Molecular mechanism of light-driven sodium pumping.

The light-driven sodium-pumping rhodopsin KR2 from Krokinobacter eikastus is the only non-proton cation active transporter with demonstrated potential for optogenetics. However, the existing structural data on KR2 correspond exclusively to its ground state, and show no sodium inside the protein, which hampers the understanding of sodium-pumping mechanism. Here we present crystal structure of the O-intermediate of the physiologically relevant pentameric form of KR2 at the resolution of 2.1 Å, revealing a sodium ion near the retinal Schiff base, coordinated by N112 and D116 of the characteristic NDQ triad. We also obtained crystal structures of D116N and H30A variants, conducted metadynamics simulations and measured pumping activities of putative pathway mutants to demonstrate that sodium release likely proceeds alongside Q78 towards the structural sodium ion bound between KR2 protomers. Our findings highlight the importance of pentameric assembly for sodium pump function, and may be used for rational engineering of enhanced optogenetic tools.

Pseudomonas aeruginosa Lipoxygenase LoxA Contributes to Lung Infection by Altering the Host Immune Lipid Signaling.

Pseudomonas aeruginosa is an opportunistic bacteria and a major cause of nosocomial pneumonia. P. aeruginosa has many virulence factors contributing to its ability to colonize the host. LoxA is a lipoxygenase enzyme secreted by P. aeruginosa that oxidizes polyunsaturated fatty acids. Based on previous in vitro biochemical studies, several biological roles of LoxA have been hypothesized, including interference of the host lipid signaling, and modulation of bacterial invasion properties. However, the contribution of LoxA to P. aeruginosa lung pathogenesis per se remained unclear. In this study, we used complementary in vitro and in vivo approaches, clinical strains of P. aeruginosa as well as lipidomics technology to investigate the role of LoxA in lung infection. We found that several P. aeruginosa clinical isolates express LoxA. When secreted in the lungs, LoxA processes a wide range of host polyunsaturated fatty acids, which further results in the production of bioactive lipid mediators (including lipoxin A4). LoxA also inhibits the expression of major chemokines (e.g., MIPs and KC) and the recruitment of key leukocytes. Remarkably, LoxA promotes P. aeruginosa persistence in lungs tissues. Hence, our study suggests that LoxA-dependent interference of the host lipid pathways may contribute to P. aeruginosa lung pathogenesis.

L amino acid transporter structure and molecular bases for the asymmetry of substrate interaction.

L-amino acid transporters (LATs) play key roles in human physiology and are implicated in several human pathologies. LATs are asymmetric amino acid exchangers where the low apparent affinity cytoplasmic side controls the exchange of substrates with high apparent affinity on the extracellular side. Here, we report the crystal structures of an LAT, the bacterial alanine-serine-cysteine exchanger (BasC), in a non-occluded inward-facing conformation in both apo and substrate-bound states. We crystallized BasC in complex with a nanobody, which blocks the transporter from the intracellular side, thus unveiling the sidedness of the substrate interaction of BasC. Two conserved residues in human LATs, Tyr 236 and Lys 154, are located in equivalent positions to the Na1 and Na2 sites of sodium-dependent APC superfamily transporters. Functional studies and molecular dynamics (MD) calculations reveal that these residues are key for the asymmetric substrate interaction of BasC and in the homologous human transporter Asc-1.

Structural asymmetry and disulfide bridges among subunits modulate the activity of human malonyl-CoA decarboxylase.

Decarboxylation of malonyl-CoA to acetyl-CoA by malonyl-CoA decarboxylase (MCD; EC 4.1.1.9) is an essential facet in the regulation of fatty acid metabolism. The structure of human peroxisomal MCD reveals a molecular tetramer that is best described as a dimer of structural heterodimers, in which the two subunits present markedly different conformations. This molecular organization is consistent with half-of-the-sites reactivity. Each subunit has an all-helix N-terminal domain and a catalytic C-terminal domain with an acetyltransferase fold (GNAT superfamily). Intersubunit disulfide bridges, Cys-206-Cys-206 and Cys-243-Cys-243, can link the four subunits of the tetramer, imparting positive cooperativity to the catalytic process. The combination of a half-of-the-sites mechanism within each structural heterodimer and positive cooperativity in the tetramer produces a complex regulatory picture that is further complicated by the multiple intracellular locations of the enzyme. Transport into the peroxisome has been investigated by docking human MCD onto the peroxisomal import protein peroxin 5, which revealed interactions that extend beyond the C-terminal targeting motif.

