowed cell spreading. An ASAP1 mutant lacking the BAR domain had no effect. CONCLUSIONS: The N-terminal BAR domain of ASAP1 mediates membrane bending and is necessary for ASAP1 function. The Arf dependence of the bending activity is consistent with ASAP1 functioning as an Arf effector.

Mutational analysis of the Arf1*GTP/Arf GAP interface reveals an Arf1 mutant that selectively affects the Arf GAP ASAP1.

Arf1 is a GTP binding protein that functions at a number of cellular sites to control membrane traffic and actin remodeling. Arf1 is regulated by site-specific GTPase-activating proteins (GAPs). The combined results of crystallographic and biochemical studies have led to the proposal that Arf1 GAPs differ in the specific interface formed with Arf1. To test this hypothesis, we have used mutagenesis to examine the interaction of three Arf GAPs (ASAP1, AGAP1, and ArfGAP1) with switch 1, switch 2, and alpha helix3 of Arf1. The GAPs were similar in being affected by mutations in switch 1 and 2. However, effects of a mutation within alpha helix3 and specific mutations within switch 1 and 2 differed among the GAPs. The largest differences were observed with a change of isoleucine 46 to aspartate ([I46D]Arf1), which reduced ASAP1-induced catalysis by approximately 10,000-fold but had a 3-fold effect on AGAP1. The reduction was due to an isolated effect on the catalytic rate, k(cat). In vivo [I46D]Arf1 had no detectable effect on the Golgi apparatus but, instead, functioned as a constitutively active mutant in the cell periphery, affecting the localization of ASAP1 and paxillin. Based on our results, we conclude that the contribution of specific residues within switch 1 of Arf to binding and achieving a transition state toward GTP hydrolysis differs among Arf GAPs.

Kinetic analysis of GTP hydrolysis catalysed by the Arf1-GTP-ASAP1 complex.

Arf (ADP-ribosylation factor) GAPs (GTPase-activating proteins) are enzymes that catalyse the hydrolysis of GTP bound to the small GTP-binding protein Arf. They have also been proposed to function as Arf effectors and oncogenes. We have set out to characterize the kinetics of the GAP-induced GTP hydrolysis using a truncated form of ASAP1 [Arf GAP with SH3 (Src homology 3) domain, ankyrin repeats and PH (pleckstrin homology) domains 1] as a model. We found that ASAP1 used Arf1-GTP as a substrate with a k(cat) of 57+/-5 s(-1) and a K(m) of 2.2+/-0.5 microM determined by steady-state kinetics and a kcat of 56+/-7 s(-1) determined by single-turnover kinetics. Tetrafluoroaluminate (AlF4-), which stabilizes complexes of other Ras family members with their cognate GAPs, also stabilized a complex of Arf1-GDP with ASAP1. As anticipated, mutation of Arg-497 to a lysine residue affected kcat to a much greater extent than K(m). Changing Trp-479, Iso-490, Arg-505, Leu-511 or Asp-512 was predicted, based on previous studies, to affect affinity for Arf1-GTP. Instead, these mutations primarily affected the k(cat). Mutants that lacked activity in vitro similarly lacked activity in an in vivo assay of ASAP1 function, the inhibition of dorsal ruffle formation. Our results support the conclusion that the Arf GAP ASAP1 functions in binary complex with Arf1-GTP to induce a transition state towards GTP hydrolysis. The results have led us to speculate that Arf1-GTP-ASAP1 undergoes a significant conformational change when transitioning from the ground to catalytically active state. The ramifications for the putative effector function of ASAP1 are discussed.

(m-Phenyl-enedimethyl-ene)diammonium p-nitro-phenyl-phosphate perchlorate.

