Bovine chymotrypsinogen A X-ray crystal structure analysis and refinement of a new crystal form at 1.8 A resolution

The X-ray structure of a new crystal form of chymotrypsinogen A grown from ethanol/water has been determined at 1.8 A resolution using Patterson search techniques. The crystals are of orthorhombic space group P212121 and contain two molecules in the asymmetric unit. Both independent molecules (referred to as A and B) have been crystallographically refined to a final R value of 0.173 with reflection data to 1.8 A resolution. Owing to different crystal contacts, both independent molecules show at various sites conformational differences, especially in segments 33-38, 142-153 and 215-222. If these three loops are omitted in a comparison, the root-mean-square (r.m.s.) deviation of the main-chain atoms of molecules A and B is 0.32 A. If segments 70-79, 143-152 and 215-221 are omitted, a comparison of either molecule A or molecule B with the chymotrypsinogen model of Freer et al. (1970) reveals an r.m.s. deviation of the alpha-carbon atoms of about 0.7 A. Compared with the active enzyme, four spatially adjacent peptide segments, in particular, are differently organized in the zymogen: the amino-terminal segment 11-19 runs in a rigid but strained conformation along the molecular surface due to the covalent linkage through Cys1; also segment 184-194 is in a rigid unique conformation due to several mutually stabilizing interactions with the amino-terminal segment; segment 216-222, which also lines the specificity pocket, adapts to different crystal contacts and exists in both chymotrypsinogen molecules in different, but defined conformations; in particular, disulfide bridge 191-220, which covalently links both latter segments, has opposite handedness in molecules A and B; finally, the autolysis loop 142 to 153 is organized in a variety of ways and in its terminal part is completely disordered. Thus, the allosteric activation domain (Huber & Bode, 1978) is organized in defined although different conformations in chymotrypsinogen molecules A and B, in contrast to trypsinogen, where all four homologous segments of the activation domain are disordered. This reflects the structural variability and deformability of the activation domain in serine proteinase proenzymes. If the aforementioned peptide segments are omitted, a comparison of our chymotrypsinogen models with gamma-chymotrypsin (Cohen et al., 1981) yields an r.m.s. deviation for alpha-carbon atoms of about 0.5 A. The residues of the "active site triad" are arranged similarly, but the oxyanion hole is lacking in chymotrypsinogen.(ABSTRACT TRUNCATED AT 400 WORDS)     

Complex formation by bovine trypsin and a tetrapeptide (Leu-Arg-Pro-Gly-NH2): X-ray structure analysis of the complex in the orthorhombic crystal form with low molecular packing density

The title tetrapeptide, Leu-Arg-Pro-Gly-NH₂, forms a complex with trypsin in a novel orthorhombic crystal form with low molecular packing density. The complex formation was directly evidenced by X-ray crystallography. The crystal structure at 1.8 Å resolution was refined to anR-factor of 20.5% for 13,923 reflection data, which were measured with synchrotron radiation. The tetrapeptide is bound to trypsin at the active site, and the binding mode is very similar to that of a bovine pancreatic trypsin inhibitor (BPTI):trypsin complex. The tetrapeptide:trypsin complex is the first observation that a peptide forms a stable complex with trypsin.     

Intermolecular interactions and characterization of the novel factor Xa exosite involved in macromolecular recognition and inhibition: crystal structure of human Gla-domainless factor Xa complexed with the anticoagulant protein NAPc2 from the hematophagous nematode Ancylostoma caninum

NAPc2, an anticoagulant protein from the hematophagous nematode Ancylostoma caninum evaluated in phase-II/IIa clinical trials, inhibits the extrinsic blood coagulation pathway by a two step mechanism, initially interacting with the hitherto uncharacterized factor Xa exosite involved in macromolecular recognition and subsequently inhibiting factor VIIa (K(i)=8.4 pM) of the factor VIIa/tissue factor complex. NAPc2 is highly flexible, becoming partially ordered and undergoing significant structural changes in the C terminus upon binding to the factor Xa exosite. In the crystal structure of the ternary factor Xa/NAPc2/selectide complex, the binding interface consists of an intermolecular antiparallel beta-sheet formed by the segment of the polypeptide chain consisting of residues 74-80 of NAPc2 with the residues 86-93 of factor Xa that is additional maintained by contacts between the short helical segment (residues 67-73) and a turn (residues 26-29) of NAPc2 with the short C-terminal helix of factor Xa (residues 233-243). This exosite is physiologically highly relevant for the recognition and inhibition of factor X/Xa by macromolecular substrates and provides a structural motif for the development of a new class of inhibitors for the treatment of deep vein thrombosis and angioplasty.     