Molecular basis of substrate-induced permeation by an amino acid antiporter.

Transporters of the amino acid, polyamine and organocation (APC) superfamily play essential roles in cell redox balance, cancer, and aminoacidurias. The bacterial L-arginine/agmatine antiporter, AdiC, is the main APC structural paradigm and shares the "5 + 5 inverted repeat" fold found in other families like the Na(+)-coupled neurotransmitter transporters. The available AdiC crystal structures capture two states of its transport cycle: the open-to-out apo and the outward-facing Arg(+)-bound occluded. However, the role of Arg(+) during the transition between these two states remains unknown. Here, we report the crystal structure at 3.0 Å resolution of an Arg(+)-bound AdiC mutant (N101A) in the open-to-out conformation, completing the picture of the major conformational states during the transport cycle of the 5 + 5 inverted repeat fold-transporters. The N101A structure is an intermediate state between the previous known AdiC conformations. The Arg(+)-guanidinium group in the current structure presents high mobility and delocalization, hampering substrate occlusion and resulting in a low translocation rate. Further analysis supports that proper coordination of this group with residues Asn101 and Trp293 is required to transit to the occluded state, providing the first clues on the molecular mechanism of substrate-induced fit in a 5 + 5 inverted repeat fold-transporter. The pseudosymmetry found between repeats in AdiC, and in all fold-related transporters, restraints the conformational changes, in particular the transmembrane helices rearrangements, which occur during the transport cycle. In AdiC these movements take place away from the dimer interface, explaining the independent functioning of each subunit.

Essential role of proximal histidine-asparagine interaction in mammalian peroxidases.

In heme enzymes belonging to the peroxidase-cyclooxygenase superfamily the proximal histidine is in close interaction with a fully conserved asparagine. The crystal structure of a mixture of glycoforms of myeloperoxidase (MPO) purified from granules of human leukocytes prompted us to revise the orientation of this asparagine and the protonation status of the proximal histidine. The data we present contrast with previous MPO structures, but are strongly supported by molecular dynamics simulations. Moreover, comprehensive analysis of published lactoperoxidase structures suggest that the described proximal heme architecture is a general structural feature of animal heme peroxidases. Its importance is underlined by the fact that the MPO variant N421D, recombinantly expressed in mammalian cell lines, exhibited modified spectral properties and diminished catalytic activity compared with wild-type recombinant MPO. It completely lost its ability to oxidize chloride to hypochlorous acid, which is a characteristic feature of MPO and essential for its role in host defense. The presented crystal structure of MPO revealed further important differences compared with the published structures including the extent of glycosylation, interaction between light and heavy polypeptides, as well as heme to protein covalent bonds. These data are discussed with respect to biosynthesis and post-translational maturation of MPO as well as to its peculiar biochemical and biophysical properties.

The structures and electronic configuration of compound I intermediates of Helicobacter pylori and Penicillium vitale catalases determined by X-ray crystallography and QM/MM density functional theory calculations.

The structures of Helicobacter pylori (HPC) and Penicillium vitale (PVC) catalases, each with two subunits in the crystal asymmetric unit, oxidized with peroxoacetic acid are reported at 1.8 and 1.7 A resolution, respectively. Despite the similar oxidation conditions employed, the iron-oxygen coordination length is 1.72 A for PVC, close to what is expected for a Fe=O double bond, and 1.80 and 1.85 A for HPC, suggestive of a Fe-O single bond. The structure and electronic configuration of the oxoferryl heme and immediate protein environment is investigated further by QM/MM density functional theory calculations. Four different active site electronic configurations are considered, Por*+-FeIV=O, Por*+-FeIV=O...HisH+, Por*+-FeIV-OH+ and Por-FeIV-OH (a protein radical is assumed in the latter configuration). The electronic structure of the primary oxidized species, Por*+-FeIV=O, differs qualitatively between HPC and PVC with an A2u-like porphyrin radical delocalized on the porphyrin in HPC and a mixed A1u-like "fluctuating" radical partially delocalized over the essential distal histidine, the porphyrin, and, to a lesser extent, the proximal tyrosine residue. This difference is rationalized in terms of HPC containing heme b and PVC containing heme d. It is concluded that compound I of PVC contains an oxoferryl Por*+-FeIV=O species with partial protonation of the distal histidine and compound I of HPC contains a hydroxoferryl Por-FeIV-OH with the second oxidation equivalent delocalized as a protein radical. The findings support the idea that there is a relation between radical migration to the protein and protonation of the oxoferryl bond in catalase.

