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The molecular replacement method was used to study the crystal structure of a chitinase from Carica papaya which belongs to family 19 of the glycosyl hydrolases (GH). The results provided insights into the significant role of water-mediated hydrogen bonding in substrate binding and the catalytic mechanism of the GH family 19 chitinases and also identified a new sequence motif for family 19 chitinases between residues Tyr111 and Tyr125.

Haemophilus influenzae HxuA is a cell-surface protein with haem-haemopexin binding activity which is key to haem acquisition from haemopexin and thus is one of the potential sources of haem for this microorganism. HxuA is secreted by its specific transporter HxuB. HxuA/HxuB belongs to the so-called two-partner secretion systems (TPSs) that are characterized by a conserved N-terminal domain in the secreted protein which is essential for secretion. Here, the 1.5 Å resolution structure of the secretion domain of HxuA, HxuA301, is reported. The structure reveals that HxuA301 folds into a β-helix domain with two extra-helical motifs, a four-stranded β-sheet and an N-terminal cap. Comparisons with other structures of TpsA secretion domains are reported. They reveal that despite limited sequence identity, strong structural similarities are found between the β-helix motifs, consistent with the idea that the TPS domain plays a role not only in the interaction with the specific TpsB partners but also as the scaffold initiating progressive folding of the TpsA proteins at the bacterial surface. [ABSTRACT FROM AUTHOR]

Abstract: Par27 from Bordetella pertussis belongs to a newly discovered class of dimeric peptidyl-prolyl isomerase (PPIase)/chaperones from the parvulin family. It is a tripartite protein with a central PPIase domain surrounded by N- and C-terminal sub-domains (NTD and CTD). Here, the Par27 structure was characterized by X-ray crystallography, small-angle X-ray scattering and template-based modeling. In the crystal lattice, Par27 consists of alternating well ordered and poorly ordered domains. The PPIase domains gave rise to diffuse scattering and could not be solved, whereas a 2.2Å resolution crystal structure was obtained for the NTD and CTD, revealing a cradle-shaped dimeric platform. Despite a lack of sequence similarity with corresponding sub-domains, the topology of the peptide chain in the NTD/CTD core is similar to that of other monomeric PPIase/chaperones such as SurA and trigger factor from Escherichia coli. In Par27, dimerization occurs by sub-domain swapping. Because of the strong amino acid sequence similarity to other parvulin domains, a model for the Par27 PPIase domain was built by template-based modeling and validated against small-angle X-ray scattering (SAXS) data. A model of the full-length dimeric Par27 structure was built by rigid-body modeling and filtering against SAXS data using the partial crystal structure of the NTD/CTD core and the template-based PPIase model. The flexibility of protein was accounted for by representing the structure as an ensemble of different conformations that collectively reproduce the scattering data. The refined models exhibit a cradle-like shape reminiscent of other PPIase/chaperones, and the variability in the orientation of the PPIase domains relative to the NTD/CTD core platform observed in the different models suggests inter-domain flexibility that could be important for the biological activity of this protein. [Copyright &y& Elsevier]

Isopentenyl diphosphate:dimethylallyl diphosphate (IPP:DMAPP) isomerase catalyses a crucial activation step in the isoprenoid biosynthesis pathway. This enzyme is responsible for the isomerization of the carbon–carbon double bond of IPP to create the potent electrophile DMAPP. DMAPP then alkylates other molecules, including IPP, to initiate the extraordinary variety of isoprenoid compounds found in nature. The crystal structures of free and metal‐bound Escherichia coli IPP isomerase reveal critical active site features underlying its catalytic mechanism. The enzyme requires one Mn2+ or Mg2+ ion to fold in its active conformation, forming a distorted octahedral metal coordination site composed of three histidines and two glutamates and located in the active site. Two critical residues, C67 and E116, face each other within the active site, close to the metal‐binding site. The structures are compatible with a mechanism in which the cysteine initiates the reaction by protonating the carbon–carbon double bond, with the antarafacial rearrangement ultimately achieved by one of the glutamates involved in the metal coordination sphere. W161 may stabilize the highly reactive carbocation generated during the reaction through quadrupole– charge interaction. [ABSTRACT FROM AUTHOR]

