X-Ray Crystallography for Macromolecular Complexes.

X-ray crystallography has for most of the last century been the standard technique to determine the high-resolution structure of biological macromolecules, including multi-subunit protein-protein and protein-nucleic acids as large as the ribosome and viruses. As such, the successful application of X-ray crystallography to many biological problems revolutionized biology and biomedicine by solving the structures of small molecules and vitamins, peptides and proteins, DNA and RNA molecules, and many complexes-affording a detailed knowledge of the structures that clarified biological and chemical mechanisms, conformational changes, interactions, catalysis and the biological processes underlying DNA replication, translation, and protein synthesis. Now reaching well into the first quarter of the twenty-first century, X-ray crystallography shares the structural biology stage with cryo-electron microscopy and other innovative structure determination methods, as relevant and central to our understanding of biological function and structure as ever. In this chapter, we provide an overview of modern X-ray crystallography and how it interfaces with other mainstream structural biology techniques, with an emphasis on macromolecular complexes.

Antitumor effect of a small-molecule inhibitor of KRASG12D in xenograft models of mucinous appendicular neoplasms.

Pseudomyxoma peritonei (PMP) is a rare disease characterized by a massive accumulation of mucus in the peritoneal cavity. The only effective treatment is aggressive surgery, aimed at removing all visible tumors. However, a high percentage of patients relapse, with subsequent progression and death. Recently, there has been an increase in therapies that target mutated oncogenic proteins. In this sense, KRAS has been reported to be highly mutated in PMP, with KRASG12D being the most common subtype. Here, we tested the efficacy of a small-molecule KRASG12D inhibitor, MRTX1133, in a high-grade PMP xenograft mouse model carrying a KRASG12D mutation. The results obtained in this work showed a profound inhibition of tumor growth, which was associated with a reduction in cell proliferation, an increase in apoptosis, and a reduction in the MAPK and PI3K/AKT/mTOR signaling pathways. In conclusion, these results demonstrate the high potency and efficacy of MRTX1133 in KRASG12D-PMP tumors and provide a rationale for clinical trials.

The structure of Leptospira interrogans GAPDH sheds light into an immunoevasion factor that can target the anaphylatoxin C5a of innate immunity.

Leptospirosis is a neglected worldwide zoonosis involving farm animals and domestic pets caused by the Gram-negative spirochete Leptospira interrogans. This bacterium deploys a variety of immune evasive mechanisms, some of them targeted at the complement system of the host's innate immunity. In this work, we have solved the X-ray crystallographic structure of L. interrogans glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to 2.37-Å resolution, a glycolytic enzyme that has been shown to exhibit moonlighting functions that potentiate infectivity and immune evasion in various pathogenic organisms. Besides, we have characterized the enzyme's kinetic parameters toward the cognate substrates and have proven that the two natural products anacardic acid and curcumin are able to inhibit L. interrogans GAPDH at micromolar concentration through a noncompetitive inhibition modality. Furthermore, we have established that L. interrogans GAPDH can interact with the anaphylatoxin C5a of human innate immunity in vitro using bio-layer interferometry and a short-range cross-linking reagent that tethers free thiol groups in protein complexes. To shed light into the interaction between L. interrogans GAPDH and C5a, we have also carried out cross-link guided protein-protein docking. These results suggest that L. interrogans could be placed in the growing list of bacterial pathogens that exploit glycolytic enzymes as extracellular immune evasive factors. Analysis of the docking results indicates a low affinity interaction that is consistent with previous evidence, including known binding modes of other α-helical proteins with GAPDH. These findings allow us to propose L. interrogans GAPDH as a potential immune evasive factor targeting the complement system.

The crystal structure of iC3b-CR3 αI reveals a modular recognition of the main opsonin iC3b by the CR3 integrin receptor.

Complement activation on cell surfaces leads to the massive deposition of C3b, iC3b, and C3dg, the main complement opsonins. Recognition of iC3b by complement receptor type 3 (CR3) fosters pathogen opsonophagocytosis by macrophages and the stimulation of adaptive immunity by complement-opsonized antigens. Here, we present the crystallographic structure of the complex between human iC3b and the von Willebrand A inserted domain of the α chain of CR3 (αI). The crystal contains two composite interfaces for CR3 αI, encompassing distinct sets of contiguous macroglobulin (MG) domains on the C3c moiety, MG1-MG2 and MG6-MG7 domains. These composite binding sites define two iC3b-CR3 αI complexes characterized by specific rearrangements of the two semi-independent modules, C3c moiety and TED domain. Furthermore, we show the structure of iC3b in a physiologically-relevant extended conformation. Based on previously available data and novel insights reported herein, we propose an integrative model that reconciles conflicting facts about iC3b structure and function and explains the molecular basis for iC3b selective recognition by CR3 on opsonized surfaces.