Two alternative substrate paths for compound I formation and reduction in catalase-peroxidase KatG from Burkholderia pseudomallei.

Five residues in the multifunctional catalase-peroxidase KatG of Burkholderia pesudomallei are essential for catalase, but not peroxidase, activity. Asp141 is the only one of these catalase-specific residues not related with the covalent adduct found in KatGs that when replaced with a nonacidic residue reduces catalase activity to 5% of native levels. Replacing the nearby catalytic residue Arg108 causes a reduction in catalase activity to 35% of native levels, whereas a variant with both Asp141 and Arg108 replaced exhibits near normal catalase activity (82% of native), suggesting a synergism in the roles of the two residues in support of catalase activity in the enzyme. Among the Asp141 variants, D141E is unique in retaining normal catalase activity but with modified kinetics, suggesting more favorable compound I formation and less favorable compound I reduction. The crystal structure of the D141E variant has been determined at 1.8-A resolution, revealing that the carboxylate of Glu141 is moved only slightly compared with Asp141, but retains its hydrogen bond interaction with the main chain nitrogen of Ile237. In contrast, the low temperature ferric Electron Paramagnetic Resonance spectra of the D141A, R108A, and R108A/D141A variants are consistent with modifications of the water matrix and/or the relative positioning of the distal residue side chains. Such changes explain the reduction in catalase activity in all but the double variant R108A/D141A. Two pathways of hydrogen bonded solvent lead from the entrance channel into the heme active site, one running between Asp141 and Arg108 and the second between Asp141 and the main chain atoms of residues 237-239. It is proposed that binding of substrate H(2)O(2) to Asp141 and Arg108 controls H(2)O(2) access to the heme active site, thereby modulating the catalase reaction.

Roles for Arg426 and Trp111 in the modulation of NADH oxidase activity of the catalase-peroxidase KatG from Burkholderia pseudomallei inferred from pH-induced structural changes.

Crystals of Burkholderia pseudomallei KatG retain their ability to diffract X-rays at high resolution after adjustment of the pH from 5.6 to 4.5, 6.5, 7.5, and 8.5, providing a unique view of the effect of pH on protein structure. One significant pH-sensitive change lies in the appearance of a perhydroxy group attached to the indole nitrogen of the active site Trp111 above pH 7, similar to a modification originally observed in the Ser324Thr variant of the enzyme at pH 5.6. The modification forms rapidly from molecular oxygen in the buffer with 100% occupancy after one minute of soaking of the crystal at room temperature and pH 8.5. The low temperature (4 K) ferric EPR spectra of the resting enzyme, being very sensitive to changes in the heme iron microenvironment, confirm the presence of the modification above pH 7 in native enzyme and variants lacking Arg426 or Met264 and its absence in variants lacking Trp111 or Tyr238. The indole-perhydroxy group is very likely the reactive intermediate of molecular oxygen in the NADH oxidase reaction, and Arg426 is required for its reduction. The second significant pH-sensitive change involves the buried side chain of Arg426 that changes from one predominant conformation at low pH to a second at high pH. The pH profiles of the peroxidase, catalase, and NADH oxidase reactions can be correlated with the distribution of Arg426 conformations. Other pH-induced structural changes include a number of surface-situated side chains, but there is only one change involving a displacement of main chain atoms triggered by the protonation of His53 in a deep pocket in the vicinity of the molecular 2-fold axis.

