Structural and functional characterization of a conserved pair of bacterial cellulose-oxidizing lytic polysaccharide monooxygenases.

For decades, the enzymatic conversion of cellulose was thought to rely on the synergistic action of hydrolytic enzymes, but recent work has shown that lytic polysaccharide monooxygenases (LPMOs) are important contributors to this process. We describe the structural and functional characterization of two functionally coupled cellulose-active LPMOs belonging to auxiliary activity family 10 (AA10) that commonly occur in cellulolytic bacteria. One of these LPMOs cleaves glycosidic bonds by oxidation of the C1 carbon, whereas the other can oxidize both C1 and C4. We thus demonstrate that C4 oxidation is not confined to fungal AA9-type LPMOs. X-ray crystallographic structures were obtained for the enzyme pair from Streptomyces coelicolor, solved at 1.3 Å (ScLPMO10B) and 1.5 Å (CelS2 or ScLPMO10C) resolution. Structural comparisons revealed differences in active site architecture that could relate to the ability to oxidize C4 (and that also seem to apply to AA9-type LPMOs). Despite variation in active site architecture, the two enzymes exhibited similar affinities for Cu(2+) (12-31 nM), redox potentials (242 and 251 mV), and electron paramagnetic resonance spectra, with only the latter clearly different from those of chitin-active AA10-type LPMOs. We conclude that substrate specificity depends not on copper site architecture, but rather on variation in substrate binding and orientation. During cellulose degradation, the members of this LPMO pair act in synergy, indicating different functional roles and providing a rationale for the abundance of these enzymes in biomass-degrading organisms.

The high catalytic rate of the cold-active Vibrio alkaline phosphatase requires a hydrogen bonding network involving a large interface loop.

The role of surface loops in mediating communication through residue networks is still a relatively poorly understood part in the study of cold adaptation of enzymes, especially in terms of their quaternary interactions. Alkaline phosphatase (AP) from the psychrophilic marine bacterium Vibrio splendidus (VAP) is characterized by an analogous large surface loop in each monomer, referred to as the large loop, that hovers over the active site of the other monomer. It presumably has a role in the high catalytic efficiency of VAP which accompanies its extremely low thermal stability. Here, we designed several different variants of VAP with the aim of removing intersubunit interactions at the dimer interface. Breaking the intersubunit contacts from one residue in particular (Arg336) reduced the temperature stability of the catalytically potent conformation and caused a 40% drop in catalytic rate. The high catalytic rates of enzymes from cold-adapted organisms are often associated with increased dynamic flexibility. Comparison of the relative B-factors of the R336L crystal structure to that of the wild-type confirmed surface flexibility was increased in a loop on the opposite monomer, but not in the large loop. The increase in flexibility resulted in a reduced catalytic rate. The large loop increases the area of the interface between the subunits through its contacts and may facilitate an alternating structural cycle demanded by a half-of-sites reaction mechanism through stronger ties, as the dimer oscillates between high affinity (active) or low phosphoryl group affinity (inactive).

The 1.4 A crystal structure of the large and cold-active Vibrio sp. alkaline phosphatase.

Alkaline phosphatase (AP) from the cold-adapted Vibrio strain G15-21 is among the AP variants with the highest known k(cat) value. Here the structure of the enzyme at 1.4 A resolution is reported and compared to APs from E. coli, human placenta, shrimp and the Antarctic bacterium strain TAB5. The Vibrio AP is a dimer although its monomers are without the long N-terminal helix that embraces the other subunit in many other APs. The long insertion loop, previously noted as a special feature of the Vibrio AP, serves a similar function. The surface does not have the high negative charge density as observed in shrimp AP, but a positively charged patch is observed around the active site that may be favourable for substrate binding. The dimer interface has a similar number of non-covalent interactions as other APs and the "crown"-domain is the largest observed in known APs. Part of it slopes over the catalytic site suggesting that the substrates may be small molecules. The catalytic serines are refined with multiple conformations in both monomers. One of the ligands to the catalytic zinc ion in binding site M1 is directly connected to the crown-domain and is closest to the dimer interface. Subtle movements in metal ligands may help in the release of the product and/or facilitate prior dephosphorylation of the covalent intermediate. Intersubunit interactions may be a major factor for promoting active site geometries that lead to the high catalytic activity of Vibrio AP at low temperatures.

Crystal structures of g-type lysozyme from Atlantic cod shed new light on substrate binding and the catalytic mechanism.

