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Preview: Acta Crystallographica Section F

Acta Crystallographica Section F

Acta Crystallographica Section F: Structural Biology Communications is a rapid all-electronic journal, which provides a home for short communications on the crystallization and structure of biological macromolecules. Structures determined through structur

Published: 2018-01-30


The Pex4p–Pex22p complex from Hansenula polymorpha: biophysical analysis, crystallization and X-ray diffraction characterization


Peroxisomes are a major cellular compartment of eukaryotic cells, and are involved in a variety of metabolic functions and pathways according to species, cell type and environmental conditions. Their biogenesis relies on conserved genes known as PEX genes that encode peroxin proteins. Peroxisomal membrane proteins and peroxisomal matrix proteins are generated in the cytosol and are subsequently imported into the peroxisome post-translationally. Matrix proteins containing a peroxisomal targeting signal type 1 (PTS1) are recognized by the cycling receptor Pex5p and transported to the peroxisomal lumen. Pex5p docking, release of the cargo into the lumen and recycling involve a number of peroxins, but a key player is the Pex4p–Pex22p complex described in this manuscript. Pex4p from the yeast Saccharomyces cerevisiae is a ubiquitin-conjugating enzyme that is anchored on the cytosolic side of the peroxisomal membrane through its binding partner Pex22p, which acts as both a docking site and a co-activator of Pex4p. As Pex5p undergoes recycling and release, the Pex4p–Pex22p complex is essential for monoubiquitination at the conserved cysteine residue of Pex5p. The absence of Pex4p–Pex22p inhibits Pex5p recycling and hence PTS1 protein import. This article reports the crystallization of Pex4p and of the Pex4p–Pex22p complex from the yeast Hansenula polymorpha, and data collection from their crystals to 2.0 and 2.85 Å resolution, respectively. The resulting structures are likely to provide important insights to understand the molecular mechanism of the Pex4p–Pex22p complex and its role in peroxisome biogenesis.

Human CCL5 trimer: expression, purification and initial crystallographic studies


The chemokine CCL5 is considered to be a potential therapeutic target because of its ability to recruit immune cells to inflammatory sites. CCL5 aggregates under physiological conditions, and high-order oligomer formation is considered to be significant for cell migration, immune-cell activation and HIV cell entry. The structure of the high-order oligomer is unknown and the mechanism by which the oligomer is derived has yet to be established. Here, a CCL5 mutant (CCL5-E66S) which is deficient in oligomer formation was mixed with native CCL5 to prepare a protein trimer. At an optimized ratio the trimeric CCL5 crystallized, and the crystal belonged to the tetragonal space group P41212, with unit-cell parameters a = 56.6, b = 56.6, c = 154.1 Å. The Matthews coefficient (VM) of the crystal is 2.58 Å3 Da−1 (three molecules in the asymmetric unit), with a solvent content of 52.32%. Diffraction data were collected to a resolution of 1.87 Å and the statistics indicated satisfactory data quality. The new structure will reveal the interfaces in the CCL5 oligomer, therefore assisting in understanding the mechanism of CCL5 oligomerization.

X-ray crystallographic and high-speed AFM studies of peroxiredoxin 1 from Chlamydomonas reinhardtii


Peroxiredoxins (PRXs) are a group of antioxidant enzymes that are found in all organisms, including plants and green algae. The 2-Cys PRX from Chlamydomonas reinhardtii (CrPRX1) is a chloroplast-localized protein that is critical for clearing reactive oxygen species in chloroplasts. CrPRX1 is reduced by thioredoxins or calredoxin (CrCRX), a recently identified calcium-dependent redox protein. The molecular interaction between PRXs and thioredoxin/CrCRX is functionally important, but discussion has been limited owing to a lack of structural information on CrPRX1, especially regarding its oligomeric state. In this study, high-speed atomic force microscopy (HS-AFM) images of CrPRX1 and an X-ray crystallographic analysis have enabled examination of the oligomeric state of CrPRX1. Diffraction data from a crystal of the Cys174Ser mutant of CrPRX1 indicate the existence of noncrystallographic fivefold symmetry. HS-AFM images of CrPRX1 further show that CrPRX1 particles form rings with pentagonal rotational symmetry. On the basis of these findings, the oligomeric state of CrPRX1 is discussed and it is concluded that this PRX exists in a ring-shaped decameric form comprising a pentamer of dimers.

Structure of bovine cytochrome c oxidase in the ligand-free reduced state at neutral pH


Cytochrome c oxidase (CcO), the terminal oxidase in cellular respiration, couples proton pumping to O2 reduction. Mammalian CcO resides in the inner mitochondrial membrane. Previously, a model of H-pathway proton pumping was proposed based on various CcO crystal structures. However, all previously determined structures were solved using crystals obtained at pH 5.7, which differs from the environmental pH of CcO in the inner membrane. The structures of fully oxidized and ligand-free reduced CcO at pH 7.3 have now been determined. Structural comparison between the oxidized and reduced states revealed that the structural alterations that occurred upon redox change at pH 5.7 in Asp51, the magnesium-containing cluster, haem groups and helix X, which provide important structural evidence for the H-pathway proton-pumping proposal, also occur at pH 7.3. These structural alterations were restricted to a local region of CcO; no domain movement was detected, nor were significant structural alterations detected in peripheral regions at either pH value. These observations indicate that the small and precise structural alterations that occur over the course of the reaction cycle are not affected by pH change, and that isolated CcO precisely performs proton pumping via the H-pathway over a wide pH range. Because the pH is not uniform across the molecular surface of CcO, the fact that the overall structure of CcO is not affected by pH changes ensures the high enzymatic efficiency of this protein in the mitochondria.