The title compound, C(8)H(14)N(2) (2+)·C(12)H(8)N(2)O(8)P(-)·ClO(4) (-), was formed by the reaction of α,α-bis-m-xylenediamine and sodium bis-p-nitro-phenyl-phosphate in the presence of Zn(ClO(4))·6H(2)O in methanol solution. The two amine groups of the m-xylenediammonium ion are each protonated and each hydrogen-bonded to two O atoms of the phosphate anion, which acts as a 1,3-bridge. The ammonium groups are arranged matched face to face and each pair is doubly bridged by two perchlorate ions through hydrogen bonding. In addition, there are also weak C-H⋯O inter-actions. Both the N-H⋯O and C-H⋯O inter-actions are contained in a channel down the a axis. The perchlorate oxygen atoms are disordered over two positions with site occupancy factors of ca 0.7 and 0.3.

Purification, crystallization and preliminary X-ray diffraction of human S100A15.

Human S100A15 is a novel member of the S100 family of EF-hand calcium-binding proteins and was recently identified in psoriasis, where it is significantly upregulated in lesional skin. The protein is implicated as an effector in calcium-mediated signal transduction pathways. Although its biological function is unclear, the association of the 11.2 kDa S100A15 with psoriasis suggests that it contributes to the pathogenesis of the disease and could provide a molecular target for therapy. To provide insight into the function of S100A15, the protein was crystallized to visualize its structure and to further the understanding of how the many similar calcium-binding mediator proteins in the cell distinguish their cognate target molecules. The S100A15 protein has been cloned, expressed and purified to homogeneity and produced two crystal forms. Crystals of form I are triclinic, with unit-cell parameters a = 33.5, b = 44.3, c = 44.8 angstroms, alpha = 71.2, beta = 68.1, gamma = 67.8 degrees and an estimated two molecules in the asymmetric unit, and diffract to 1.7 angstroms resolution. Crystals of form II are monoclinic, with unit-cell parameters a = 82.1, b = 33.6, c = 52.2 angstroms, beta = 128.2 degrees and an estimated one molecule in the asymmetric unit, and diffract to 2.0 angstroms resolution. This structural analysis of the human S100A15 will further aid in the phylogenic comparison between the other members of the S100 protein family, especially the highly homologous paralog S100A7.

Self-healing collodion baby: a dynamic phenotype explained by a particular transglutaminase-1 mutation.

Spontaneous healing with no or only very mild ichthyosis distinguishes the "self-healing collodion baby" from other congenital ichthyoses. In two self-healing collodion baby siblings with markedly diminished epidermal transglutaminase 1 activity we found the compound heterozygous transglutaminase 1 mutations G278R and D490G. Molecular modeling and biochemical assays of mutant proteins under elevated hydrostatic pressure suggest significantly reduced activity in G278R and a chelation of water molecules in D490G that locks the mutated enzyme in an inactive trans conformation in utero. After birth these water molecules are removed and the enzyme is predicted to isomerize back to a partially active cis form, explaining the dramatic improvement of this skin condition.

Cell-cell adhesion and RhoA-mediated actin polymerization are independent phenomena in microtubule disrupted keratinocytes.

E-cadherin-mediated adherens junction formation and maintenance are thought to involve actin filament rearrangements through the action of small GTPases. Recently, we demonstrated that microtubule disruption in normal human epidermal keratinocytes grown in low calcium media conditions induces cell-cell adhesion by redistribution of endogenous E-cadherin, and it promotes stress fiber formation. This actin rearrangement was apparently mediated by RhoA activation. This model system therefore provides a tool with which to dissect relationships between cell-cell adhesion and Rho-mediated stress fiber formation. In this study, we have demonstrated in normal human epidermal keratinocytes that disruption of actin structures including stress fibers does not interfere with E-cadherin redistribution during microtubule-induced cell-cell adhesion. Moreover, this cell-cell adhesion could not be blocked by RhoA inactivation at the level for inhibition of stress fiber formation. Additionally, in the immortalized HaCaT keratinocyte cell line, which does not undergo cell-cell adhesion after microtubule disruption in low calcium conditions, expression of dominant-active RhoA could induce stress fiber formation without inducing adhesion. On the other hand, a variant of the HaCaT cell line, HC-R1, showed microtubule-disruption-induced cell-cell adhesion without stress fiber formation. Together, our results suggest that, in keratinocytes, the process of cell adhesion can occur independently of RhoA-mediated stress fiber formation.