Crystal structures of Atlantic cod lysozyme have been solved with and without ligand bound in the active site to 1.7 and 1.9 A resolution, respectively. The structures reveal the presence of NAG in the substrate binding sites at both sides of the catalytic Glu73, hence allowing the first crystallographic description of the goose-type (g-type) lysozyme E-G binding sites. In addition, two aspartic acid residues suggested to participate in catalysis (Asp101 and Asp90) were mutated to alanine. Muramidase activity data for two single mutants and one double mutant demonstrates that both residues are involved in catalysis, but Asp101 is the more critical of the two. The structures and activity data suggest that a water molecule is the nucleophile completing the catalytic reaction, and the roles of the aspartic acids are to ensure proper positioning of the catalytic water.

X-ray crystal structure of Vibrio alkaline phosphatase with the non-competitive inhibitor cyclohexylamine.

BACKGROUND: Para-nitrophenyl phosphate, the common substrate for alkaline phosphatase (AP), is available as a cyclohexylamine salt. Here, we report that cyclohexylamine is a non-competitive inhibitor of APs. METHODS: Cyclohexylamine inhibited four different APs. Co-crystallization with the cold-active Vibrio AP (VAP) was performed and the structure solved. RESULTS: Inhibition of VAP fitted a non-competitive kinetic model (Km unchanged, Vmax reduced) with IC50 45.3 mM at the pH optimum 9.8, not sensitive to 0.5 M NaCl, and IC50 27.9 mM at pH 8.0, where the addition of 0.5 M NaCl altered the inhibition to the level observed at pH 9.8. APs from E. coli and calf intestines were less sensitive to cyclohexylamine, whereas an Antarctic bacterial AP was similar to VAP in this respect. X-ray crystallography at 2.3 Å showed two binding sites, one in the active site channel and another at the surface close to dimer interface. Antarctic bacterial AP and VAP have Trp274 in common in their active-sites, that takes part in binding cyclohexylamine. VAP variants W274A, W274K, and W274H gave IC50 values of 179 mM, 188 mM and 187 mM, respectively, at pH 9.8. CONCLUSIONS: The binding of cyclohexylamine in locations at the dimeric interface and/or in the active site of APs may delay product release or reduce the rate of catalytic step(s) involving conformational changes and intersubunit communications. GENERAL SIGNIFICANCE: Cyclohexylamine is a common chemical in industries and used as a counterion in substrates for alkaline phosphatase, a clinically important and common enzyme in the biosphere.

Structural insights of the enzymes from the chitin utilization locus of Flavobacterium johnsoniae.

Chitin is one of the most abundant renewable organic materials found on earth. The chitin utilization locus in Flavobacterium johnsoniae, which encodes necessary proteins for complete enzymatic depolymerization of crystalline chitin, has recently been characterized but no detailed structural information on the enzymes was provided. Here we present protein structures of the F. johnsoniae chitobiase (FjGH20) and chitinase B (FjChiB). FjGH20 is a multi-domain enzyme with a helical domain not before observed in other chitobiases and a domain organization reminiscent of GH84 (ß-N-acetylglucosaminidase) family members. The structure of FjChiB reveals that the protein lacks loops and regions associated with exo-acting activity in other chitinases and instead has a more solvent accessible substrate binding cleft, which is consistent with its endo-chitinase activity. Additionally, small angle X-ray scattering data were collected for the internal 70 kDa region that connects the N- and C-terminal chitinase domains of the unique 158 kDa multi-domain chitinase A (FjChiA). The resulting model of the molecular envelope supports bioinformatic predictions of the region comprising six domains, each with similarities to either Fn3-like or Ig-like domains. Taken together, the results provide insights into chitin utilization by F. johnsoniae and reveal structural diversity in bacterial chitin metabolism.

Structure of a highly stable mutant of human fibroblast growth factor 1.