Ribokinase from Leishmania donovani: purification, characterization and X-ray crystallographic analysis


Leishmania is an auxotrophic protozoan parasite which acquires d-ribose by transporting it from the host cell and also by the hydrolysis of nucleosides. The enzyme ribokinase (RK) catalyzes the first step of ribose metabolism by phosphorylating d-ribose using ATP to produce d-ribose-5-phosphate. To understand its structure and function, the gene encoding RK from L. donovani was cloned, expressed and purified using affinity and size-exclusion chromatography. Circular-dichroism spectroscopy of the purified protein showed comparatively more α-helix in the secondary-structure content, and thermal unfolding revealed the Tm to be 317.2 K. Kinetic parameters were obtained by functional characterization of L. donovani RK, and the Km values for ribose and ATP were found to be 296 ± 36 and 116 ± 9.0 µM, respectively. Crystals obtained by the hanging-drop vapour-diffusion method diffracted to 1.95 Å resolution and belonged to the hexagonal space group P61, with unit-cell parameters a = b = 100.25, c = 126.77 Å. Analysis of the crystal content indicated the presence of two protomers in the asymmetric unit, with a Matthews coefficient (VM) of 2.45 Å3 Da−1 and 49.8% solvent content. Further study revealed that human counterpart of this protein could be used as a template to determine the first three-dimensional structure of the RK from trypanosomatid parasites.

Structural characterization of Porphyromonas gingivalis enoyl-ACP reductase II (FabK)


Enoyl-acyl carrier protein (ACP) reductase II (FabK) is a critical rate-limiting enzyme in the bacterial type II fatty-acid synthesis (FAS II) pathway. FAS II pathway enzymes are markedly disparate from their mammalian analogs in the FAS I pathway in both structure and mechanism. Enzymes involved in bacterial fatty-acid synthesis represent viable drug targets for Gram-negative pathogens, and historical precedent exists for targeting them in the treatment of diseases of the oral cavity. The Gram-negative organism Porphyromonas gingivalis represents a key causative agent of the costly and highly prevalent disease known as chronic periodontitis, and exclusively expresses FabK as its enoyl reductase enzyme in the FAS-II pathway. Together, these characteristics distinguish P. gingivalis FabK (PgFabK) as an attractive and novel narrow-spectrum antibacterial target candidate. PgFabK is a flavoenzyme that is dependent on FMN and NADPH as cofactors for the enzymatic reaction, which reduces the enoyl substrate via a ping-pong mechanism. Here, the structure of the PgFabK enzyme as determined using X-ray crystallography is reported to 1.9 Å resolution with endogenous FMN fully resolved and the NADPH cofactor partially resolved. PgFabK possesses a TIM-barrel motif, and all flexible loops are visible. The determined structure has allowed insight into the structural basis for the NADPH dependence observed in PgFabK and the role of a monovalent cation that has been observed in previous studies to be stringently required for FabK activity. The PgFabK structure and the insights gleaned from its analysis will facilitate structure-based drug-discovery efforts towards the prevention and treatment of P. gingivalis infection.

Xylanase B from Clostridium cellulovorans 743B: overexpression, purification, crystallization and X-ray diffraction analysis


Clostridium cellulovorans produces multi-enzyme complexes called cellulosomes capable of efficiently degrading cellulosic biomass. There are three xylanase genes containing a sequence corresponding to a dockerin domain that are necessary for constructing cellulosomes in the genome. Among the xylanases encoded by these genes, xylanase B (XynB) contains a catalytic domain belonging to glycoside hydrolase family 10 and a carbohydrate-binding module (CBM) at the N-terminus, making it a member of CBM family 22. In this study, XynB was cloned, overexpressed, purified and crystallized. XynB was crystallized using the hanging-drop vapour-diffusion method in the presence of 0.2 M sodium acetate trihydrate, 0.1 M Tris–HCl pH 8.5, 32%(w/v) PEG 4000 at 293 K. X-ray diffraction analysis revealed that the crystal diffracted to 1.95 Å resolution and belonged to space group P212121, with unit-cell parameters a = 74.28, b = 77.55, c = 88.20 Å, α = β = γ = 90°. The data-evaluation statistics revealed high quality of the collected data, thereby establishing a solid basis for determination of the structure of cellulosomal xylanase from C. cellulovorans.

A thermostable and alkaline GDSL-motif esterase from Bacillus sp. K91: crystallization and X-ray crystallographic analysis


The esterase Est8 from the thermophilic bacterium Bacillus sp. K91 belongs to the GDSL family and is active on a variety of acetylated compounds, including 7-aminocephalosporanic acid. In contrast to other esterases of the GDSL family, the catalytic residues Asp182 and His185 were more pivotal for the catalytic activity of Est8 than the Ser11 residue. To better understand the biochemical and enzymatic properties of Est8, recombinant Est8 protein was purified and crystallized. Crystals of Est8 were obtained by the hanging-drop vapour-diffusion method using 2.0 M ammonium sulfate, 5%(v/v) 2-propanol as the crystallization solution. X-ray diffraction data were collected to a resolution of 2.30 Å with an Rmerge of 16.4% from a crystal belonging to space group P41212 or P43212, with unit-cell parameters a = b = 68.50, c = 79.57 Å.