Prodomain-driven enzyme dimerization: a pH-dependent autoinhibition mechanism that controls Plasmodium Sub1 activity before merozoite egress.

Malaria symptoms are associated with the asexual multiplication of Plasmodium falciparum within human red blood cells (RBCs) and fever peaks coincide with the egress of daughter merozoites following the rupture of the parasitophorous vacuole (PV) and the RBC membranes. Over the last two decades, it has emerged that the release of competent merozoites is tightly regulated by a complex cascade of events, including the unusual multi-step activation mechanism of the pivotal subtilisin-like protease 1 (Sub1) that takes place in three different cellular compartments and remains poorly understood. Following an initial auto-maturation in the endoplasmic reticulum (ER) between its pro- and catalytic domains, the Sub1 prodomain (PD) undergoes further cleavages by the parasite aspartic protease plasmepsin X (PmX) within acidic secretory organelles that ultimately lead to full Sub1 activation upon discharge into the PV. Here, we report the crystal structure of full-length P. falciparum Sub1 (PfS1FL) and demonstrate, through structural, biochemical, and biophysical studies, that the atypical Plasmodium-specific Sub1 PD directly promotes the assembly of inactive enzyme homodimers at acidic pH, whereas Sub1 is primarily monomeric at neutral pH. Our results shed new light into the finely tuned Sub1 spatiotemporal activation during secretion, explaining how PmX processing and full activation of Sub1 can occur in different cellular compartments, and uncover a robust mechanism of pH-dependent subtilisin autoinhibition that plays a key role in P. falciparum merozoites egress from infected host cells.IMPORTANCEMalaria fever spikes are due to the rupture of infected erythrocytes, allowing the egress of Plasmodium sp. merozoites and further parasite propagation. This fleeting tightly regulated event involves a cascade of enzymes, culminating with the complex activation of the subtilisin-like protease 1, Sub1. Differently than other subtilisins, Sub1 activation strictly depends upon the processing by a parasite aspartic protease within acidic merozoite secretory organelles. However, Sub1 biological activity is required in the pH neutral parasitophorous vacuole, to prime effectors involved in the rupture of the vacuole and erythrocytic membranes. Here, we show that the unusual, parasite-specific Sub1 prodomain is directly responsible for its acidic-dependent dimerization and autoinhibition, required for protein secretion, before its full activation at neutral pH in a monomeric form. pH-dependent Sub1 dimerization defines a novel, essential regulatory element involved in the finely tuned spatiotemporal activation of the egress of competent Plasmodium merozoites.

Acetylation regulates the oligomerization state and activity of RNase J, the Helicobacter pylori major ribonuclease.

In the gastric pathogen Helicobacter pylori, post-transcriptional regulation relies strongly on the activity of the essential ribonuclease RNase J. Here, we elucidated the crystal and cryo-EM structures of RNase J and determined that it assembles into dimers and tetramers in vitro. We found that RNase J extracted from H. pylori is acetylated on multiple lysine residues. Alanine substitution of several of these residues impacts on H. pylori morphology, and thus on RNase J function in vivo. Mutations of Lysine 649 modulates RNase J oligomerization in vitro, which in turn influences ribonuclease activity in vitro. Our structural analyses of RNase J reveal loops that gate access to the active site and rationalizes how acetylation state of K649 can influence activity. We propose acetylation as a regulatory level controlling the activity of RNase J and its potential cooperation with other enzymes of RNA metabolism in H. pylori.

Molecular insights into RmcA-mediated c-di-GMP consumption: Linking redox potential to biofilm morphogenesis in Pseudomonas aeruginosa.