Fibroblast growth factors (FGFs) are involved in diverse cellular processes such as cell migration, angiogenesis, osteogenesis, wound healing and embryonic and foetal development. Human acidic fibroblast growth factor (FGF-1) is the only member of the FGF family that binds with high affinity to all four FGF receptors and thus is considered to be the human mitogen with the broadest specificity. However, pharmacological applications of FGF-1 are limited owing to its low stability. It has previously been reported that the introduction of single mutations can significantly improve the stability of FGF-1 and its resistance to proteolytic degradation. Here, the structure of the Q40P/S47I/H93G triple mutant of FGF-1, which exhibits much higher stability, a prolonged half-life and enhanced mitogenic activity, is presented. Compared with the wild-type structure, three localized conformational changes in the stable triple mutant were observed, which is in agreement with the perfect energetic additivity of the single mutations described in a previous study. The huge change in FGF-1 stability (the denaturation temperature increased by 21.5 K, equivalent to DeltaDeltaG(den) = 24.3 kJ mol(-1)) seems to result from the formation of a short 3(10)-helix (position 40), an improvement in the propensity of amino acids to form beta-sheets (position 47) and the rearrangement of a local hydrogen-bond network (positions 47 and 93).

The crystal structure of haemoglobin from Atlantic cod.

The crystal structure of haemoglobin from Atlantic cod has been solved to 2.54 Šresolution. The structure consists of two tetramers in the crystallographic asymmetric unit. The structure of haemoglobin obtained from one individual cod suggests polymorphism in the tetrameric assembly.

Features and structure of a cold active N-acetylneuraminate lyase.

N-acetylneuraminate lyases (NALs) are enzymes that catalyze the reversible cleavage and synthesis of sialic acids. They are therefore commonly used for the production of these high-value sugars. This study presents the recombinant production, together with biochemical and structural data, of the NAL from the psychrophilic bacterium Aliivibrio salmonicida LFI1238 (AsNAL). Our characterization shows that AsNAL possesses high activity and stability at alkaline pH. We confirm that these properties allow for the use in a one-pot reaction at alkaline pH for the synthesis of N-acetylneuraminic acid (Neu5Ac, the most common sialic acid) from the inexpensive precursor N-acetylglucosamine. We also show that the enzyme has a cold active nature with an optimum temperature for Neu5Ac synthesis at 20°C. The equilibrium constant for the reaction was calculated at different temperatures, and the formation of Neu5Ac acid is favored at low temperatures, making the cold active enzyme a well-suited candidate for use in such exothermic reactions. The specific activity is high compared to the homologue from Escherichia coli at three tested temperatures, and the enzyme shows a higher catalytic efficiency and turnover number for cleavage at 37°C. Mutational studies reveal that amino acid residue Asn 168 is important for the high kcat. The crystal structure of AsNAL was solved to 1.65 Å resolution and reveals a compact, tetrameric protein similar to other NAL structures. The data presented provides a framework to guide further optimization of its application in sialic acid production and opens the possibility for further design of the enzyme.

Effects of salt on the kinetics and thermodynamic stability of endonuclease I from Vibrio salmonicida and Vibrio cholerae.

Adaptation to extreme environments affects the stability and catalytic efficiency of enzymes, often endowing them with great industrial potential. We compared the environmental adaptation of the secreted endonuclease I from the cold-adapted marine fish pathogen Vibrio salmonicida (VsEndA) and the human pathogen Vibrio cholerae (VcEndA). Kinetic analysis showed that VsEndA displayed unique halotolerance. It retained a considerable amount of activity from low concentrations to at least 0.6 m NaCl, and was adapted to work at higher salt concentrations than VcEndA by maintaining a low K(m) value and increasing k(cat). In differential scanning calorimetry, salt stabilized both enzymes, but the effect on the calorimetric enthalpy and cooperativity of unfolding was larger for VsEndA, indicating salt dependence. Mutation of DNA binding site residues (VsEndA, Q69N and K71N; VcEndA, N69Q and N71K) affected the kinetic parameters. The VsEndA Q69N mutation also increased the T(m) value, whereas other mutations affected mainly DeltaH(cal). The determined crystal structure of VcEndA N69Q revealed the loss of one hydrogen bond present in native VcEndA, but also the formation of a new hydrogen bond involving residue 69 that could possibly explain the similar T(m) values for native and N69Q-mutated VcEndA. Structural analysis suggested that the stability, catalytic efficiency and salt tolerance of EndA were controlled by small changes in the hydrogen bonding networks and surface electrostatic potential. Our results indicate that endonuclease I adaptation is closely coupled to the conditions of the habitats of natural Vibrio, with VsEndA displaying a remarkable salt tolerance unique amongst the endonucleases characterized so far.

Structural insight into a CE15 esterase from the marine bacterial metagenome.