A molecular switch and electronic circuit modulate catalase activity in catalase-peroxidases.

The catalase reaction of catalase-peroxidases involves catalase-specific features built into a peroxidase core. An arginine, 20 A from the active-site heme, acts as a molecular switch moving between two conformations, one that activates heme oxidation and one that activates oxoferryl heme reduction by H(2)O(2), facilitating the catalatic pathway in a peroxidase. The influence of the arginine is imparted to the heme through its association with or dissociation from a tyrosinate that modulates reactivity through a Met-Tyr-Trp crosslinked adduct and a pi electron interaction of the heme with the adduct Trp.

Crystal structure of a putative type I restriction-modification S subunit from Mycoplasma genitalium.

The crystal structure of the eubacteria Mycoplasma genitalium ORF MG438 polypeptide, determined by multiple anomalous dispersion and refined at 2.3 A resolution, reveals the organization of S subunits from the Type I restriction and modification system. The structure consists of two globular domains, with about 150 residues each, separated by a pair of 40 residue long antiparallel alpha-helices. The globular domains correspond to the variable target recognition domains (TRDs), as previously defined for S subunits on sequence analysis, while the two helices correspond to the central (CR1) and C-terminal (CR2) conserved regions, respectively. The structure of the MG438 subunit presents an overall cyclic topology with an intramolecular 2-fold axis that superimposes the N and the C-half parts, each half containing a globular domain and a conserved helix. TRDs are found to be structurally related with the small domain of the Type II N6-adenine DNA MTase TaqI. These relationships together with the structural peculiarities of MG438, in particular the presence of the intramolecular quasi-symmetry, allow the proposal of a model for S subunits recognition of their DNA targets in agreement with previous experimental results. In the crystal, two subunits of MG438 related by a crystallographic 2-fold axis present a large contact area mainly involving the symmetric interactions of a cluster of exposed hydrophobic residues. Comparison with the recently reported structure of an S subunit from the archaea Methanococcus jannaschii highlights the structural features preserved despite a sequence identity below 20%, but also reveals important differences in the globular domains and in their disposition with respect to the conserved regions.

Characterization of a large subunit catalase truncated by proteolytic cleavage.

The large subunit catalase HPII from Escherichia coli can be truncated by proteolysis to a structure similar to small subunit catalases. Mass spectrometry analysis indicates that there is some heterogeneity in the precise cleavage sites, but approximately 74 N-terminal residues, 189 C-terminal residues, and a 9-11-residue internal fragment, including residues 298-308, are removed. Crystal structure refinement at 2.8 A reveals that the tertiary and quaternary structure of the native enzyme is retained with only very subtle changes despite the loss of 36% of the sequence. The truncated variant exhibits a 1.8 times faster turnover rate and enhanced sensitivity to high concentrations of H(2)O(2), consistent with easier access of the substrate to the active site. In addition, the truncated variant is more sensitive to inhibition, particularly by reagents such as aminotriazole and azide which are larger than substrate H(2)O(2). The main channel leading to the heme cavity is largely unaffected by the truncation, but the lateral channel is shortened and its entrance widened by removal of the C-terminal domain, providing an explanation for easier access to the active site. Opening of the entrance to the lateral channel also opens the putative NADPH binding site, but NADPH binding could not be demonstrated. Despite the lack of bound NADPH, the compound I species of both native and truncated HPII are reduced back to the resting state with compound II being evident in the absorbance spectrum only of the heme b-containing H392A variant.

Structural characterization of the Ser324Thr variant of the catalase-peroxidase (KatG) from Burkholderia pseudomallei.