The ability of many bacteria to form biofilms contributes to their resilience and makes infections more difficult to treat. Biofilm growth leads to the formation of internal oxygen gradients, creating hypoxic subzones where cellular reducing power accumulates, and metabolic activities can be limited. The pathogen Pseudomonas aeruginosa counteracts the redox imbalance in the hypoxic biofilm subzones by producing redox-active electron shuttles (phenazines) and by secreting extracellular matrix, leading to an increased surface area-to-volume ratio, which favors gas exchange. Matrix production is regulated by the second messenger bis-(3',5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP) in response to different environmental cues. RmcA (Redox modulator of c-di-GMP) from P. aeruginosa is a multidomain phosphodiesterase (PDE) that modulates c-di-GMP levels in response to phenazine availability. RmcA can also sense the fermentable carbon source arginine via a periplasmic domain, which is linked via a transmembrane domain to four cytoplasmic Per-Arnt-Sim (PAS) domains followed by a diguanylate cyclase (DGC) and a PDE domain. The biochemical characterization of the cytoplasmic portion of RmcA reported in this work shows that the PAS domain adjacent to the catalytic domain tunes RmcA PDE activity in a redox-dependent manner, by differentially controlling protein conformation in response to FAD or FADH2. This redox-dependent mechanism likely links the redox state of phenazines (via FAD/FADH2 ratio) to matrix production as indicated by a hyperwrinkling phenotype in a macrocolony biofilm assay. This study provides insights into the role of RmcA in transducing cellular redox information into a structural response of the biofilm at the population level. Conditions of resource (i.e. oxygen and nutrient) limitation arise during chronic infection, affecting the cellular redox state and promoting antibiotic tolerance. An understanding of the molecular linkages between condition sensing and biofilm structure is therefore of crucial importance from both biological and engineering standpoints.

Raynals, an online tool for the analysis of dynamic light scattering.

Dynamic light scattering (DLS) is routinely employed to assess the homogeneity and size-distribution profile of samples containing microscopic particles in suspension or solubilized polymers. In this work, Raynals, user-friendly software for the analysis of single-angle DLS data that uses the Tikhonov-Phillips regularization, is introduced. Its performance is evaluated on simulated and experimental data generated by different DLS instruments for several proteins and gold nanoparticles. DLS data can easily be misinterpreted and the simulation tools available in Raynals allow the limitations of the measurement and its resolution to be understood. It was designed as a tool to address the quality control of biological samples during sample preparation and optimization and it helps in the detection of aggregates, showing the influence of large particles. Lastly, Raynals provides flexibility in the way that the data are presented, allows the export of publication-quality figures, is free for academic use and can be accessed online on the eSPC data-analysis platform at https://spc.embl-hamburg.de/.

Insight into the role of the Bateman domain at the molecular and physiological levels through engineered IMP dehydrogenases.

Inosine 5'-monophosphate (IMP) dehydrogenase (IMPDH) is an ubiquitous enzyme that catalyzes the NAD+ -dependent oxidation of inosine 5'-monophosphate into xanthosine 5'-monophosphate. This enzyme is formed of two distinct domains, a core domain where the catalytic reaction occurs, and a less-conserved Bateman domain. Our previous studies gave rise to the classification of bacterial IMPDHs into two classes, according to their oligomeric and kinetic properties. MgATP is a common effector but cause to different effects when it binds within the Bateman domain: it is either an allosteric activator for Class I IMPDHs or a modulator of the oligomeric state for Class II IMPDHs. To get insight into the role of the Bateman domain in the dissimilar properties of the two classes, deleted variants of the Bateman domain and chimeras issued from the interchange of the Bateman domain between the three selected IMPDHs have been generated and characterized using an integrative structural biology approach. Biochemical, biophysical, structural, and physiological studies of these variants unveil the Bateman domain as being the carrier of the molecular behaviors of both classes.

Interactions of the protein tyrosine phosphatase PTPN3 with viral and cellular partners through its PDZ domain: insights into structural determinants and phosphatase activity.

The human protein tyrosine phosphatase non-receptor type 3 (PTPN3) is a phosphatase containing a PDZ (PSD-95/Dlg/ZO-1) domain that has been found to play both tumor-suppressive and tumor-promoting roles in various cancers, despite limited knowledge of its cellular partners and signaling functions. Notably, the high-risk genital human papillomavirus (HPV) types 16 and 18 and the hepatitis B virus (HBV) target the PDZ domain of PTPN3 through PDZ-binding motifs (PBMs) in their E6 and HBc proteins respectively. This study focuses on the interactions between the PTPN3 PDZ domain (PTPN3-PDZ) and PBMs of viral and cellular protein partners. We solved the X-ray structures of complexes between PTPN3-PDZ and PBMs of E6 of HPV18 and the tumor necrosis factor-alpha converting enzyme (TACE). We provide new insights into key structural determinants of PBM recognition by PTPN3 by screening the selectivity of PTPN3-PDZ recognition of PBMs, and by comparing the PDZome binding profiles of PTPN3-recognized PBMs and the interactome of PTPN3-PDZ. The PDZ domain of PTPN3 was known to auto-inhibit the protein's phosphatase activity. We discovered that the linker connecting the PDZ and phosphatase domains is involved in this inhibition, and that the binding of PBMs does not impact this catalytic regulation. Overall, the study sheds light on the interactions and structural determinants of PTPN3 with its cellular and viral partners, as well as on the inhibitory role of its PDZ domain on its phosphatase activity.