The family 15 carbohydrate esterase (CE15) MZ0003, which derives from a marine Arctic metagenome, has a broader substrate scope than other members of this family. Here we report the crystal structure of MZ0003, which reveals that residues comprising the catalytic triad differ from previously-characterized fungal homologs, and resolves three large loop regions that are unique to this bacterial sub-clade. The catalytic triad of the bacterial CE15, which includes Asp 332 as its third member, closely resembles that of family 1 carbohydrate esterases (CE1), despite the overall lower structural similarity with members of this family. Two of the three loop regions form a subdomain that deepens the active site pocket and includes several basic residues that contribute to the high positive charge surrounding the active site. Docking simulations predict specific interactions with the sugar moiety of glucuronic-acid substrates, and with aromatically-substituted derivatives that serve as model compounds for the lignin-carbohydrate complex of plant cell walls. Molecular dynamics simulations indicate considerable flexibility of the sub-domain in the substrate-bound form, suggesting plasticity to accommodate different substrates is possible. The findings from this first reported structure of a bacterial member of the CE15 family provide insight into the basis of its broader substrate specificity.

Comparative sequence and structure analysis reveal features of cold adaptation of an enzyme in the thermolysin family.

Knowledge about the structural features underlying cold adaptation is important for designing enzymes of different industrial relevance. Vibriolysin from Antarctic bacterium strain 643 (VAB) is at present the only enzyme of the thermolysin family from an organism that thrive in extremely cold climate. In this study comparative sequence-structure analysis and molecular dynamics (MD) simulations were used to reveal the molecular features of cold adaptation of VAB. Amino acid sequence analysis of 44 thermolysin enzymes showed that VAB compared to the other enzymes has: (1) fewer arginines, (2) a lower Arg/(Lys + Arg) ratio, (3) a lower fraction of large aliphatic side chains, expressed by the (Ile + Leu)/(Ile + Leu + Val) ratio, (4) more methionines, (5) more serines, and (6) more of the thermolabile amino acid asparagine. A model of the catalytic domain of VAB was constructed based on homology with pseudolysin. MD simulations for 3 ns of VAB, pseudolysin, and thermolysin supported the assumption that cold-adapted enzymes have a more flexible three-dimensional (3D) structure than their thermophilic and mesophilic counterparts, especially in some loop regions. The structural analysis indicated that VAB has fewer intramolecular cation-pi electron interactions and fewer hydrogen bonds than its mesophilic (pseudolysin) and thermophilic (thermolysin) counterparts. Lysine is the dominating cationic amino acids involved in salt bridges in VAB, while arginine is dominating in thermolysin and pseudolysin. VAB has a greater volume of inaccessible cavities than pseudolysin and thermolysin. The electrostatic potentials on the surface of the catalytic domain were also more negative for VAB than for thermolysin and pseudolysin. Thus, the MD simulations, the structural patterns, and the amino acid composition of VAB relative to other enzymes of the thermolysin family suggest that VAB possesses the biophysical properties generally following adaptation to cold climate.

The 1.8 A crystal structure of a proteinase K-like enzyme from a psychrotroph Serratia species.

Proteins from organisms living in extreme conditions are of particular interest because of their potential for being templates for redesign of enzymes both in biotechnological and other industries. The crystal structure of a proteinase K-like enzyme from a psychrotroph Serratia species has been solved to 1.8 A. The structure has been compared with the structures of proteinase K from Tritirachium album Limber and Vibrio sp. PA44 in order to reveal structural explanations for differences in biophysical properties. The Serratia peptidase shares around 40 and 64% identity with the Tritirachium and Vibrio peptidases, respectively. The fold of the three enzymes is essentially identical, with minor exceptions in surface loops. One calcium binding site is found in the Serratia peptidase, in contrast to the Tritirachium and Vibrio peptidases which have two and three, respectively. A disulfide bridge close to the S2 site in the Serratia and Vibrio peptidases, an extensive hydrogen bond network in a tight loop close to the substrate binding site in the Serratia peptidase and different amino acid sequences in the S4 sites are expected to cause different substrate specificity in the three enzymes. The more negative surface potential of the Serratia peptidase, along with a disulfide bridge close to the S2 binding site of a substrate, is also expected to contribute to the overall lower binding affinity observed for the Serratia peptidase. Clear electron density for a tripeptide, probably a proteolysis product, was found in the S' sites of the substrate binding cleft.

Characterization of a recombinantly expressed proteinase K-like enzyme from a psychrotrophic Serratia sp.

