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Neutrophil proteinase 3 (PR3) is an important drug target for inflammatory lung diseases such as chronic obstructive pulmonary disease and cystic fibrosis. Drug discovery efforts targeting PR3 require active enzyme for in vitro characterization, such as inhibitor screening, enzymatic assays, and structural studies. Recombinant expression of active PR3 overcomes the need for enzyme supplies from human blood and in addition allows studies on the influence of mutations on enzyme activity and ligand binding. Here, we report the expression of recombinant PR3 (rPR3) using a baculovirus expression system. The purification and activation process described resulted in highly pure and active PR3. The activity of rPR3 in the presence of commercially available inhibitors was compared with human PR3 by using a fluorescence-based enzymatic assay. Purified rPR3 had comparable activity to the native human enzyme, thus being a suitable alternative for enzymatic studies in vitro. Further, we established a surface plasmon resonance-based assay to determine binding affinities and kinetics of PR3 ligands. These methods provide valuable tools for early drug discovery aiming towards treatment of lung inflammation. [ABSTRACT FROM AUTHOR]

Actins are filament-forming, highly-conserved proteins in eukaryotes. They are involved in essential processes in the cytoplasm and also have nuclear functions. Malaria parasites (Plasmodium spp.) have two actin isoforms that differ from each other and from canonical actins in structure and filament-forming properties. Actin I has an essential role in motility and is fairly well characterized. The structure and function of actin II are not as well understood, but mutational analyses have revealed two essential functions in male gametogenesis and in the oocyst. Here, we present expression analysis, high-resolution filament structures, and biochemical characterization of Plasmodium actin II. We confirm expression in male gametocytes and zygotes and show that actin II is associated with the nucleus in both stages in filament-like structures. Unlike actin I, actin II readily forms long filaments in vitro, and near-atomic structures in the presence or absence of jasplakinolide reveal very similar structures. Small but significant differences compared to other actins in the openness and twist, the active site, the D-loop, and the plug region contribute to filament stability. The function of actin II was investigated through mutational analysis, suggesting that long and stable filaments are necessary for male gametogenesis, while a second function in the oocyst stage also requires fine-tuned regulation by methylation of histidine 73. Actin II polymerizes via the classical nucleation-elongation mechanism and has a critical concentration of ~0.1 μM at the steady-state, like actin I and canonical actins. Similarly to actin I, dimers are a stable form of actin II at equilibrium. Author summary: Malaria is a parasitic infection caused by Plasmodium spp., which belong to the phylum Apicomplexa. In 2020, approximately 627000 people died from nearly 241 million malaria cases registered worldwide. In contrast to other apicomplexan parasites, Plasmodium spp. encode two actin isoforms. While actin I is part of the glideosome complex, essential for locomotion, actin II is present in the mosquito stages, where it has functions in gametogenesis and in the oocyst. The exact function of actin II is still unclear, and information at the molecular level is limited. We show here that actin II is associated with the nucleus in gametocytes and zygotes and performs specific functions requiring both long filaments and fine-tuning by methylation of histidine 73. We determined the structures of the filamentous form of actin II at near-atomic resolution and characterized its polymerization properties in vitro. Our study provides a molecular basis for the differences between actins of the malaria parasite and humans, as well as between the two parasite actin isoforms. Furthermore, the in vivo studies provide insights into the function of actin II in the parasite. [ABSTRACT FROM AUTHOR]