A model for the reaction mechanism of the transglutaminase 3 enzyme.

Transglutaminase enzymes (TGases) catalyze the calcium dependent formation of an isopeptide bond between protein-bound glutamine and lysine substrates. Previously we have shown that activated TGase 3 acquires two additional calcium ions at site two and three. The calcium ion at site three results in the opening of a channel. At this site, the channel opening and closing could modulate, depending on which metal is bound. Here we propose that the front of the channel could be used by the two substrates for enzyme reaction. We propose that the glutamine substrate is directed from Trp236 into the enzyme, shown by molecular docking. Then a lysine substrate approaches the opened active site to engage Trp327, leading to formation of the isopeptide bond. Further, direct comparisons of the structures of TGase 3 with other TGases have allowed us to identify several residues that might potentially be involved in generic and specific recognition of the glutamine and lysine substrates.

Inactive and highly active, proteolytically processed transglutaminase-5 in epithelial cells.

Transglutaminases (TGs) are Ca(2+)-dependent enzymes capable of catalyzing transamidation of glutamine residues to form intermolecular isopeptide bonds. These enzymes are involved in various biological phenomena, including blood coagulation, wound healing, cell death, tissue repair, and terminal differentiation of keratinocytes. Among the TG-family members, TG5 is one of the latest identified enzymes and therefore the less characterized at the functional level. In this work, we reported that TG5 is proteolytically processed in the baculovirus expression system and in mammal epithelial cells. Similar to other members of the TG family-TG1, TG3, and factor XIIIa -, TG5 full-length enzyme has very low enzymatic activity, while the 53-kDa proteolytically processed form is highly active.

A three-dimensional model of the human transglutaminase 1: insights into the understanding of lamellar ichthyosis.

The stratum corneum, the outer layer of the epidermis, serves as a protective barrier to isolate the skin from the external environment. Keratinocyte transglutaminase 1 (TGase 1) catalyzes amide crosslinking between glutamine and lysine residues on precursor proteins forming the impermeable layers of the epidermal cell envelopes (CE), the highly insoluble membranous structures of the stratum corneum. Patients with the autosomal recessive skin disorder lamellar ichthyosis (LI) appear to have deficient cross-linking of the cell envelope due to mutations identified in TGase 1, linking this enzyme to LI. In the absence of a crystal structure, molecular modeling was used to generate the structure of TGase 1. We have mapped the known mutations of TGase 1 from our survey obtained from a search of PubMed and successfully predicted the impact of these mutations on LI. Furthermore, we have identified Ca(2+) binding sites and propose that Ca(2+) induces a cis to trans isomerization in residues near the active site as part of the enzyme transamidation activation. Docking experiments suggest that substrate binding subsequently induces the reverse cis to trans isomerization, which may be a significant part of the catalytic process. These results give an interpretation at the molecular level of previously reported mutations and lead to further insights into the structural model of TGase 1, providing a new basis for understanding LI.

Purification, crystallization and preliminary X-ray diffraction analysis of the phage T4 vertex protein gp24 and its mutant forms.

The study of bacteriophage T4 assembly has revealed regulatory mechanisms pertinent not only to viruses but also to macromolecular complexes. The capsid of bacteriophage T4 is composed of the major capsid protein gp23, and a minor capsid protein gp24, which is arranged as pentamers at the vertices of the capsid. In this study the T4 capsid protein gp24 and its mutant forms were overexpressed and purified to homogeneity. The overexpression from plasmid vectors of all the constructs in Escherichia coli yields biologically active protein in vivo as determined by assembly of active virus following infection with inactivated gene 24 mutant viruses. The gp24 mutant was subjected to surface entropy reduction by mutagenesis and reductive alkylation in order to improve its crystallization properties and diffraction quality. To determine if surface mutagenesis targeting would result in diffractable crystals, two glutamate to alanine mutations (E89A,E90A) were introduced. We report here the biochemical observations and consequent mutagenesis experiment that resulted in improvements in the stability, crystallizability and crystal quality of gp24 without affecting the overall folding. Rational modification of the protein surface to achieve crystallization appears promising for improving crystallization behavior and crystal diffracting qualities. The crystal of gp24(E89A,E90A) diffracted to 2.6A resolution compared to wild-type gp24 at 3.80A resolution under the same experimental conditions. Surface mutation proved to be a better method than reductive methylation for improving diffraction quality of the gp24 crystals.