The gene encoding a peptidase that belongs to the proteinase K family of serine peptidases has been identified from a psychrotrophic Serratia sp., and cloned and expressed in Escherichia coli. The gene has 1890 base pairs and encodes a precursor protein of 629 amino acids with a theoretical molecular mass of 65.5 kDa. Sequence analysis suggests that the peptidase consists of a prepro region, a catalytic domain and two C-terminal domains. The enzyme is recombinantly expressed as an active approximately 56 kDa peptidase and includes both C-terminal domains. Purified enzyme is converted to the approximately 34 kDa form by autolytic cleavage when incubated at 50 degrees C for 30 min, but retains full activity. In the present work, the Serratia peptidase (SPRK) is compared with the family representative proteinase K (PRK) from Tritirachium album Limber. Both enzymes show a relatively high thermal stability and a broad pH stability profile. SPRK possess superior stability towards SDS at 50 degrees C compared to PRK. On the other hand, SPRK is considerably more labile to removal of calcium ions. The activity profiles against temperature and pH differ for the two enzymes. SPRK shows both a broader pH optimum as well as a higher temperature optimum than PRK. Analysis of the catalytic properties of SPRK and PRK using the synthetic peptide succinyl-Ala-Ala-Pro-Phe-pNA as substrate showed that SPRK possesses a 3.5-4.5-fold higher kcat at the temperature range 12-37 degrees C, but a fivefold higher Km results in a slightly lower catalytic efficiency (kcat/Km) of SPRK compared to PRK.

Crystallization and preliminary X-ray diffraction analysis of a cold-adapted catalase from Vibrio salmonicida.

Catalase (EC 1.11.1.6) catalyses the breakdown of hydrogen peroxide to water and molecular oxygen. Recombinant Vibrio salmonicida catalase (VSC) possesses typical cold-adapted features, with higher catalytic efficiency, lower thermal stability and a lower temperature optimum than its mesophilic counterpart from Proteus mirabilis. Crystals of VSC were produced by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 98.15, b = 217.76, c = 99.28 A, beta = 110.48 degrees. Data were collected to 1.96 A and a molecular-replacement solution was found with eight molecules in the asymmetric unit.

Multiple specialised goose-type lysozymes potentially compensate for an exceptional lack of chicken-type lysozymes in Atlantic cod.

Previous analyses of the Atlantic cod genome showed unique combinations of lacking and expanded number of genes for the immune system. The present study examined lysozyme activity, lysozyme gene distribution and expression in cod. Enzymatic assays employing specific bacterial lysozyme inhibitors provided evidence for presence of g-type, but unexpectedly not for c-type lysozyme activity. Database homology searches failed to identify any c-type lysozyme gene in the cod genome or in expressed sequence tags from cod. In contrast, we identified four g-type lysozyme genes (LygF1a-d) constitutively expressed, although differentially, in all cod organs examined. The active site glutamate residue is replaced by alanine in LygF1a, thus making it enzymatic inactive, while LygF1d was found in two active site variants carrying alanine or glutamate, respectively. In vitro and in vivo infection by the intracellular bacterium Francisella noatunensis gave a significantly reduced LygF1a and b expression but increased expression of the LygF1c and d genes as did also the interferon gamma (IFNγ) cytokine. These results demonstrate a lack of c-type lysozyme that is unprecedented among vertebrates. Our results further indicate that serial gene duplications have produced multiple differentially regulated cod g-type lysozymes with specialised functions potentially compensating for the lack of c-type lysozymes.

Crystal structures of five bovine chymotrypsin complexes with P1 BPTI variants.

The bovine chymotrypsin-bovine pancreatic trypsin inhibitor (BPTI) interaction belongs to extensively studied models of protein-protein recognition. The accommodation of the inhibitor P1 residue in the S1 binding site of the enzyme forms the hot spot of this interaction. Mutations introduced at the P1 position of BPTI result in a more than five orders of magnitude difference of the association constant values with the protease. To elucidate the structural aspects of the discrimination between different P1 residues, crystal structures of five bovine chymotrypsin-P1 BPTI variant complexes have been determined at pH 7.8 to a resolution below 2 A. The set includes polar (Thr), ionizable (Glu, His), medium-sized aliphatic (Met) and large aromatic (Trp) P1 residues and complements our earlier studies of the interaction of different P1 side-chains with the S1 pocket of chymotrypsin. The structures have been compared to the complexes of proteases with similar and dissimilar P1 preferences, including Streptomyces griseus proteases B and E, human neutrophil elastase, crab collagenase, bovine trypsin and human thrombin. The S1 sites of these enzymes share a common general shape of significant rigidity. Large and branched P1 residues adapt in their complexes similar conformations regardless of the polarity and size differences between their S1 pockets. Conversely, long and flexible residues such as P1 Met are present in the disordered form and display a conformational diversity despite similar inhibitory properties with respect to most enzymes studied. Thus, the S1 specificity profiles of the serine proteases appear to result from the precise complementarity of the P1-S1 interface and minor conformational adjustments occurring upon the inhibitor binding.