Malaria is responsible for half a million deaths annually and poses a huge economic burden on the developing world. The mosquito-borne parasites (Plasmodium spp.) that cause the disease depend upon an unconventional actomyosin motor for both gliding motility and host cell invasion. The motor system, often referred to as the glideosome complex, remains to be understood in molecular terms and is an attractive target for new drugs that might block the infection pathway. Here, we present the high-resolution structure of the actomyosin motor complex from Plasmodium falciparum. The complex includes the malaria parasite actin filament (PfAct1) complexed with the class XIV myosin motor (PfMyoA) and its two associated light-chains. The high-resolution core structure reveals the PfAct1:PfMyoA interface in atomic detail, while at lower-resolution, we visualize the PfMyoA light-chain binding region, including the essential light chain (PfELC) and the myosin tail interacting protein (PfMTIP). Finally, we report a bare PfAct1 filament structure at improved resolution. Author summary: We present the structure of the malaria parasite motor complex; actin 1 (PfAct1) and myosin A (PfMyoA) with its two light chains. We also report a high-resolution structure of filamentous PfAct1 that reveals new atomic details of the ATPase site, including a solvent-filled cavity. PfAct1 undergoes no conformational changes upon PfMyoA binding. The PfMyoA structure in the complex superimposes with a recent crystal structure of PfMyoA although small, but significant, structural rearrangements occur at the actomyosin binding interface. [ABSTRACT FROM AUTHOR]

The science of X‐ray free‐electron lasers (XFELs) critically depends on the performance of the X‐ray laser and on the quality of the samples placed into the X‐ray beam. The stability of biological samples is limited and key biomolecular transformations occur on short timescales. Experiments in biology require a support laboratory in the immediate vicinity of the beamlines. The XBI BioLab of the European XFEL (XBI denotes XFEL Biology Infrastructure) is an integrated user facility connected to the beamlines for supporting a wide range of biological experiments. The laboratory was financed and built by a collaboration between the European XFEL and the XBI User Consortium, whose members come from Finland, Germany, the Slovak Republic, Sweden and the USA, with observers from Denmark and the Russian Federation. Arranged around a central wet laboratory, the XBI BioLab provides facilities for sample preparation and scoring, laboratories for growing prokaryotic and eukaryotic cells, a Bio Safety Level 2 laboratory, sample purification and characterization facilities, a crystallization laboratory, an anaerobic laboratory, an aerosol laboratory, a vacuum laboratory for injector tests, and laboratories for optical microscopy, atomic force microscopy and electron microscopy. Here, an overview of the XBI facility is given and some of the results of the first user experiments are highlighted. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum causes the most lethal form of malaria. The cooperation of heat shock protein (Hsp) 70 and 90 is thought to facilitate folding of select group of cellular proteins that are crucial for cyto-protection and development of the parasites. Hsp70 and Hsp90 are brought into a functional complex that allows substrate exchange by stress inducible protein 1 (STI1), also known as Hsp70-Hsp90 organising protein (Hop). P. falciparum Hop (PfHop) co-localises and occurs in complex with the parasite cytosolic chaperones, PfHsp70-1 and PfHsp90. Here, we characterised the structure of recombinant PfHop using synchrotron radiation circular dichroism (SRCD) and small-angle X-ray scattering. Structurally, PfHop is a monomeric, elongated but folded protein, in agreement with its predicted TPR domain structure. Using SRCD, we established that PfHop is unstable at temperatures higher than 40°C. This suggests that PfHop is less stable at elevated temperatures compared to its functional partner, PfHsp70-1, that is reportedly stable at temperatures as high as 80°C. These findings contribute towards our understanding of the role of the Hop-mediated functional partnership between Hsp70 and Hsp90. [ABSTRACT FROM AUTHOR]

Plasmodium actins form very short filaments and have a noncanonical link between ATP hydrolysis and polymerization. Long filaments are detrimental to the parasites, but the structural factors constraining Plasmodium microfilament lengths have remained unknown. Using high-resolution crystallography, we show that magnesium binding causes a slight flattening of the Plasmodium actin I monomer, and subsequent phosphate release results in a more twisted conformation. Thus, the Mg-bound monomer is closer in conformation to filamentous (F) actin than the Ca form, and this likely facilitates polymerization. A coordinated potassium ion resides in the active site during hydrolysis and leaves together with the phosphate, a process governed by the position of the Arg178/Asp180-containing A loop. Asp180 interacts with either Lys270 or His74, depending on the protonation state of the histidine, while Arg178 links the inner and outer domains (ID and OD) of the actin protomer. Hence, the A loop acts as a switch between stable and unstable filament conformations, the latter leading to fragmentation. Our data provide a comprehensive model for polymerization, ATP hydrolysis and phosphate release, and fragmentation of parasite microfilaments. Similar mechanisms may well exist in canonical actins, although fragmentation is much less favorable due to several subtle sequence differences as well as the methylation of His73, which is absent on the corresponding His74 in Plasmodium actin I. [ABSTRACT FROM AUTHOR]