DNA-binding mechanism and evolution of replication protein A.

Replication Protein A (RPA) is a heterotrimeric single stranded DNA-binding protein with essential roles in DNA replication, recombination and repair. Little is known about the structure of RPA in Archaea, the third domain of life. By using an integrative structural, biochemical and biophysical approach, we extensively characterize RPA from Pyrococcus abyssi in the presence and absence of DNA. The obtained X-ray and cryo-EM structures reveal that the trimerization core and interactions promoting RPA clustering on ssDNA are shared between archaea and eukaryotes. However, we also identified a helical domain named AROD (Acidic Rpa1 OB-binding Domain), and showed that, in Archaea, RPA forms an unanticipated tetrameric supercomplex in the absence of DNA. The four RPA molecules clustered within the tetramer could efficiently coat and protect stretches of ssDNA created by the advancing replisome. Finally, our results provide insights into the evolution of this primordial replication factor in eukaryotes.

The c-di-AMP-binding protein CbpB modulates the level of ppGpp alarmone in Streptococcus agalactiae.

Cyclic di-AMP is an essential signalling molecule in Gram-positive bacteria. This second messenger regulates the osmotic pressure of the cell by interacting directly with the regulatory domains, either RCK_C or CBS domains, of several potassium and osmolyte uptake membrane protein systems. Cyclic di-AMP also targets stand-alone CBS domain proteins such as DarB in Bacillus subtilis and CbpB in Listeria monocytogenes. We show here that the CbpB protein of Group B Streptococcus binds c-di-AMP with a very high affinity. Crystal structures of CbpB reveal the determinants of binding specificity and significant conformational changes occurring upon c-di-AMP binding. Deletion of the cbpB gene alters bacterial growth in low potassium conditions most likely due to a decrease in the amount of ppGpp caused by a loss of interaction between CbpB and Rel, the GTP/GDP pyrophosphokinase.

Deciphering the Molecular Interaction Between the Adhesion G Protein-Coupled Receptor ADGRV1 and its PDZ-Containing Regulator PDZD7.

Hearing relies on the transduction of sound-evoked vibrations into electrical signals, occurring in the stereocilia bundle of inner ear hair cells. The G protein-coupled receptor (GPCR) ADGRV1 and the multi-PDZ protein PDZD7 play a critical role in the formation and function of stereocilia through their scaffolding and signaling properties. During hair cell development, the GPCR activity of ADGRV1 is specifically inhibited by PDZD7 through an unknown mechanism. Here, we describe the key interactions mediated by the two N-terminal PDZ domains of PDZD7 and the cytoplasmic domain of ADGRV1. Both PDZ domains can bind to the C-terminal PDZ binding motif (PBM) of ADGRV1 with the critical contribution of atypical C-terminal ß extensions. The two PDZ domains form a supramodule in solution, stabilized upon PBM binding. Interestingly, we showed that the stability and binding properties of the PDZ tandem are affected by two deafness-causing mutations located in the binding grooves of PDZD7 PDZ domains.

Dengue virus NS1 protein conveys pro-inflammatory signals by docking onto high-density lipoproteins.

The dengue virus nonstructural protein 1 (NS1) is a secreted virulence factor that modulates complement, activates immune cells and alters endothelial barriers. The molecular basis of these events remains incompletely understood. Here we describe a functional high affinity complex formed between NS1 and human high-density lipoproteins (HDL). Collapse of the soluble NS1 hexamer upon binding to the lipoprotein particle leads to the anchoring of amphipathic NS1 dimeric subunits into the HDL outer layer. The stable complex can be visualized by electron microscopy as a spherical HDL with rod-shaped NS1 dimers protruding from the surface. We further show that the assembly of NS1-HDL complexes triggers the production of pro-inflammatory cytokines in human primary macrophages while NS1 or HDL alone do not. Finally, we detect NS1 in complex with HDL and low-density lipoprotein (LDL) particles in the plasma of hospitalized dengue patients and observe NS1-apolipoprotein E-positive complexes accumulating overtime. The functional reprogramming of endogenous lipoprotein particles by NS1 as a means to exacerbate systemic inflammation during viral infection provides a new paradigm in dengue pathogenesis.

Development of a highly specific and sensitive VHH-based sandwich immunoassay for the detection of the SARS-CoV-2 nucleoprotein.