Exploring the effect of two different teaching strategies on first-year nursing students' understanding of nutritional concepts: A mixed-method approach.

AIM: The aim of the study was twofold. First, to explore understanding difficulties in nutritional concepts among nursing students and second, to analyse the effect of two teaching strategies on the level of understanding. BACKGROUND: Previous studies have pointed out the need to improve the nutritional care education offered to undergraduates in health sciences. As nurses play a crucial role in lifestyle counselling and nutrition assessment, it is essential to identify the learning difficulties that can impair their learning capacity and the more effective teaching strategy in the nutrition area during the nursing degree. DESIGN: A pretest-posttest study was conducted with a mixed-method conversion design. METHODS: 146 first-year-nursing students were included. Two teaching methods, debate-based learning and lecture-based learning were used for instruction on the effect of macronutrient intake on an individual's state of health. Each group completed two open-ended questions before and after the intervention. Firstly, phenomenographic analysis was performed to identify the learning categories representing variations in students' understanding. Secondly, qualitative data were quantised and statistically analysed to address differences in learning improvement. RESULTS: More than 40% of students struggled with the idea that proteins are associated with weight gain independently of the teaching strategy applied. In the first evaluation question, the debate-based learning teaching method improved students' learning, achieving higher levels of understanding (p < 0.05). In the second evaluation question, no significant differences were found between the two groups. CONCLUSIONS: Areas of problematic knowledge in nutrition education have been identified for the first time that will allow nurse educators to design research-based teaching strategies. Further studies are necessary not only on how to address these misconceptions in nursing students, but also to investigate how nurse educators can contribute to deeper levels of student learning.

Peroxisomal catalases from the yeasts Pichia pastoris and Kluyveromyces lactis as models for oxidative damage in higher eukaryotes.

Catalases are among the main scavengers of reactive oxygen species (ROS) present in the peroxisome, thereby preventing oxidative cellular and tissular damage. In human, multiple diseases are associated with malfunction of these organelles, which causes accumulation of ROS species and consequently the inefficient detoxification of cells. Despite intense research, much remains to be clarified about the precise molecular role of catalase in cellular homeostasis. Yeast peroxisomes and their peroxisomal catalases have been used as eukaryotic models for oxidative metabolism, ROS generation and detoxification, and associated pathologies. In order to provide reliable models for oxidative metabolism research, we have determined the high-resolution crystal structures of peroxisomal catalase from two important biotechnology and basic biology yeast models, Pichia pastoris and Kluyveromyces lactis. We have performed an extensive functional, biochemical and stability characterization of both enzymes in order to establish their differential activity profiles. Furthermore, we have analyzed the role of the peroxisomal catalase under study in the survival of yeast to oxidative burst challenges combining methanol, water peroxide, and sodium chloride. Interestingly, whereas catalase activity was induced 200-fold upon challenging the methylotrophic P. pastoris cells with methanol, the increase in catalase activity in the non-methylotrophic K. lactis was only moderate. The inhibitory effect of sodium azide and ß-mercaptoethanol over both catalases was analyzed, establishing IC50 values for both compounds that are consistent with an elevated resistance of both enzymes toward these inhibitors. Structural comparison of these two novel catalase structures allows us to rationalize the differential susceptibility to inhibitors and oxidative bursts. The inherent worth and validity of the P. pastoris and K. lactis yeast models for oxidative damage will be strengthened by the availability of reliable structural-functional information on these enzymes, which are central to our understanding of peroxisomal response toward oxidative stress.

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N 6-threonylcarbamoyladenosine TcdA Function.

We demonstrate methods for the expression and purification of tRNA(UUU) in Escherichia coli and the analysis by gel retardation assays of the binding of tRNA(UUU) to TcdA, an N6-threonylcarbamoyladenosine (t6A) dehydratase, which cyclizes the threonylcarbamoyl side chain attached to A37 in the anticodon stem loop (ASL) of tRNAs to cyclic t6A (ct6A). Transcription of the synthetic gene encoding tRNA(UUU) is induced in E. coli with 1 mM isopropyl ß-D-1-thiogalactopyranoside (IPTG) and the cells containing tRNA are harvested 24 h post-induction. The RNA fraction is purified using the acid phenol extraction method. Pure tRNA is obtained by a gel filtration chromatography that efficiently separates the small-sized tRNA molecules from larger intact or fragmented nucleic acids. To analyze TcdA binding to tRNA(UUU), TcdA is mixed with tRNA(UUU) and separated on a native agarose gel at 4 °C. The free tRNA(UUU) migrates faster, while the TcdA-tRNA(UUU) complexes undergo a mobility retardation that can be observed upon staining of the gel. We demonstrate that TcdA is a tRNA(UUU)-binding enzyme. This gel retardation assay can be used to study TcdA mutants and the effects of additives and other proteins on binding.