Tyrosine hydroxylase (TH) is a multi‐domain, homo‐oligomeric enzyme that catalyses the rate‐limiting step of catecholamine neurotransmitter biosynthesis. Missense variants of human TH are associated with a recessive neurometabolic disease with low levels of brain dopamine and noradrenaline, resulting in a variable clinical picture, from progressive brain encephalopathy to adolescent onset DOPA‐responsive dystonia (DRD). We expressed isoform 1 of human TH (hTH1) and its dystonia‐associated missense variants in E. coli, analysed their quaternary structure and thermal stability using size‐exclusion chromatography, circular dichroism, multi‐angle light scattering, transmission electron microscopy, small‐angle X‐ray scattering and assayed hydroxylase activity. Wild‐type (WT) hTH1 was a mixture of enzymatically stable tetramers (85.6%) and octamers (14.4%), with little interconversion between these species. We also observed small amounts of higher order assemblies of long chains of enzyme by transmission electron microscopy. To investigate the role of molecular assemblies in the pathogenesis of DRD, we compared the structure of WT hTH1 with the DRD‐associated variants R410P and D467G that are found in vicinity of the predicted subunit interfaces. In contrast to WT hTH1, R410P and D467G were mixtures of tetrameric and dimeric species. Inspection of the available structures revealed that Arg‐410 and Asp‐467 are important for maintaining the stability and oligomeric structure of TH. Disruption of the normal quaternary enzyme structure by missense variants is a new molecular mechanism that may explain the loss of TH enzymatic activity in DRD. Unstable missense variants could be targets for pharmacological intervention in DRD, aimed to re‐establish the normal oligomeric state of TH. Missense mutations of the tyrosine hydroxylase (TH) gene, encoding a multi‐domain, homo‐oligomeric enzyme, are associated with a recessive neurometabolic disorder (Dopa‐responsive dystonia). We performed a detailed in vitro structural and functional characterization of the recombinant human protein in solution, including disease‐associated variants affecting subunit interfaces. We suggest that while wild‐type full‐length TH exists in octameric and tetrameric forms, some disease‐related mutations can disrupt the dimer–dimer interface, causing the breakdown of the proper oligomeric structure of the enzyme. Size‐exclusion chromatography (left) shows the separation of the different oligomeric species, together with their respective molecular structures (right). [ABSTRACT FROM AUTHOR]

Cadmium ions can be effectively used to promote crystal growth and for experimental phasing. Here, the use of cadmium ions as a suitable anomalous scatterer at the standard wavelength of 1 Å is demonstrated. The structures of three different proteins were determined using cadmium single-wavelength anomalous dispersion (SAD) phasing. Owing to the strong anomalous signal, the structure of lysozyme could be automatically phased and built using a very low anomalous multiplicity (1.1) and low-completeness (77%) data set. Additionally, it is shown that cadmium ions can easily substitute divalent ions in ATP-divalent cation complexes. This property could be generally applied for phasing experiments of a wide range of nucleotide-binding proteins. Improvements in crystal growth and quality, good anomalous signal at standard wavelengths ( i.e. no need to change photon energy) and rapid phasing and refinement using a single data set are benefits that should allow cadmium ions to be widely used for experimental phasing. [ABSTRACT FROM AUTHOR]