The two-component system BvgAS controls the virulence regulon in Bordetella pertussis. BvgS is the prototype of a family of sensor histidine kinases harboring periplasmic Venus flytrap (VFT) domains. The VFT domains are connected to the cytoplasmic kinase moiety by helical linkers separated by a Per-ARNT-Sim (PAS) domain. Antagonism between the two linkers, as one forms a coiled coil when the other is dynamic and vice versa, regulates BvgS activity. Here, we solved the structure of the intervening PAS domain by X-ray crystallography. Two forms were obtained that notably differ by the connections between the PAS core domain and the flanking helical linkers. Structure-guided mutagenesis indicated that those connections participate in the regulation of BvgS activity. Thus, the PAS domain appears to function as a switch facilitator module whose conformation determines the output of the system. As many BvgS homologs have similar architectures, the mechanisms unveiled here are likely to generally apply to the regulation of sensor histidine kinases of that family. [ABSTRACT FROM AUTHOR]

In living systems, the intramolecular cyclization of N-terminal glutamine residues is accomplished by glutaminyl cyclase enzymes (EC 2.3.2.5). While in mammals these enzymes are involved in the synthesis of hormonal and neurotransmitter peptides, the physiological role played by the corresponding plant enzymes still remains to be unravelled. Papaya glutaminyl cyclase (PQC), a 33 kDa enzyme found in the latex of the tropical tree Carica papaya, displays an exceptional resistance to chemical and thermal denaturation as well as to proteolysis. In order to elucidate its enzymatic mechanism and to gain insights into the structural determinants underlying its remarkable stability, PQC was isolated from papaya latex, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 62.82, b = 81.23, c = 108.17 Å and two molecules per asymmetric unit. Diffraction data have been collected at ESRF beamline BM14 and processed to a resolution of 1.7 Å. [ABSTRACT FROM AUTHOR]

Escherichia coli isopentenyl diphosphate isomerase, an enzyme catalyzing a key step in isoprenoid biosynthesis, has been produced in selenomethionyl form. The protein was purified and crystallized by the hanging-drop vapour-diffusion method. Crystals display trigonal symmetry, with unit-cell parameters a = b = 71.3, c = 61.7 Å, and diffract to 1.45 Å resolution. [ABSTRACT FROM AUTHOR]

Glutaminyl cyclases (QCs) (EC 2.3.2.5) catalyze the intramolecular cyclization of protein N-terminal glutamine residues into pyroglutamic acid with the concomitant liberation of ammonia. QCs may be classified in two groups containing, respectively, the mammalian enzymes, and the enzymes from plants, bacteria, and parasites. The crystal structure of the QC from the latex of Carica papaya (PQC) has been determined at 1.7Å resolution. The structure was solved by the single wavelength anomalous diffraction technique using sulfur and zinc as anomalous scatterers. The enzyme folds into a five-bladed β-propeller, with two additional α-helices and one β hairpin. The propeller closure is achieved via an original molecular velcro, which links the last two blades into a large eight stranded β-sheet. The zinc ion present in the PQC is bound via an octahedral coordination into an elongated cavity located along the pseudo 5-fold axis of the β-propeller fold. This zinc ion presumably plays a structural role and may contribute to the exceptional stability of PQC, along with an extended hydrophobic packing, the absence of long loops, the three-joint molecular velcro and the overall folding itself. Multiple sequence alignments combined with structural analyses have allowed us to tentatively locate the active site, which is filled in the crystal structure either by a Tris molecule or an acetate ion. These analyses are further supported by the experimental evidence that Tris is a competitive inhibitor of PQC. The active site is located at the C-terminal entrance of the PQC central tunnel. W83, W110, W169, Q24, E69, N155, K225, F22 and F67 are highly conserved residues in the C-terminal entrance, and their putative role in catalysis is discussed. The PQC structure is representative of the plants, bacterial and parasite enzymes and contrasts with that of mammalian enzymes, that may possibly share a conserved scaffold of the bacterial aminopeptidase. [Copyright &y& Elsevier]

