[This corrects the article DOI: 10.1371/journal.ppat.1012176.].

Babesiosis is a tick-borne disease that poses a significant threat to animal health worldwide. In addition, climate change and the risk of human-to-human transmission through blood transfusion have made babesiosis an emerging disease in humans. Babesiosis is caused by the intraerythrocytic development of protozoan parasites from the genus Babesia, which belongs to the apicomplexan phylum that notably includes the more-widely studied causative agent of malaria, Plasmodium falciparum. Of the several hundred Babesia species identified so far, only a few are known to infect humans, with B. microti being the most prevalent and responsible for most of the clinical cases reported to date. There is no licensed vaccine for B. microti, and the development of a reliable serological diagnostic test would contribute to ensuring the safety of blood transfusions. The identification and characterization of parasite surface proteins are important steps in achieving this aim. One such protein is the GPI-anchored Major Surface Antigen BmSA1 (also known as BmGPI12), which is expressed at high levels at the surface of the merozoite. We present here the high-resolution solution structure of the 28 kDa structured core of BmSA1 (∆∆BmSA1) obtained through NMR spectroscopy. The structure of BmSA1 appears unrelated to the previously published structures of the major surface antigens of B. divergens (Bd37) or of B. canis (Bc28.1), which are thought to play a similar role in parasite invasion. We also define the erythrocyte binding function of ∆∆BmSA1, using NMR spectroscopy to map the binding interface. Finally, we used bioinformatic tools to map the potential epitopes of antibodies at the surface of the structured core of BmSA1.

Fungal effectors play crucial roles in plant infection. Despite low sequence identity, they were recently discovered to belong to families with similar three-dimensional structures. In this study, we elucidated the structures of Zt-NIP1 and Mycgr3-91409-2 effectors of the wheat fungal pathogen Zymoseptoria tritici using X-ray crystallography and NMR. These effectors displayed a structural homology with, respectively, KP4 and KP6α killer toxins from UmV dsRNA viruses of the maize fungal pathogen Ustilago maydis. Consequently, Zt-NIP1 and Mycgr3-91409-2 were renamed Zt-KP4-1 and Zt-KP6-1. Orthologues and paralogues of Zt-KP4-1 and Zt-KP6-1 were identified in Zymoseptoria, but not in other fungi, except ECP2 effectors related to Zt-KP4-1. Assessment of the biological activities of Zt-KP6-1 and Zt-KP4-1 revealed their ability to inhibit fungal growth, but they were unable to induce wheat leaf necrosis. A novel pipeline relying on cysteine-pattern constrained HMM searches and Foldseek analysis of AlphaFold2 predicted structures from Uniprot generated a comprehensive inventory of KP4 and KP6 proteins in fungi and plants. Their structure-based classification revealed four KP4 and three KP6 structural superfamilies and provided far-reaching hypotheses on their biological function and evolution. This framework highlights the power of structure determination and modelling for the classification of effectors and their functional investigation.

Proteins unfold under different environmental insults, among which are heat, cold, high pressure, and chaotropic agents. Understanding the mechanisms that determine unfolding under each of these conditions is an important problem that directly relates to the physical forces that determine the three-dimensional structure of a protein. Here, we studied a residue-specific description of the unfolding transitions of marginally stable yeast protein Yfh1 using high-pressure nuclear magnetic resonance. We compared the cold, heat, and pressure unfolded states and demonstrated what has up to now been only a hypothesis: the pressure-unfolded spectrum at room temperature shares features in common with that at low but not at high temperature and room pressure, suggesting a tighter similarity of the mechanisms and a similar role of hydration in these two processes. By exploring the phase diagram of the protein and mapping unfolding onto the three-dimensional structure of the protein, we also show that the pressure-induced unfolding pathways at low and high temperatures differ, suggesting a synergic mechanism between pressure- and temperature-induced denaturation. Our observations help us to reconstruct the structural events determining unfolding and distinguish the mechanisms that rule the different processes of unfolding.

The majority of the Earth's microbial biomass is found in high pressure environments, raising the question of how protein sequences adapt to such environments. Pressure adaptation is more complex, and less well-understood, than adaptation to extreme temperatures since in high pressure environments, these two thermodynamic parameters are often coupled, as in the cold deep-sea or at hydrothermal vents. To begin to address this question, we investigated the functional and folding properties of an exonuclease, Cnase, from the first Gram positive piezophile to be sequenced, Carnobacterium sp. AT7, isolated at 2500 m depth and at ∼2 °C in the Aleutian trench in the North Pacific. We find that Cnase is a bonafide exonuclease, despite its high negative charge. We also find that Cnase largely conserves the structure and folding mechanism of its mesophilic and well-studied homolog, staphylococcal nuclease, Snase, despite significant differences in their sequences.