Profilin is an actin monomer binding protein that provides ATP-actin for incorporation into actin filaments. In contrast to higher eukaryotic cells with their large filamentous actin structures, apicomplexan parasites typically contain only short and highly dynamic microfilaments. In apicomplexans, profilin appears to be the main monomer-sequestering protein. Compared to classical profilins, apicomplexan profilins contain an additional arm-like β-hairpin motif, which we show here to be critically involved in actin binding. Through comparative analysis using two profilin mutants, we reveal this motif to be implicated in gliding motility of Plasmodium berghei sporozoites, the rapidly migrating forms of a rodent malaria parasite transmitted by mosquitoes. Force measurements on migrating sporozoites and molecular dynamics simulations indicate that the interaction between actin and profilin fine-tunes gliding motility. Our data suggest that evolutionary pressure to achieve efficient high-speed gliding has resulted in a unique profilin-actin interface in these parasites. [ABSTRACT FROM AUTHOR]

Background: Eukaryotic tRNA splicing is an essential process in the transformation of a primary tRNA transcript into a mature functional tRNA molecule. 5'-phosphate ligation involves two steps: a healing reaction catalyzed by polynucleotide kinase (PNK) in association with cyclic phosphodiesterase (CPDase), and a sealing reaction catalyzed by an RNA ligase. The enzymes that catalyze tRNA healing in yeast and higher eukaryotes are homologous to the members of the 2H phosphoesterase superfamily, in particular to the vertebrate myelin enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase). Results: We employed different biophysical and biochemical methods to elucidate the overall structural and functional features of the tRNA healing enzymes yeast Trl1 PNK/CPDase and lancelet PNK/CPDase and compared them with vertebrate CNPase. The yeast and the lancelet enzymes have cyclic phosphodiesterase and polynucleotide kinase activity, while vertebrate CNPase lacks PNK activity. In addition, we also show that the healing enzymes are structurally similar to the vertebrate CNPase by applying synchrotron radiation circular dichroism spectroscopy and small-angle X-ray scattering. Conclusions: We provide a structural analysis of the tRNA healing enzyme PNK and CPDase domains together. Our results support evolution of vertebrate CNPase from tRNA healing enzymes with a loss of function at its N-terminal PNK-like domain. [ABSTRACT FROM AUTHOR]

Rhoptries are club-shaped, regulated secretory organelles that cluster at the apical pole of apicomplexan parasites. Their discharge is essential for invasion and the establishment of an intracellular lifestyle. Little is known about rhoptry biogenesis and recycling during parasite division. In Toxoplasma gondii, positioning of rhoptries involves the armadillo repeats only protein (ARO) and myosin F (MyoF). Here, we show that two ARO partners, ARO-interacting protein (AIP) and adenylate cyclase β (ACβ) localize to a rhoptry subcompartment. In absence of AIP, ACβ disappears from the rhoptries. By assessing the contribution of each ARO armadillo (ARM) repeat, we provide evidence that ARO is multifunctional, participating not only in positioning but also in clustering of rhoptries. Structural analyses show that ARO resembles the myosin-binding domain of the Caenorhabditis elegans myosin chaperone UNC-45. A conserved patch of aromatic and acidic residues denotes the putative MyoF-binding site, and the overall arrangement of the ARM repeats explains the dramatic consequences of deleting each of them. Finally, Plasmodium falciparum ARO functionally complements ARO depletion and interacts with the same partners, highlighting the conservation of rhoptry biogenesis in Apicomplexa. [ABSTRACT FROM AUTHOR]

Apicomplexan parasites are the causative agents of notorious human and animal diseases that give rise to considerable human suffering and economic losses worldwide. The most prominent parasites of this phylum are the malaria-causing Plasmodium species, which are widespread in tropical and subtropical regions, and Toxoplasma gondii, which infects one third of the world's population. These parasites share a common form of gliding motility which relies on an actin-myosin motor. The components of this motor and the actin-regulatory proteins in Apicomplexa have unique features compared with all other eukaryotes. This, together with the crucial roles of these proteins, makes them attractive targets for structure-based drug design. In recent years, several structures of glideosome components, in particular of actins and actin regulators from apicomplexan parasites, have been determined, which will hopefully soon allow the creation of a complete molecular picture of the parasite actin-myosin motor and its regulatory machinery. Here, current knowledge of the function of this motor is reviewed from a structural perspective. [ABSTRACT FROM AUTHOR]