The Ser315Thr variant of the catalase-peroxidase KatG from Mycobacterium tuberculosis imparts resistance to the pro-drug isonicotinic acid hydrazide (isoniazid) through a failure to convert it to the active drug, isonicotinoyl-NAD. The equivalent variant in KatG from Burkholderia pseudomallei, Ser324Thr, has been constructed, revealing catalase and peroxidase activities that are similar to those of the native enzyme. The other activities of the variant protein, including the NADH oxidase, the isoniazid hydrazinolysis and isonicotinoyl-NAD synthase activities are reduced by 60-70%. The crystal structure of the variant differs from that of the native enzyme in having the methyl group of Thr324 situated in the entrance channel to the heme cavity, in a modified water matrix in the entrance channel and heme cavity, in lacking the putative perhydroxy modification on the heme, in the multiple locations of a few side-chains, and in the presence of an apparent perhydroxy modification on the indole nitrogen atom of the active-site Trp111. The position of the methyl group of Thr324 creates a constriction or narrowing of the channel leading to the heme cavity, providing an explanation for the lower reactivity towards isoniazid and the slower rate of isonicotinoyl-NAD synthesis.

Structure of the C-terminal domain of the catalase-peroxidase KatG from Escherichia coli.

Catalase-peroxidases or KatGs, the apparent in vivo activators of the anti-tubercular pro-drug isoniazid, are active as homodimers, each subunit having two distinct but sequence- and structure-related domains. The N-terminal domain contains the haem group and is catalytically active, while the C-terminal domain lacks the cofactor. The C-terminal domain of KatG from Escherichia coli is expressed as a soluble protein which has been crystallized in triclinic, orthorhombic and tetragonal crystal forms. Packing in the orthorhombic crystals, with eight molecules in the asymmetric unit, follows the pattern of commensurate modulated structures, which explains the diversity of pseudo-origin peaks observed in the native Patterson map. The different crystal forms arise from variations in the length and sequence of the N-terminal extensions in the different constructs. Despite the variability in the N-terminal region, the overall domain conformations beginning with Pro437 are very similar both to each other and to the C-terminal domains within the native structures of the KatGs from Haloarcula marismortui and Burkholderia pseudomallei. Some structural reorganization in the C-terminal domain relative to the N-terminal domain has evolved to compensate for the absence of the haem group. A high percentage of the residues in the C-terminal domains of KatG proteins from different sources are highly conserved and these residues are spread uniformly throughout the domain. The easily folded nature and retention of structure in the C-terminal domain suggests that it may serve as a platform for the folding of the N-terminal domain and for stabilization of the molecular dimer.

Crystallization and preliminary X-ray analysis of the catalase-peroxidase KatG from Burkholderia pseudomallei.

The bifunctional catalase-peroxidase KatG encoded by the katG gene of Burkholderia pseudomallei has a predicted subunit size of 81.6 kDa. It shows high sequence similarity to other catalase-peroxidases of bacterial, archaebacterial and fungal origin, including 64% identity to KatG from Mycobacterium tuberculosis and lesser sequence similarity to members of the plant peroxidase family. Crystals from this protein were grown in 16-20% PEG 4000, 20% 2-methyl-2,4-pentanediol and 0.1 M sodium citrate pH 5.6 by the hanging-drop vapour-diffusion method at 293 K. These crystals diffracted beyond 1.8 A resolution and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 100.9, b = 115.6, c = 175.2 A. The data are consistent with either a monomer or a dimer in the crystal asymmetric unit.

Crystallization and preliminary X-ray analysis of the hydroperoxidase I C-terminal domain from Escherichia coli.

Hydroperoxidases (HP) are normally large haem-containing bifunctional enzymes capable of acting as both catalases and peroxidases. The C-terminal domain of HPI from Escherichia coli (KatG), extending from residue Tyr422 to Leu726, was found to be resistant to trypsin proteolysis. The segment of katG encoding this domain was cloned and overexpressed to produce a haemless protein that is soluble even at concentrations above 30 mg ml(-1). This protein shows a 25% sequence identity with cytochrome c peroxidase (CCP) from Saccharomyces cerevisae, despite lacking the characteristic catalytic and iron-binding residues. Crystals from this protein were grown in 0.6 M sodium citrate buffered to pH 7.5 with HEPES by the hanging-drop vapour-diffusion method at 293 K. These crystals diffracted beyond 2.0 A resolution and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 84.2, b = 98.7, c = 302.8 A. Three pseudo-origin peaks in the Patterson maps indicate an unusual packing compatible with the presence of three molecules in the crystal asymmetric unit and a solvent content of about 80% by volume.