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The adenosine di-phosphate (ADP) ribosylation factor (Arf) small guanosine tri-phosphate (GTP)ases function as molecular switches to activate signaling cascades that control membrane organization in eukaryotic cells. In Arf1, the GDP/GTP switch does not occur spontaneously but requires guanine nucleotide exchange factors (GEFs) and membranes. Exchange involves massive conformational changes, including disruption of the core β-sheet. The mechanisms by which this energetically costly switch occurs remain to be elucidated. To probe the switch mechanism, we coupled pressure perturbation with nuclear magnetic resonance (NMR), Fourier Transform infra-red spectroscopy (FTIR), small-angle X-ray scattering (SAXS), fluorescence, and computation. Pressure induced the formation of a classical molten globule (MG) ensemble. Pressure also favored the GDP to GTP transition, providing strong support for the notion that the MG ensemble plays a functional role in the nucleotide switch. We propose that the MG ensemble allows for switching without the requirement for complete unfolding and may be recognized by GEFs. An MG-based switching mechanism could constitute a pervasive feature in Arfs and Arf-like GTPases, and more generally, the evolutionarily related (Ras-like small GTPases) Rags and Gα GTPases. [ABSTRACT FROM AUTHOR]

NCYM is a cis-antisense gene of MYCN oncogene and encodes an oncogenic protein that stabilizes MYCN via inhibition of GSK3b. High NCYM expression levels are associated with poor clinical outcomes in human neuroblastomas, and NCYM overexpression promotes distant metastasis in animal models of neuroblastoma. Using vacuum-ultraviolet circular dichroism and small-angle X-ray scattering, we previously showed that NCYM has high flexibility with partially folded structures; however, further structural characterization is required for the design of anti-cancer agents targeting NCYM. Here we report the 1H, 15N and 13C nuclear magnetic resonance assignments of NCYM. Secondary structure prediction using Secondary Chemical Shifts and TALOS-N analysis demonstrates that the structure of NCYM is essentially disordered, even though residues in the central region of the peptide clearly present a propensity to adopt a dynamic helical structure. This preliminary study provides foundations for further analysis of interaction between NCYM and potential partners. [ABSTRACT FROM AUTHOR]

Magnaporthe AVRs and ToxB-like (MAX) effectors constitute a family of secreted virulence proteins in the fungus Pyricularia oryzae (syn. Magnaporthe oryzae), which causes blast disease on numerous cereals and grasses. In spite of high sequence divergence, MAX effectors share a common fold characterized by a ß-sandwich core stabilized by a conserved disulfide bond. In this study, we investigated the structural landscape and diversity within the MAX effector repertoire of P. oryzae. Combining experimental protein structure determination and in silico structure modeling we validated the presence of the conserved MAX effector core domain in 77 out of 94 groups of orthologs (OG) identified in a previous population genomic study. Four novel MAX effector structures determined by NMR were in remarkably good agreement with AlphaFold2 (AF2) predictions. Based on the comparison of the AF2-generated 3D models we propose a classification of the MAX effectors superfamily in 20 structural groups that vary in the canonical MAX fold, disulfide bond patterns, and additional secondary structures in N- and C-terminal extensions. About one-third of the MAX family members remain singletons, without strong structural relationship to other MAX effectors. Analysis of the surface properties of the AF2 MAX models also highlights the high variability within the MAX family at the structural level, potentially reflecting the wide diversity of their virulence functions and host targets. Author summary: MAX effectors are a family of virulence proteins from the plant pathogenic fungus Pyricularia (syn. Magnaporthe) oryzae that share a similar 3D structure despite very low amino-acid sequence identity. Characterizing the function and evolution of these proteins requires a detailed understanding of their structural diversity. With this in mind, we have determined the NMR structures of four new MAX effectors and shown a near-perfect match with the corresponding AlphaFold2 (AF2) models. We then applied a prediction pipeline based on similarity searches with structural modeling using the AF2 software to predict MAX effectors in a collection of 120 P. oryzae genomes. The resulting models and experimental structures revealed that the MAX core while preserved is highly permissive to secondary structure variations and may coexists with extensive structural diversity in terms of structured N- or C-terminal extensions permitting their classification. For a subset of AF2 models, we have also analyzed the physico-chemical properties of the core domain surfaces, adding another, more functional perspective, notably surface electrostatics and stickiness. This work constitutes a major step in understanding the relationships among MAX effectors by analyzing their structural landscape and cataloguing specific physico-chemical properties. It also provides valuable insights for guiding research into the putative targets of these effectors in infected plant hosts. [ABSTRACT FROM AUTHOR]

Human babesiosis is a vector-borne zoonotic infection caused mostly by the Apicomplexan parasite Babesia microti, distributed worldwide. The infection can result in severe symptoms such as hemolytic anemia, especially in immunodeficient patients. Also, asymptomatic patients continue transmission as unscreened blood donors, and represent a risk for Public Health. Early host-parasite interactions are mediated by BmSA1, the major surface antigen of Babesia microti, crucial for invasion and immune escape. Hence, a structural and functional characterization of the BmSA1 protein constitutes a first strategic milestone toward the development of innovative tools to control infection. Knowledge of the 3D structure of such an important antigen is crucial for the development of vaccines or new diagnostic tests. Here, we report the 1H, 15N and 13C NMR resonance assignment of ∆∆BmSA1, a truncated recombinant version of BmSA1 without the N-terminal signal peptide and the hydrophobic C-terminal GPI-anchor. Secondary structure prediction using CSI.3 and TALOS-N demonstrates a high content of alpha-helical structure. This preliminary study provides foundations for further structural characterization of BMSA1. [ABSTRACT FROM AUTHOR]

Effectors are small and very diverse proteins secreted by fungi and translocated in plant cells during infection. Among them, MAX effectors (for Magnaporthe Avrs and ToxB) were identified as a family of effectors that share an identical fold topology despite having highly divergent sequences. They are mostly secreted by ascomycetes from the Magnaporthe genus, a fungus that causes the rice blast, a plant disease leading to huge crop losses. As rice is the first source of calories in many countries, especially in Asia and Africa, this constitutes a threat for world food security. Hence, a better understanding of these effectors, including structural and functional characterization, constitutes a strategic milestone in the fight against phytopathogen fungi and may give clues for the development of resistant varieties of rice. We report here the near complete 1H, 15 N and 13C NMR resonance assignment of three new putative MAX effectors (MAX47, MAX60 and MAX67). Secondary structure determination using TALOS-N and CSI.3 demonstrates a high content of β-strands in all the three proteins, in agreement with the canonic ß-sandwich structure of MAX effectors. This preliminary study provides foundations for further structural characterization, that will help in turn to improve sequence predictions of other MAX effectors through data mining. [ABSTRACT FROM AUTHOR]

A study was conducted to examine the synthesis of disulfide bonds in endothelin-1 by determining a salt bridge and its possible role in the disulfide bond formation. Distance geometry computations were performed using a HP735 workstation supported by the DIANA program while a SANDER module was utilized to carry out energy minimization and molecular dynamic simulations. Results suggested that the cleavage of Arg52-Cys54 amide bond after the formation of native disulfide bonds.