Acta crystallographica. Section F, Structural biology communications最新文献

Clinical and environmental isolates of Stenotrophomonas maltophilia produce an enterobactin-like siderophore that promotes bacterial growth under low-iron conditions. Although prior mutational and bioinformatic analyses indicated that most of the enzymes encoded by the S. maltophilia entCEBB′FA locus are suitably reminiscent of their counterparts in Escherichia coli and other bacteria, Stenotrophomonas EntB was unusual. In bacteria producing enterobactin-related molecules, EntB and its homologs are usually multi-domain proteins in which the amino portion acts as an isochorismatase and the carboxy domain serves as an aryl carrier protein (ArCP). However, in S. maltophilia the isochorismatase and ArCP functions are encoded by two distinct genes: entB and entB′, respectively. Current mutant analysis was used to first confirm that S. maltophilia entB is needed for siderophore activity and bacterial growth in iron-depleted media. A crystal structure of S. maltophilia EntB was then obtained. The structure aligned with the N-terminal portion of EntB from E. coli and VibB from Vibrio cholerae, affirming the protein to be a single-domain isochorismatase. However, S. maltophilia EntB also aligned with the single-domain PhzD from Pseudomonas aeruginosa, which is a key enzyme involved in the biosynthesis of the antimicrobial compound phenazine. BLASTP searches indicated that entB and its neighboring genes are fully conserved amongst S. maltophilia strains but are variably present in other Stenotrophomonas species. The closest homologs to S. maltophilia EntB outside the genus were hypothetical proteins/putative isochorismatases in some Gram-negative bacteria (for example Pseudomonas spp. and Xanthomonas spp.), Gram-positive bacteria (Streptomyces spp. and Bacillus subtilis) and fungi (for example Rhizopus arrhizus and Knufia peltigerae).

The genome of the marine bacterium Muricauda eckloniae sp. DK169 contains an extensive polysaccharide-utilization locus that targets fucoidan from brown algae. Within this locus is a gene that encodes a putative fucoidan-degrading glycoside hydrolase (locus tag AAY42_01205) assigned to glycoside hydrolase family 168, which we call MeGH168. We present the 2.0 Å resolution X-ray crystal structure of MeGH168, demonstrating a (β/α)₈-barrel fold. The eight loop regions joining each α-helix and β-strand surround the catalytic groove. A comparison with the structure of a GH168, Fun168A, in complex with a fragment of fucoidan (PDB entry 8ya7) revealed conservation of key residues in the catalytic site. However, structural variation in positive-subsite loop regions may recontour the active site to create differences in substrate specificity between the two GH168s. The present data provide additional structural insights into the GH168 family, particularly expanding on sequence and structure conservation (and the lack thereof) in relation to substrate interactions.

Crystal-based structural methods, including X-ray crystallography, are frequently utilized for the determination of high-resolution structures of biomolecules. All crystal-based diffraction methods first require the preparation of biomolecular crystals, and careful sample preparation for crystallization experiments can increase the frequency of success. In this article, strategies to optimize factors that can impact crystallization are presented, from which buffers and reducing agents are most favorable to which crystallization techniques could be used.

Surface-layer proteins (SLPs) play a crucial role in the self-assembly of bacterial surface layers, yet the structural details of their assembly domains remain largely unexplored. Here, we report the crystal structure of SlpM_C1, a structural module within the self-assembly domain of SlpM from Lactobacillus brevis. SlpM_C1 adopts a β-grasp fold, a conserved structural motif found in diverse protein families. Structural comparisons with ubiquitin and the SlpA_II domain from L. acidophilus reveal both shared and distinct features, highlighting elements of structural convergence despite sequence divergence. Furthermore, the dimerization patterns of SlpM_C1 and SlpA_II are compared and discussed. These findings provide new insights into the architecture and evolutionary adaptability of SLPs in Lactobacillus species.

Proteins of the NarJ subfamily from facultatively or obligately anaerobic bacteria play key roles as chaperones in folding and cofactor insertion for complex iron–sulfur molybdoenzymes (CISMs), which mediate energy production under anaerobic conditions. YcdY was identified as a NarJ subfamily member but was proposed to increase the catalytic activity of the non-CISM enzyme YcdX phosphatase, presumably by inserting a zinc cofactor into YcdX. To elucidate the structural features of YcdY required for its chaperone function, we determined the crystal structure of Enterobacter cloacae YcdY (enYcdY). enYcdY adopts a single-domain, curved helix-bundle structure decorated with α-helices. enYcdY contains an extensive dent on its concave side. The dent in enYcdY generally forms using hydrophobic or conserved residues. Based on comparative structural and sequence analyses, we propose that enYcdY uses the dent to recognize and fold the client protein. Interestingly, enYcdY did not increase the enzymatic activity of E. cloacae YcdX (enYcdX) in the presence or absence of Zn²⁺ ions, even for partially denatured enYCdX protein. The same results were obtained for the Escherichia coli counterparts, in contrast to a previous report. These observations suggest that YcdY functions as a chaperone for proteins other than YcdX.