Structural consequences of accommodation of four non-cognate amino acid residues in the S1 pocket of bovine trypsin and chymotrypsin.

Crystal structures of P1 Gly, Val, Leu and Phe bovine pancreatic trypsin inhibitor (BPTI) variants in complex with two serine proteinases, bovine trypsin and chymotrypsin, have been determined. The association constants for the four mutants with the two enzymes show that the enlargement of the volume of the P1 residue is accompanied by an increase of the binding energy, which is more pronounced for bovine chymotrypsin. Since the conformation of the P1 side-chains in the two S1 pockets is very similar, we suggest that the difference in DeltaG values between the enzymes must arise from the more polar environment of the S1 site of trypsin. This results mainly from the substitutions of Met192 and Ser189 observed in chymotrypsin with Gln192 and Asp189 present in trypsin. The more polar interior of the S1 site of trypsin is reflected by a much higher order of the solvent network in the empty pocket of the enzyme, as is observed in the complexes of the two enzymes with the P1 Gly BPTI variant. The more optimal binding of the large hydrophobic P1 residues by chymotrypsin is also reflected by shrinkage of the S1 pocket upon the accommodation of the cognate residues of this enzyme. Conversely, the S1 pocket of trypsin expands upon binding of such side-chains, possibly to avoid interaction with the polar residues of the walls. Further differentiation between the two enzymes is achieved by small differences in the shape of the S1 sites, resulting in an unequal steric hindrance of some of the side-chains, as observed for the gamma-branched P1 Leu variant of BPTI, which is much more favored by bovine chymotrypsin than trypsin. Analysis of the discrimination of beta-branched residues by trypsin and chymotrypsin is based on the complexes with the P1 Val BPTI variant. Steric repulsion of the P1 Val residue by the walls of the S1 pocket of both enzymes prevents the P1 Val side-chain from adopting the most optimal chi1 value.

The structure of bovine lysosomal alpha-mannosidase suggests a novel mechanism for low-pH activation.

Lysosomal alpha-mannosidase (LAM: EC 3.2.1.24) belongs to the sequence-based glycoside hydrolase family 38 (GH38). Two other mammalian GH38 members, Golgi alpha-mannosidase II (GIIAM) and cytosolic alpha-mannosidase, are expressed in all tissues. In humans, cattle, cat and guinea pig, lack of lysosomal alpha-mannosidase activity causes the autosomal recessive disease alpha-mannosidosis. Here, we describe the three-dimensional structure of bovine lysosomal alpha-mannosidase (bLAM) at 2.7A resolution and confirm the solution state dimer by electron microscopy. We present the first structure of a mammalian GH38 enzyme that offers indications for the signal areas for mannose phosphorylation, suggests a previously undetected mechanism of low-pH activation and provides a template for further biochemical studies of the family 38 glycoside hydrolases as well as lysosomal transport. Furthermore, it provides a basis for understanding the human form of alpha-mannosidosis at the atomic level. The atomic coordinates and structure factors have been deposited in the Protein Data Bank (accession codes 1o7d and r1o7dsf).

Psoriasis pathogenesis - Pso p27 constitutes a compact structure forming large aggregates.

Psoriasis is a chronic inflammatory skin disease. The absence of microbial organisms as potential causal agents has given rise to the hypothesis that the inflammation is due to an autoimmune reaction. The defined inflamed areas of the skin lesions argue for an immunological disease with a local production of a causal antigen. Pso p27 is a protein generated in mast cells in psoriatic plaques, but not in uninvolved skin. We recently demonstrated that the Pso p27 is generated by cleavage of SerpinB3 (SCCA1) in the presence of mast cell associated chymase. In this communication we demonstrate by X-ray crystallographic analysis that the cleavage products associate into a complex similar to SCCA1, but with the reactive centre loop inserted into a 5-stranded central ß-sheet. Native gel electrophoresis show that these Pso p27 complexes form large aggregates which may be of significance with respect to an immunogenic role of Pso p27.