The current COVID-19 pandemic illustrates the importance of obtaining reliable methods for the rapid detection of SARS-CoV-2. A highly specific and sensitive diagnostic test able to differentiate the SARS-CoV-2 virus from common human coronaviruses is therefore needed. Coronavirus nucleoprotein (N) localizes to the cytoplasm and the nucleolus and is required for viral RNA synthesis. N is the most abundant coronavirus protein, so it is of utmost importance to develop specific antibodies for its detection. In this study, we developed a sandwich immunoassay to recognize the SARS-CoV-2 N protein. We immunized one alpaca with recombinant SARS-CoV-2 N and constructed a large single variable domain on heavy chain (VHH) antibody library. After phage display selection, seven VHHs recognizing the full N protein were identified by ELISA. These VHHs did not recognize the nucleoproteins of the four common human coronaviruses. Hydrogen Deuterium eXchange-Mass Spectrometry (HDX-MS) analysis also showed that these VHHs mainly targeted conformational epitopes in either the C-terminal or the N-terminal domains. All VHHs were able to recognize SARS-CoV-2 in infected cells or on infected hamster tissues. Moreover, the VHHs could detect the SARS variants B.1.17/alpha, B.1.351/beta, and P1/gamma. We propose that this sandwich immunoassay could be applied to specifically detect the SARS-CoV-2 N in human nasal swabs.

Actinobacteria challenge the paradigm: A unique protein architecture for a well-known, central metabolic complex.

α-oxoacid dehydrogenase complexes are large, tripartite enzymatic machineries carrying out key reactions in central metabolism. Extremely conserved across the tree of life, they have been, so far, all considered to be structured around a high-molecular weight hollow core, consisting of up to 60 subunits of the acyltransferase component. We provide here evidence that Actinobacteria break the rule by possessing an acetyltranferase component reduced to its minimally active, trimeric unit, characterized by a unique C-terminal helix bearing an actinobacterial specific insertion that precludes larger protein oligomerization. This particular feature, together with the presence of an odhA gene coding for both the decarboxylase and the acyltransferase domains on the same polypetide, is spread over Actinobacteria and reflects the association of PDH and ODH into a single physical complex. Considering the central role of the pyruvate and 2-oxoglutarate nodes in central metabolism, our findings pave the way to both therapeutic and metabolic engineering applications.

Molecular Mechanisms Underpinning the Circulation and Cellular Uptake of Mycobacterium ulcerans Toxin Mycolactone.

Mycolactone is a diffusible lipid toxin produced by Mycobacterium ulcerans, the causative agent of Buruli ulcer disease. Altough bacterially derived mycolactone has been shown to traffic from cutaneous foci of infection to the bloodstream, the mechanisms underpinning its access to systemic circulation and import by host cells remain largely unknown. Using biophysical and cell-based approaches, we demonstrate that mycolactone specific association to serum albumin and lipoproteins is necessary for its solubilization and is a major mechanism to regulate its bioavailability. We also demonstrate that Scavenger Receptor (SR)-B1 contributes to the cellular uptake of mycolactone. Overall, we suggest a new mechanism of transport and cell entry, challenging the dogma that the toxin enters host cells via passive diffusion.

SARS-CoV-2 Nsp3 unique domain SUD interacts with guanine quadruplexes and G4-ligands inhibit this interaction.

The multidomain non-structural protein 3 (Nsp3) is the largest protein encoded by coronavirus (CoV) genomes and several regions of this protein are essential for viral replication. Of note, SARS-CoV Nsp3 contains a SARS-Unique Domain (SUD), which can bind Guanine-rich non-canonical nucleic acid structures called G-quadruplexes (G4) and is essential for SARS-CoV replication. We show herein that the SARS-CoV-2 Nsp3 protein also contains a SUD domain that interacts with G4s. Indeed, interactions between SUD proteins and both DNA and RNA G4s were evidenced by G4 pull-down, Surface Plasmon Resonance and Homogenous Time Resolved Fluorescence. These interactions can be disrupted by mutations that prevent oligonucleotides from folding into G4 structures and, interestingly, by molecules known as specific ligands of these G4s. Structural models for these interactions are proposed and reveal significant differences with the crystallographic and modeled 3D structures of the SARS-CoV SUD-NM/G4 interaction. Altogether, our results pave the way for further studies on the role of SUD/G4 interactions during SARS-CoV-2 replication and the use of inhibitors of these interactions as potential antiviral compounds.