Crystal structure of the leucine-rich repeat domain of the NOD-like receptor NLRP1: Implications for binding of muramyl dipeptide

The NOD-like receptor NLRP1 (NLR family, pyrin domain containing 1) senses the presence of the bacterial cell wall component l-muramyl dipeptide (MDP) inside the cell. We determined the crystal structure of the LRR domain of human NLRP1 in the absence of MDP to a resolution of 1.65 Å. The fold of the structure can be assigned to the ribonuclease inhibitor-like class of LRR proteins. We compared our structure with X-ray models of the LRR domains of NLRX1 and NLRC4 and a homology model of the LRR domain of NOD2. We conclude that the MDP binding site of NLRP1 is not located in the LRR domain.     

Fine tuning of coenzyme specificity in family 2 aldo-keto reductases revealed by crystal structures of the Lys-274-->Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD+ and NADP+

Aldo-keto reductases of family 2 employ single site replacement Lys-->Arg to switch their cosubstrate preference from NADPH to NADH. X-ray crystal structures of Lys-274-->Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD+ and NADP+ were determined at a resolution of 2.4 and 2.3A, respectively. Due to steric conflicts in the NADP+-bound form, the arginine side chain must rotate away from the position of the original lysine side chain, thereby disrupting a network of direct and water-mediated interactions between Glu-227, Lys-274 and the cofactor 2'-phosphate and 3'-hydroxy groups. Because anchoring contacts of its Glu-227 are lost, the coenzyme-enfolding loop that becomes ordered upon binding of NAD(P)+ in the wild-type remains partly disordered in the NADP+-bound mutant. The results delineate a catalytic reaction profile for the mutant in comparison to wild-type.     

Crystal structure of the kainate receptor GluR5 ligand-binding core in complex with (S)-glutamate

The X-ray structure of the ligand-binding core of the kainate receptor GluR5 (GluR5-S1S2) in complex with (S)-glutamate was determined to 1.95 A resolution. The overall GluR5-S1S2 structure comprises two domains and is similar to the related AMPA receptor GluR2-S1S2J. (S)-glutamate binds as in GluR2-S1S2J. Distinct features are observed for Ser741, which stabilizes a highly coordinated network of water molecules and forms an interdomain bridge. The GluR5 complex exhibits a high degree of domain closure (26 degrees) relative to apo GluR2-S1S2J. In addition, GluR5-S1S2 forms a novel dimer interface with a different arrangement of the two protomers compared to GluR2-S1S2J.     

Crystal structure and biochemical studies of Brucella melitensis 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase

The prokaryotic 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes the irreversible cleavage of the glycosidic bond in 5′-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH), a process that plays a key role in several metabolic pathways. Its absence in all mammalian species has implicated this enzyme as a promising target for antimicrobial drug design. Here, we report the crystal structure of BmMTAN in complex with its product adenine at a resolution of 2.6 Å determined by single-wavelength anomalous dispersion method. 11 key residues were mutated for kinetic characterization. Mutations of Tyr134 and Met144 resulted in the largest overall increase in Km, whereas mutagenesis of residues Glu18, Glu145 and Asp168 completely abolished activity. Glu145 and Asp168 were identified as active site residues essential for catalysis. The catalytic mechanism and implications of this structure for broad-based antibiotic design are discussed.     

Crystal structure of a soluble decoy receptor IL-22BP bound to interleukin-22

Interleukin-22 (IL-22) plays an important role in the regulation of immune and inflammatory responses in mammals. The IL-22 binding protein (IL-22BP), a soluble receptor that specifically binds IL-22, prevents the IL-22/interleukin-22 receptor 1 (IL-22R1)/interleukin-10 receptor 2 (IL-10R2) complex assembly and blocks IL-22 biological activity. Here we present the crystal structure of the IL-22/IL-22BP complex at 2.75 Å resolution. The structure reveals IL-22BP residues critical for IL-22 binding, which were confirmed by site-directed mutagenesis and functional studies. Comparison of IL-22/IL-22BP and IL-22/IL-22R1 crystal structures shows that both receptors display an overlapping IL-22 binding surface, which is consistent with the inhibitory role played by IL-22 binding protein.