Fungal effectors play crucial roles in plant infection. Despite low sequence identity, their classification into families is feasible using structural homology. In this study, we have elucidated the structures of the two effectors Mycgr3-91409 and Zt-NIP1 from the wheat fungal pathogen Zymoseptoria tritici using X-ray crystallography and NMR. These proteins shared structural homology with, respectively, KP6alpha and KP4 killer toxins, encoded by UmV dsRNA viruses infecting the corn fungal pathogen Ustilago maydis. Consequently, Zt-Mycgr3-91409 and Zt-NIP1 were renamed Zt-KP6-1 and Zt-KP4-1. Orthologs and paralogs of Zt-KP6-1 and Zt-KP4-1 were identified in Zymoseptoria species, but not in other fungi, with the exception of ECP2 effectors related to Zt-KP4-1. A novel pipeline relying on Foldseek and cysteine-pattern constrained HMM searches identified a large number of proteins structurally related to Zt-KP6-1 and Zt-KP4-1 in fungi, and to Zt-KP4-1 in plants. These proteins were classified into distinct structural super families (4 KP4 and 3 KP6) including effectors not known as structurally related to KP4 (ECP2) or KP6 (BAS4). The biological activities of Zt-KP6-1 and Zt-KP4-1 were assessed on plants and fungi. They did not induce symptoms on wheat leaves. However, they inhibited the growth of Botrytis cinerea and Z. tritici, demonstrating their toxicity to fungi. These results highlight the importance of structure determination of effectors for their classification and the prediction of their function. ### Competing Interest Statement The authors have declared no competing interest.

Proteins unfold under different environmental insults, among which heat, cold, high pressure and chaotropic agents. Understanding the mechanisms that determine unfolding under each of these condition is an important problem that directly relates to the physical forces that determine the three-dimensional structure of a protein. Here, we studied the mechanism of pressure unfolding of the marginally stable yeast protein Yfh1 using high-pressure nuclear magnetic resonance as this is the most appropriate technique to obtain a residue-specific description of the folding/unfolding transitions of a protein. We demonstrate that the pressure-unfolded spectrum shares features in common with that at low but not at high temperature and room pressure, suggesting a tighter similarity between the two processes that could be explained by a similar role of hydration in the process. This is the first time that comparison between the three infolded states could be tested experimentally, and confirms something that up to now has been only suggested as an hypothesis. By recording the phase diagram of the protein, we also show that temperature switches the pressure-induced unfolding pathway suggesting a synergic mechanism between pressure- and temperature-induced denaturation: at moderate pressures, Yfh1 unfolding at low temperature starts at a patch of negatively charged residues that we have previously demonstrated to induce electrostatic frustration and cause cold denaturation. Heat unfolding involves instead a cavity created by insufficient protection of the hydrophobic core. These observations help us to reconstruct the structural events determining unfolding and distinguish the mechanisms that rule the different processes of unfolding. ### Competing Interest Statement The authors have declared no competing interest.

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.

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.

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 1 H, 15 N and 13 C 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.

Does a similar 3D structure mean a similar folding pathway? This question is particularly meaningful when it concerns proteins sharing a similar 3D structure, but low sequence identity or homology. MAX effectors secreted by the phytopathogenic fungus Magnaporthe oryzae present such characteristics. They share a common 3D structure, a ß-sandwich with the same topology for all the family members, but an extremely low sequence identity/homology. In a previous study, we have investigated the folding of two MAX effectors, AVR-Pia and AVR-Pib, using High-Hydrostatic-Pressure NMR and found that they display a similar folding pathway, with a common folding intermediate. In the present work, we used a similar strategy to investigate the folding conformational landscape of another MAX effector, MAX60, and found a very different folding intermediate. Our analysis strongly supports that the presence of a C-terminal α-helical extension in the 3D structure of MAX60 could be responsible for its different folding pathway.

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 1 H, 15 N and 13 C 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.

In this article, using a combination of techniques (bioinformatics, structural biology, and cell biology), we identified and characterized a new class of effectors present only in intracellular mycobacteria. These proteins specifically target host cell mitochondria when ectopically expressed in cells.

ABSTRACT 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.

Multidomain kinases use many ways to integrate and process diverse stimuli. Here, we investigated the mechanism by which the protein tyrosine kinase 2-beta (PYK2) functions as a sensor and effector of cellular calcium influx. We show that the linker between the PYK2 kinase and FAT domains (KFL) encompasses an unusual calmodulin (CaM) binding element. PYK2 KFL is disordered and engages CaM through an ensemble of transient binding events. Calcium increases the association by promoting structural changes in CaM that expose auxiliary interaction opportunities. KFL also forms fuzzy dimers, and dimerization is enhanced by CaM binding. As a monomer, however, KFL associates with the PYK2 FERM-kinase fragment. Thus, we identify a mechanism whereby calcium influx can promote PYK2 self-association, and hence kinase-activating trans- autophosphorylation. Collectively, our findings describe a flexible protein module that expands the paradigms for CaM binding and self-association, and their use for controlling kinase activity.