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High-hydrostatic pressure is an alternative perturbation method that can be used to destabilize globular proteins. Generally perfectly reversible, pressure exerts local effects on regions or domains of a protein containing internal voids, contrary to heat or chemical denaturant that destabilize protein structures uniformly. When combined with NMR spectroscopy, high pressure (HP) allows one to monitor at a residue-level resolution the structural transitions occurring upon unfolding and to determine the kinetic properties of the process. The use of HP-NMR has long been hampered by technical difficulties. Owing to the recent development of commercially available high-pressure sample cells, HP-NMR experiments can now be routinely performed. This review summarizes recent advances of HP-NMR techniques for the characterization at a quasi-atomic resolution of the protein folding energy landscape.

It has been known for nearly 100 years that pressure unfolds proteins, yet the physical basis of this effect is not understood. Unfolding by pressure implies that the molar volume of the unfolded state of a protein is smaller than that of the folded state. This decrease in volume has been proposed to arise from differences between the density of bulk water and water associated with the protein, from pressure-dependent changes in the structure of bulk water, from the loss of internal cavities in the folded states of proteins, or from some combination of these three factors. Here, using 10 cavity-containing variants of staphylococcal nuclease, we demonstrate that pressure unfolds proteins primarily as a result of cavities that are present in the folded state and absent in the unfolded one. High-pressure NMR spectroscopy and simulations constrained by the NMR data were used to describe structural and energetic details of the folding landscape of staphylococcal nuclease that are usually inaccessible with existing experimental approaches using harsher denaturants. Besides solving a 100-year-old conundrum concerning the detailed structural origins of pressure unfolding of proteins, these studies illustrate the promise of pressure perturbation as a unique tool for examining the roles of packing, conformational fluctuations, and water penetration as determinants of solution properties of proteins, and for detecting folding intermediates and other structural details of protein-folding landscapes that are invisible to standard experimental approaches.

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.

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.

Multidimensional NMR intrinsically provides multiple probes that can be used for deciphering the folding pathways of proteins: NH amide and CαHα groups are strategically located on the backbone of the protein, while CH3 groups, on the side-chain of methylated residues, are involved in important stabilizing interactions in the hydrophobic core. Combined with high hydrostatic pressure, these observables provide a powerful tool to explore the conformational landscapes of proteins. In the present study, we made a comparative assessment of the NH, CαHα, and CH3 groups for analyzing the unfolding pathway of ∆+PHS Staphylococcal Nuclease. These probes yield a similar description of the folding pathway, with virtually identical thermodynamic parameters for the unfolding reaction, despite some notable differences. Thus, if partial unfolding begins at identical pressure for these observables (especially in the case of backbone probes) and concerns similar regions of the molecule, the residues involved in contact losses are not necessarily the same. In addition, an unexpected slight shift toward higher pressure was observed in the sequence of the scenario of unfolding with CαHα when compared to amide groups.

Dengue fever is a mosquito-borne endemic disease in tropical and subtropical regions, causing a significant public health problem in Southeast Asia. Domain III (ED3) of the viral envelope protein contains the two dominant putative epitopes and part of the heparin sulfate receptor binding region that drives the dengue virus (DENV)’s fusion with the host cell. Here, we used high-hydrostatic-pressure nuclear magnetic resonance (HHP-NMR) to obtain residue-specific information on the folding process of domain III from serotype 4 dengue virus (DEN4-ED3), which adopts the classical three-dimensional (3D) ß-sandwich structure known as the Ig-like fold. Interestingly, the folding pathway of DEN4-ED3 shares similarities with that of the Titin I27 module, which also adopts an Ig-like fold, but is functionally unrelated to ED3. For both proteins, the unfolding process starts by the disruption of the N- and C-terminal strands on one edge of the ß-sandwich, yielding a folding intermediate stable over a substantial pressure range (from 600 to 1000 bar). In contrast to this similarity, pressure-jump kinetics indicated that the folding transition state is considerably more hydrated in DEN4-ED3 than in Titin I27.

Resuscitation-promoting factor (RPF) proteins reactivate stationary-phase cultures of (G+C)-rich Gram-positive bacteria including the causative agent of tuberculosis, Mycobacterium tuberculosis . We report the solution structure of the RPF domain from M. tuberculosis Rv1009 (RpfB) solved by heteronuclear multidimensional NMR. Structural homology with various glycoside hydrolases suggested that RpfB cleaved oligosaccharides. Biochemical studies indicate that a conserved active site glutamate is important for resuscitation activity. These data, as well as the presence of a clear binding pocket for a large molecule, indicate that oligosaccharide cleavage is probably the signal for revival from dormancy.

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. Protein tyrosine kinase 2-beta is shown to function as a sensor and effector of cellular calcium influx through self-association.

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.