Coronins are involved in the regulation of actin dynamics in a multifaceted way, participating in cell migration and vesicular trafficking. Apicomplexan parasites, which exhibit an actin-dependent gliding motility that is essential for traversal through tissues, as well as invasion of and egress from host cells, express only a single coronin, whereas higher eukaryotes possess several isoforms. We set out to characterize the 3-D structure, biochemical function, subcellular localization, and genetic ablation of Toxoplasma gondii coronin (TgCOR), to shed light on its biological role. A combination of X-ray crystallography, small-angle scattering of X-rays, and light scattering revealed the atomic structure of the conserved WD40 domain and the dimeric arrangement of the full-length protein. TgCOR binds to F-actin and increases the rate and extent of actin polymerization. In vivo, TgCOR relocalizes transiently to the posterior pole of motile and invading parasites, independent of actin dynamics, but concomitant to microneme secretory organelle discharge. TgCOR contributes to, but is not essential for, invasion and egress. Taken together, our data point toward a role for TgCOR in stabilizing newly formed, short filaments and F-actin cross-linking, as well as functions linked to endocytosis and recycling of membranes. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is one of the most widely spread parasitic organisms in the world. Together with other apicomplexan parasites, it utilizes a special actin-myosin motor for its cellular movement, called gliding motility. This actin-based process is regulated by a small set of actin-binding proteins, which in Apicomplexa comprises only 10-15 proteins, compared with >150 in higher eukaryotes. Coronin is a highly conserved regulator of the actin cytoskeleton, but its functions, especially in parasites, have remained enigmatic. Coronins consist of an N-terminal actin-binding β-propeller WD40 domain, followed by a conserved region, and a C-terminal coiled-coil domain implicated in oligomerization. Here, the WD40 domain and the conserved region of coronin from T. gondii were produced recombinantly and crystallized. A single-wavelength diffraction data set was collected to a resolution of 1.65 Å. The crystal belonged to the orthorhombic space group C2221, with unit-cell parameters a = 55.13, b = 82.51, c = 156.98 Å. [ABSTRACT FROM AUTHOR]

Actins are highly conserved proteins and key players in central processes in all eukaryotic cells. The two actins of the malaria parasite are among the most divergent eukaryotic actins and also differ from each other more than isoforms in any other species. Microfilaments have not been directly observed in Plasmodium and are presumed to be short and highly dynamic. We show that actin I cannot complement actin II in male gametogenesis, suggesting critical structural differences. Cryo-EM reveals that Plasmodium actin I has a unique filament structure, whereas actin II filaments resemble canonical F-actin. Both Plasmodium actins hydrolyze ATP more efficiently than α-actin, and unlike any other actin, both parasite actins rapidly form short oligomers induced by ADP. Crystal structures of both isoforms pinpoint several structural changes in the monomers causing the unique polymerization properties. Inserting the canonical D-loop to Plasmodium actin I leads to the formation of long filaments in vitro. In vivo, this chimera restores gametogenesis in parasites lacking actin II, suggesting that stable filaments are required for exflagellation. Together, these data underline the divergence of eukaryotic actins and demonstrate how structural differences in the monomers translate into filaments with different properties, implying that even eukaryotic actins have faced different evolutionary pressures and followed different paths for developing their polymerization properties. [ABSTRACT FROM AUTHOR]

P2 is a fatty acid-binding protein expressed in vertebrate peripheral nerve myelin, where it may function in bilayer stacking and lipid transport. P2 binds to phospholipid membranes through its positively charged surface and a hydrophobic tip, and accommodates fatty acids inside its barrel structure. The structure of human P2 refined at the ultrahigh resolution of 0.93 Å allows detailed structural analyses, including the full organization of an internal hydrogen-bonding network. The orientation of the bound fatty-acid carboxyl group is linked to the protonation states of two coordinating arginine residues. An anion-binding site in the portal region is suggested to be relevant for membrane interactions and conformational changes. When bound to membrane multilayers, P2 has a preferred orientation and is stabilized, and the repeat distance indicates a single layer of P2 between membranes. Simulations show the formation of a double bilayer in the presence of P2, and in cultured cells wildtype P2 induces membrane-domain formation. Here, the most accurate structural and functional view to date on P2, a major component of peripheral nerve myelin, is presented, showing how it can interact with two membranes simultaneously while going through conformational changes at its portal region enabling ligand transfer. [ABSTRACT FROM AUTHOR]