Structured summary

MINT-7010533: IL-22 BP (uniprotkb:Q969J5) and IL-22 (uniprotkb:Q9GZX6) bind (MI:0407) by X-ray crystallography (MI:0114)     

Biosynthesis of riboflavin: structure and properties of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate reductase of Methanocaldococcus jannaschii

The pyrimidine reductase of the riboflavin biosynthetic pathway (MjaRED) specified by the open reading frame MJ0671 of Methanocaldococcus jannaschii was expressed in Escherichia coli using a synthetic gene. The synthetic open reading frame that was optimized for expression in E. coli directed the synthesis of abundant amounts of the enzyme with an apparent subunit mass of 25 kDa. The enzyme was purified to apparent homogeneity and was shown to catalyze the conversion of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate into 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate at a rate of 0.8 micromol min(-1) mg(-1) at pH 8.0 and at 30 degrees C. The protein is a homodimer as shown by sedimentation equilibrium analysis and sediments at an apparent velocity of 3.5 S. The structure of the enzyme in complex with the cofactor nicotinamide adenine dinucleotide phosphate was determined by X-ray crystallography at a resolution of 2.5 Angstroms. The folding pattern resembles that of dihydrofolate reductase with the Thermotoga maritima ortholog as the most similar structure. The substrate, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate, was modeled into the putative active site. The model suggests the transfer of the pro-R hydrogen of C-4 of NADPH to C-1' of the substrate.     

Three-dimensional structure determined for a subunit of human tRNA splicing endonuclease (Sen15) reveals a novel dimeric fold

Splicing of eukaryal intron-containing tRNAs requires the action of the heterotetrameric splicing endonuclease, which is composed of two catalytic subunits, Sen34 and Sen2, and two structural subunits, Sen15 and Sen54. Here we report the solution structure of the human tRNA splicing endonuclease subunit HsSen15. To facilitate the structure determination, we removed the disordered 35 N-terminal and 14 C-terminal residues of the full-length protein to produce HsSen15(36-157). The structure of HsSen15(36-157), the first for a subunit of a eukaryal splicing endonuclease, revealed that the protein possesses a novel homodimeric fold. Each monomer consists of three alpha-helices and a mixed antiparallel/parallel beta-sheet, arranged in a topology similar to that of the C-terminal domain of Methanocaldococcus jannaschii endonuclease. The dimeric interface is dominated by a beta-barrel structure, formed by face-to-face packing of two, three-stranded beta-sheets. Each of the beta-sheets results from reciprocal parallel pairing of one beta-strand from one subunit with two other beta-strands from the symmetric subunit. The structural model provides insights into the functional assembly of the human tRNA splicing endonuclease.     

Dual role of FMN in flavodoxin function: Electron transfer cofactor and modulation of the protein-protein interaction surface

Flavodoxin (Fld) replaces Ferredoxin (Fd) as electron carrier from Photosystem I (PSI) to Ferredoxin-NADP⁺ reductase (FNR). A number of Anabaena Fld (AnFld) variants with replacements at the interaction surface with FNR and PSI indicated that neither polar nor hydrophobic residues resulted critical for the interactions, particularly with FNR. This suggests that the solvent exposed benzenoid surface of the Fld FMN cofactor might contribute to it. FMN has been replaced with analogues in which its 7- and/or 8-methyl groups have been replaced by chlorine and/or hydrogen. The oxidised Fld variants accept electrons from reduced FNR more efficiently than Fld, as expected from their less negative midpoint potential. However, processes with PSI (including reduction of Fld semiquinone by PSI, described here for the first time) are impeded at the steps that involve complex re-arrangement and electron transfer (ET). The groups introduced, particularly chlorine, have an electron withdrawal effect on the pyrazine and pyrimidine rings of FMN. These changes are reflected in the magnitude and orientation of the molecular dipole moment of the variants, both factors appearing critical for the re-arrangement of the finely tuned PSI:Fld complex. Processes with FNR are also slightly modulated. Despite the displacements observed, the negative end of the dipole moment points towards the surface that contains the FMN, still allowing formation of complexes competent for efficient ET. This agrees with several alternative binding modes in the FNR:Fld interaction. In conclusion, the FMN in Fld not only contributes to the redox process, but also to attain the competent interaction of Fld with FNR and PSI.