Integral outer membrane transporters of the Omp85/TpsB superfamily mediate the translocation of proteins across, or their integration into, the outer membranes of Gram-negative bacteria, chloroplasts, and mitochondria. The Bordetella pertussis FhaC/FHA couple serves as a model for the two-partner secretion pathway in Gram-negative bacteria, with the TpsB protein, FhaC, being the specific transporter of its TpsA partner, FHA, across the outer membrane. In this work, we have investigated the structure/function relationship of FhaC by analyzing the ion channel properties of the wild type protein and a collection of mutants with varied FHA secretion activities. We demonstrated that the channel is formed by the C-terminal two-thirds of FhaC most likely folding into a β-barrel domain predicted to be conserved throughout the family. A C-proximal motif that represents the family signature appears essential for pore function. The N-terminal 200 residues of FhaC constitute a functionally distinct domain that modulates the pore properties and may participate in FHA recognition. [ABSTRACT FROM AUTHOR]

Osmoregulated periplasmic glucans (OPGs) G protein (OpgG) is required for OPGs biosynthesis. OPGs from Escherichia coli are branched glucans, with a backbone of β-1,2 glucose units and with branches attached by β-1,6 linkages. In Proteobacteria, OPGs are involved in osmoprotection, biofilm formation, virulence and resistance to antibiotics. Despite their important biological implications, enzymes synthesizing OPGs are poorly characterized. Here, we report the 2.5Å crystal structure of OpgG from E.coli. The structure was solved using a selenemethionine derivative of OpgG and the multiple anomalous diffraction method (MAD). The protein is composed of two β-sandwich domains connected by one turn of 310 helix. The N-terminal domain (residues 22–388) displays a 25-stranded β-sandwich fold found in several carbohydrate-related proteins. It exhibits a large cleft comprising many aromatic and acidic residues. This putative binding site shares some similarities with enzymes such as galactose mutarotase and glucodextranase, suggesting a potential catalytic role for this domain in OPG synthesis. On the other hand, the C-terminal domain (residues 401–512) has a seven-stranded immunoglobulin-like β-sandwich fold, found in many proteins where it is mainly implicated in interactions with other molecules. The structural data suggest that OpgG is an OPG branching enzyme in which the catalytic activity is located in the large N-terminal domain and controlled via the smaller C-terminal domain. [Copyright &y& Elsevier]

Probing the solution structure of membrane proteins represents a formidable challenge, particularly when using small-angle scattering. Detergent molecules often present residual scattering contributions even at their match point in small-angle neutron scattering (SANS) measurements. Here, we studied the conformation of FhaC, the outer-membrane, β-barrel transporter of the Bordetella pertussis filamentous hemagglutinin adhesin. SANS measurements were performed on homogeneous solutions of FhaC solubilized in n-octyl-d17-βD-glucoside and on a variant devoid of the α helix H1, which critically obstructs the FhaC pore, in two solvent conditions corresponding to the match points of the protein and the detergent, respectively. Protein-bound detergent amounted to 142 ± 10 mol/mol as determined by analytical ultracentrifugation. By using molecular modeling and starting from three distinct conformations of FhaC and its variant embedded in lipid bilayers, we generated ensembles of protein-detergent arrangement models with 120-160 detergent molecules. The scattered curves were back-calculated for each model and compared with experimental data. Good fits were obtained for relatively compact, connected detergent belts, which occasionally displayed small detergent-free patches on the outer surface of the β barrel. The combination of SANS and modeling clearly enabled us to infer the solution structure of FhaC, with H1 inside the pore as in the crystal structure. We believe that our strategy of combining explicit atomic detergent modeling with SANS measurements has significant potential for structural studies of other detergent-solubilized membrane proteins., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Proteins with both peptidylprolyl isomerase (PPIase) and chaperone activities play a crucial role in protein folding in the periplasm of Gram-negative bacteria. Few such proteins have been structurally characterized and to date only the crystal structure of SurA from Escherichia coli has been reported. Par27, the prototype of a new group of parvulins, has recently been identified. Par27 exhibits both chaperone and PPIase activities in vitro and is the first identified parvulin protein that forms dimers in solution. Par27 has been expressed in E. coli. The protein was purified using affinity and gel-filtration chromatographic techniques and crystallized in two different crystal forms. Form A, which belongs to space group P2 (unit-cell parameters a = 42.2, b = 142.8, c = 56.0 A, beta = 95.1 degrees ), diffracts to 2.8 A resolution, while form B, which belongs to space group C222 (unit-cell parameters a = 54.6, b = 214.1, c = 57.8 A), diffracts to 2.2 A resolution. Preliminary diffraction data analysis agreed with the presence of one monomer in the asymmetric unit of the orthorhombic crystal form and two in the monoclinic form.