Helminthic parasites of the genus Schistosoma contain a tegumental membrane, which is of crucial importance for modulation of the host immune response and parasite survival. The actin cytoskeleton plays an important role in the function of the tegument. Profilins are among the most important proteins regulating actin dynamics. Schistosoma japonicum possesses one profilin-like protein, which has been characterized as a potential vaccine candidate. Notably, profilins are highly immunogenic molecules in many organisms. Here, the profilin from S. japonicum was expressed, purified and crystallized. A native data set to 1.91 Å resolution and a single-wavelength anomalous diffraction (SAD) data set to a resolution of 2.2 Å were collected. The crystals belonged to space group P212121, with unit-cell parameters a = 31.82, b = 52.17, c = 59.79 Å and a = 35.29, b = 52.15, c = 59.82 Å, respectively. [ABSTRACT FROM AUTHOR]

The soil bacterium Streptomyces reticuli secretes the octameric protein HbpS that acts as a sensory component of the redox-signalling pathway HbpS-SenS-SenR. This system modulates a genetic response on iron- and haem-mediated oxidative stress. Moreover, HbpS alone provides this bacterium with a defence mechanism to the presence of high concentrations of iron ions and haem. While the protection against haem has been related to its haem-binding and haem-degrading activity, the interaction with iron has not been studied in detail. In this work, we biochemically analyzed the iron-binding activity of a set of generated HbpS mutant proteins and present evidence showing the involvement of one internal and two exposed D/EXXE motifs in binding of high quantities of ferrous iron, with the internal E78XXE81 displaying the tightest binding. We additionally show that HbpS is able to oxidize ferrous to ferric iron ions. Based on the crystal structure of both the wild-type and the mutant HbpS-D78XXD81, we conclude that the local arrangement of the side chains from the glutamates in E78XXE81 within the octameric assembly is a pre-requisite for interaction with iron. The data obtained led us to propose that the exposed and the internal motif build a highly specific route that is involved in the transport of high quantities of iron ions into the core of the HbpS octamer. Furthermore, physiological studies using Streptomyces transformants secreting either wild-type or HbpS mutant proteins and different redox-cycling compounds led us to conclude that the iron-sequestering activity of HbpS protects these soil bacteria from the hazardous side effects of peroxide- and iron-based oxidative stress. [ABSTRACT FROM AUTHOR]

The ADF/cofilin family has been characterized as a group of actin-binding proteins critical for controlling the assembly of actin within the cells. In this study, the solution structure of the ADF/cofilin from Trypanosoma brucei (TbCof) was determined by NMR spectroscopy. TbCof adopts the conserved ADF/cofilin fold with a central b-sheet composed of six b- strands surrounded by five a-helices. Isothermal titration calorimetry experiments denoted a submicromolar affinity between TbCof and G-actin, and the affinity between TbCof and ADP-G-actin was five times higher than that between TbCof and ATP-G-actin at low ionic strength. The results obtained from electron microscopy and actin filament sedimentation assays showed that TbCof depolymerized but did not co-sediment with actin filaments and its ability of F- actin depolymerization was pH independent. Similar to actin, TbCof was distributed throughout the cytoplasm. All our data indicate a structurally and functionally conserved ADF/cofilin from Trypanosoma brucei. [ABSTRACT FROM AUTHOR]