ARAP2 effects on the actin cytoskeleton are dependent on Arf6-specific GTPase-activating-protein activity and binding to RhoA-GTP.

ARAP2 is a protein that contains both ArfGAP and RhoGAP domains. We found that it is a phosphatidylinositol (3,4,5)-trisphosphate-dependent Arf6 GAP that binds RhoA-GTP but lacks RhoGAP activity. In agreement with the hypothesis that ARAP2 mediates effects of RhoA, endogenous ARAP2 associated with focal adhesions (FAs) and reduction of ARAP2 expression, by RNAi, resulted in fewer FAs and actin stress fibers (SFs). In cells with reduced levels of endogenous ARAP2, FAs and SFs could be restored with wild-type recombinant ARAP2 but not mutants lacking ArfGAP or Rho-binding activity. Constitutively active Arf6 also caused a loss of SFs. The Rho effector ROKalpha was ineffective in restoring FAs. Conversely, overexpression of ARAP2 did not restore SFs in cells treated with a ROK inhibitor but induced punctate accumulations of paxillin. We conclude that ARAP2 is an Arf6GAP that functions downstream of RhoA to regulate focal adhesion dynamics.

Bathing suit ichthyosis is caused by transglutaminase-1 deficiency: evidence for a temperature-sensitive phenotype.

Bathing suit ichthyosis (BSI) is a striking and unique clinical form of autosomal recessive congenital ichthyosis characterized by pronounced scaling on the bathing suit areas but sparing of the extremities and the central face. Here we report on a series of 10 BSI patients. Our genetic, ultrastructural and biochemical investigations show that BSI is caused by transglutaminase-1 (TGase-1) deficiency. Altogether, we identified 13 mutations in TGM1-among them seven novel missense mutations and one novel nonsense mutation. Structural modeling for the Tyr276Asn mutation reveals that the residue is buried in the hydrophobic interior of the enzyme and that the hydroxyl side chain of Tyr276 is exposed to solvent in a cavity of the enzyme. Cryosections of healthy skin areas demonstrated an almost normal TGase activity, in contrast to the affected BSI skin, which only showed a cytoplasmic and clearly reduced TGase-1 activity. The distribution of TGase-1 substrates in the epidermis of affected skin corresponded to the situation in TGase-1 deficiency. Interestingly, the expression of TGase-3 and cathepsin D was reduced. Digital thermography validated a striking correlation between warmer body areas and presence of scaling in patients suggesting a decisive influence of the skin temperature. In situ TGase testing in skin of BSI patients demonstrated a marked decrease of enzyme activity when the temperature was increased from 25 to 37 degrees C. We conclude that BSI is caused by TGase-1 deficiency and suggest that it is a temperature-sensitive phenotype.

Structural and functional similarities between the capsid proteins of bacteriophages T4 and HK97 point to a common ancestry.

Gene product (gp) 24 of bacteriophage T4 forms the pentameric vertices of the capsid. Using x-ray crystallography, we found the principal domain of gp24 to have a polypeptide fold similar to that of the HK97 phage capsid protein plus an additional insertion domain. Fitting gp24 monomers into a cryo-EM density map of the mature T4 capsid suggests that the insertion domain interacts with a neighboring subunit, effecting a stabilization analogous to the covalent crosslinking in the HK97 capsid. Sequence alignment and genetic data show that the folds of gp24 and the hexamer-forming capsid protein, gp23*, are similar. Accordingly, models of gp24* pentamers, gp23* hexamers, and the whole capsid were built, based on a cryo-EM image reconstruction of the capsid. Mutations in gene 23 that affect capsid shape map to the capsomer's periphery, whereas mutations that allow gp23 to substitute for gp24 at the vertices modify the interactions between monomers within capsomers. Structural data show that capsid proteins of most tailed phages, and some eukaryotic viruses, may have evolved from a common ancestor.