The focused issue on Empowering education through structural genomics is introduced. The virtual issue is available at https://journals.iucr.org/special_issues/2024/educationsg.

Collagen lysyl hydroxylases catalyze the hydroxylation of collagen lysine residues during collagen synthesis in animals and mimiviruses. Lysyl hydroxylation is crucial for collagen fibrogenesis and function. We previously demonstrated that recombinant mimiviral and human collagen lysyl hydroxylases, isolated from bacterial and mammalian cells, have Fe²⁺ in their active sites, suggesting that lysyl hydroxylases have a high affinity for Fe²⁺. We found that Fe²⁺ binding stabilizes lysyl hydroxylase dimers, although the underlying mechanism remains unclear. Crystal structure analysis of mimiviral lysyl hydroxylase revealed that Fe²⁺ is coordinated by a 2His–1Asp (His825/His877/Asp827) triad, with a nearby highly conserved histidine residue (His869) involved in an alternative 2His–1Asp triad (His869/His877/Asp827). This unique structural architecture suggests that the alternative 2His–1Asp triad may also bind Fe²⁺. To investigate whether the alternative 2His–1Asp triad binds Fe²⁺ and how Fe²⁺ binding regulates lysyl hydroxylase dimerization, we crystallized the mimiviral lysyl hydroxylase mutant His825Ala, which lacks one 2His–1Asp (His825/His877/Asp827) triad but retains the alternative triad (His869/His877/Asp827). Despite providing Fe²⁺ during crystallization, we found no electron density near the alternative 2His–1Asp triad in the His825Ala mutant, indicating that the alternative 2His–1Asp triad does not bind Fe²⁺ with high affinity. Although the His825Ala mutant forms a dimer similar to the wild-type enzyme, conformational changes occur in residues near Ala825, including Leu873, which is critical for dimerization. These structural findings provide new insights into the function and regulation of collagen lysyl hydroxylases.

A lectin-like protein was discovered in Agaricus bisporus as part of the mushroom tyrosinase complex. The protein has a β-trefoil fold, which is typical of the ricin B-like-type lectin family. The structure of the recombinant protein has been elucidated, and its specific sugar-binding affinity towards mannose and mannitol has also been reported; therefore, the protein was named A. bisporus mannose-binding protein (Abmb). Although the sugar-binding site of Abmb is predicted to be close to the C-terminus, the sugar-binding site has not yet been determined. In this study, a variant of recombinant Abmb with a longer C-terminal region including a 6×His-tag was constructed and its structure was solved at 1.51 and 2.34 Å resolution in an orthorhombic and a monoclinic space group, respectively. The overall structure showed a β-trefoil fold as previously reported; however, several surface loop regions including the C-terminal region showed high flexibility. In addition, a glycan-search assay of this variant showed weak binding affinity towards β-d-galactose but no affinity towards α-d-mannose. The plasticity of the C-terminal tail could be related to the differences in the carbohydrate-binding affinity of Abmb.

Entamoeba histolytica causes amebiasis, a neglected disease that kills ∼100 000 people globally each year. Due to emerging drug resistance, E. histolytica is one of the target organisms for structure-based drug discovery by the Seattle Structural Genomics Center for Infectious Disease (SSGCID). Purification, crystallization and three structures of the putative drug target endoribonuclease L-PSP from E. histolytica (EhL-PSP) are presented. EhL-PSP has a two-layer α/β-sandwich with structural homology to endoribonuclease L-PSP. All three structures reveal the prototypical YjgF/YER057c/UK114 family trimer topology with accessible allosteric active sites. Citrate molecules from the crystallization solution are bound to the allosteric site in two of the three reported structures. The large allosteric site of EhL-PSP is well conserved with bacterial YjgF/YER057c/UK114 family members and could be targeted for inhibition, drug discovery or repurposing.

DNA ligases are foundational molecular-biological tools used for cloning and sequencing workflows, and are essential replicative enzymes for all cellular life forms as well as many viruses and bacteriophage. There is considerable interest in structurally and functionally characterizing novel DNA ligases and profiling their suitability for molecular-biological applications. Here, we report the crystal structure of the ATP-dependent DNA ligase from the Rhizobium phage vB_RleM_P10VF bound to a nicked DNA duplex determined to 2.2 Å resolution. The enzyme crystallized in the DNA-encircling conformation, arrested as a step 2 intermediate in the catalytic cycle with the adenylating cofactor transferred to the 5′-phosphate of the DNA nick. The overall structure of the DNA ligase closely resembles that of the T4 DNA ligase, including an α-helical globular DNA-binding domain. Several secondary-structural elements are abbreviated in the P10VF DNA ligase relative to the T4 DNA ligase enzyme, which may account for its lower specific activity, especially on DNA substrates containing double-stranded breaks.