Apicomplexan parasites, such as the malaria-causing Plasmodium species, utilize a unique way of locomotion and host cell invasion. This substrate-dependent gliding motility requires rapid cycling of actin between the monomeric state and very short, unbranched filaments. Despite the crucial role of actin polymerization for the survival of the malaria parasite, the majority of Plasmodium cellular actin is present in the monomeric form. Plasmodium lacks most of the canonical actin nucleators, and formins are essentially the only candidates for this function in all Apicomplexa. The malaria parasite has two formins, containing conserved formin homology (FH) 2 and rudimentary FH1 domains. Here, we show that Plasmodium falciparum formin 1 associates with and nucleates both mammalian and Plasmodium actin filaments. Although Plasmodium profilin alone sequesters actin monomers, thus inhibiting polymerization, its monomer-sequestering activity does not compete with the nucleating activity of formin 1 at an equimolar profilin-actin ratio. We have determined solution structures of P. falciparum formin 1 FH2 domain both in the presence and absence of the lasso segment and the FH1 domain, and show that the lasso is required for the assembly of functional dimers. [ABSTRACT FROM AUTHOR]

The malaria parasite Plasmodium depends on its actin-based motor system for motility and host-cell invasion. Actin-depolymerization factors are important regulatory proteins that affect the rate of actin turnover. Plasmodium has two actin-depolymerization factors which seem to have different functions and display low sequence homology to the higher eukaryotic family members. Plasmodium actin-depolymerization factors 1 and 2 have been crystallized. The crystals diffracted X-rays to maximum resolutions of 2.0 and 2.1 Å and belonged to space groups P3121 or P3221, with unit-cell parameters a = b = 68.8, c = 76.0 Å, and P21212, with unit-cell parameters a = 111.6, b = 57.9, c = 40.5 Å, respectively, indicating the presence of one or two molecules per asymmetric unit in both cases. [ABSTRACT FROM AUTHOR]

The myelin sheath is a tightly packed multilayered membrane structure insulating selected axons in the central and the peripheral nervous systems. Myelin is a biochemically unique membrane, containing a specific set of proteins. In this study, we expressed and purified recombinant human myelin P2 protein and determined its crystal structure to a resolution of 1.85 Å. A fatty acid molecule, modeled as palmitate based on the electron density, was bound inside the barrel-shaped protein. Solution studies using synchrotron radiation indicate that the crystal structure is similar to the structure of the protein in solution. Docking experiments using the high-resolution crystal structure identified cholesterol, one of the most abundant lipids in myelin, as a possible ligand for P2, a hypothesis that was proven by fluorescence spectroscopy. In addition, electrostatic potential surface calculations supported a structural role for P2 inside the myelin membrane. The potential membrane-binding properties of P2 and a peptide derived from its N terminus were studied. Our results provide an enhanced view into the structure and function of the P2 protein from human myelin, which is able to bind both monomeric lipids inside its cavity and membrane surfaces. [ABSTRACT FROM AUTHOR]

The crystal structure of aclacinomycin oxidoreductase (AknOx), a tailoring enzyme involved in the biosynthesis of the polyketide antibiotic aclacinomycin, was determined to 1.65 Å resolution by multiwavelength anomalous diffraction using data from selenomethionine-substituted crystals. The crystals belong to space group P21, with unit-cell parameters a = 68.2, b = 264.5, c = 68.2 Å, β = 119°. Analysis of the intensity statistics clearly showed the presence of pseudomerohedral twinning. The data set could also be indexed and scaled with an Rsym of 0.072 in the orthorhombic space group C2221 (unit-cell parameters a = 69.7, b = 117.5, c = 264.4 Å), indicating the possibility of pseudomerohedral twinning along the diagonal between the monoclinic a and c directions. Refinement using this twin operator resulted in an Rfree of 24.2%. A monoclinic lattice with a = c and β close to 120° can emulate a hexagonal metric, with the possibility of a threefold twin operator along the b axis and three twin domains. Refinement assuming three-domain twinning gave a final Rfree of 26.5%. The structure of AknOx can be thus refined with comparable Rfree values using either of the twin operators separately, suggesting the possibility that crystals of AknOx contain six twin domains generated by the twofold and threefold twin operators perpendicular to each other. Both twin operators coincide with noncrystallographic symmetry axes that may promote twinning. [ABSTRACT FROM AUTHOR]