Crystal structure of transglutaminase 3 in complex with GMP: structural basis for nucleotide specificity.

Epidermal-type Transglutaminase 3 (TGase 3) is a Ca(2+)-dependent enzyme involved in the cross-linking of structural proteins required in the assembly of the cell envelope. We have recently shown that calcium-activated TGase 3, like TGase 2, can bind, hydrolyze, and is inhibited by GTP despite lacking structural homology with other GTP-binding proteins. Here we report the crystal structure determined at 2.0 A resolution of TGase 3 in complex with GMP to elucidate the structural features required for nucleotide recognition. Binding affinities for various nucleotides were found by fluorescence displacement to be as follows: guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) (0.4 microm), GTP (0.6 microm), GDP (1.0 microm), GMP (0.4 microm), and ATP (28.0 microm). Furthermore, we found that GMP binds as a reversible, noncompetitive inhibitor of TGase 3 transamidation activity, similar to GTPgammaS and GDP. A genetic algorithm similarity program (GASP) approach (virtual ligand screening) identified three compounds from the Lead Quest trade mark data base (Tripos Inc.) based on superimposition of GTPgammaS, GDP, and GMP guanine nucleotides from our crystal structures to generate the minimum align flexible fragment. These three were nucleotide analogs without a phosphate group containing the minimal binding motif for TGase 3 that includes a nucleoside recognition groove. Binding affinities were measured as follows: TP349915 (K(d) = 4.1 microm), TP395289 (K(d) = 38.5 microm), TP394305 (K(d) = 1.0 mm). Remarkably, these compounds do not inhibit but instead activate TGase 3 transamidation by about 10-fold. These results suggest that the nucleotide binding pocket in TGase 3 may be exploited to either enhance or inhibit the enzymatic activity as required for different therapeutic approaches.

The emerging structural understanding of transglutaminase 3.

Transglutaminases (TGase; protein-glutamine: amine gamma-glutamyl-transferase) are a family of calcium-dependent acyl-transfer enzymes ubiquitously expressed in mammalian cells and responsible for catalyzing covalent cross-links between proteins or peptides. A series of recent crystal structures have revealed the overall architecture of TGase enzymes, and provided a deep look at their active site, calcium and magnesium ions, and the manner by which guanine nucleotides interact with this enzyme. These structures, backed with extensive biochemical studies, are providing new insights as to how access to the enzyme's active site may be gated through the coordinated changes in cellular calcium and magnesium concentrations and GTP/GDP. Calcium-activated TGase 3 can bind, hydrolyze, and is inhibited by GTP, despite lacking structural homology with other GTP binding proteins. A structure based sequence homology among the TGase enzyme family shows that these essential structural features are shared among other members of the TGase family.

Crystallography and mutagenesis point to an essential role for the N-terminus of human mitochondrial ClpP.

We have determined a 2.1 A crystal structure for human mitochondrial ClpP (hClpP), the proteolytic component of the ATP-dependent ClpXP protease. HClpP has a structure similar to that of the bacterial enzyme, with the proteolytic active sites sequestered within an aqueous chamber formed by face-to-face assembly of the two heptameric rings. The hydrophobic N-terminal peptides of the subunits are bound within the narrow (12 A) axial channel, positioned to interact with unfolded substrates translocated there by the associated ClpX chaperone. Mutation or deletion of these residues causes a drastic decrease in ClpX-mediated protein and peptide degradation. Residues 8-16 form a mobile loop that extends above the ring surface and is also required for activity. The 28 amino acid C-terminal domain, a unique feature of mammalian ClpP proteins, lies on the periphery of the ring, with its proximal portion forming a loop that extends out from the ring surface. Residues at the start of the C-terminal domain impinge on subunit interfaces within the ring and affect heptamer assembly and stability. We propose that the N-terminal peptide of ClpP is a structural component of the substrate translocation channel and may play an important functional role as well.