PDZ Sample Quality Assessment by Biochemical and Biophysical Characterizations.

PDZ domains are small globular domains involved in protein-protein interactions. They participate in a wide range of critical cellular processes. These domains, very abundant in the human proteome, are widely studied by high-throughput interactomics approaches and by biophysical and structural methods. However, the quality of the results is strongly related to the optimal folding and solubility of the domains. We provide here a detailed description of protocols for a strict quality assessment of the PDZ constructs. We describe appropriate experimental approaches that have been selected to overcome the small size of such domains to check the purity, identity, homogeneity, stability, and folding of samples.

A High-Affinity Calmodulin-Binding Site in the CyaA Toxin Translocation Domain is Essential for Invasion of Eukaryotic Cells.

The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells are still a matter of intense research. The adenylate cyclase (CyaA) toxin from Bordetella pertussis displays a unique intoxication pathway in which its catalytic domain is directly translocated across target cell membranes. The CyaA translocation region contains a segment, P454 (residues 454-484), which exhibits membrane-active properties related to antimicrobial peptides. Herein, the results show that this peptide is able to translocate across membranes and to interact with calmodulin (CaM). Structural and biophysical analyses reveal the key residues of P454 involved in membrane destabilization and calmodulin binding. Mutational analysis demonstrates that these residues play a crucial role in CyaA translocation into target cells. In addition, calmidazolium, a calmodulin inhibitor, efficiently blocks CyaA internalization. It is proposed that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin. Trapping of CyaA by the CaM:P454 interaction in the cytosol may assist the entry of the N-terminal catalytic domain by converting the stochastic motion of the polypeptide chain through the membrane into an efficient vectorial chain translocation into host cells.

RNase R is associated in a functional complex with the RhpA DEAD-box RNA helicase in Helicobacter pylori.

Ribonucleases are central players in post-transcriptional regulation, a major level of gene expression regulation in all cells. Here, we characterized the 3'-5' exoribonuclease RNase R from the bacterial pathogen Helicobacter pylori. The 'prototypical' Escherichia coli RNase R displays both exoribonuclease and helicase activities, but whether this latter RNA unwinding function is a general feature of bacterial RNase R had not been addressed. We observed that H. pylori HpRNase R protein does not carry the domains responsible for helicase activity and accordingly the purified protein is unable to degrade in vitro RNA molecules with secondary structures. The lack of RNase R helicase domains is widespread among the Campylobacterota, which include Helicobacter and Campylobacter genera, and this loss occurred gradually during their evolution. An in vivo interaction between HpRNase R and RhpA, the sole DEAD-box RNA helicase of H. pylori was discovered. Purified RhpA facilitates the degradation of double stranded RNA by HpRNase R, showing that this complex is functional. HpRNase R has a minor role in 5S rRNA maturation and few targets in H. pylori, all included in the RhpA regulon. We concluded that during evolution, HpRNase R has co-opted the RhpA helicase to compensate for its lack of helicase activity.

Quality control of purified proteins to improve data quality and reproducibility: results from a large-scale survey.

As the scientific community strives to make published results more transparent and reliable, it has become obvious that poor data reproducibility can often be attributed to insufficient quality control of experimental reagents. In this context, proteins and peptides reagents require much stricter quality controls than those routinely performed on them in a significant proportion of research laboratories. Members of the ARBRE-MOBIEU and the P4EU networks have combined their expertise to generate guidelines for the evaluation of purified proteins used in life sciences and medical trials. These networks, representing more than 150 laboratories specialized in protein production and/or protein molecular biophysics, have implemented such guidelines in their respective laboratories. Over a one-year period, the network members evaluated the contribution these guidelines made toward obtaining more productive, robust and reproducible research by correlating the applied quality controls to given samples with the reliability and reproducibility of the scientific data obtained using these samples in follow-up experiments. The results indicate that QC guideline implementation facilitates the optimization of the protein purification process and improves the reliability of downstream experiments. It seems, therefore, that investing in protein QC might be advantageous to all the stakeholders in life sciences (researchers, editors, and funding agencies alike), because this practice improves data veracity and minimizes loss of valuable time and resources. In the light of these conclusions, the network members suggest that the implementation of these simple QC guidelines should become minimal reporting practice in the publication of data derived from the use of protein and peptide reagents.

Assessing and Improving Protein Sample Quality.

One essential prerequisite of any experiment involving a purified protein, such as interaction studies or structural and biophysical characterization, is to work with a "good-quality" sample in order to ensure reproducibility and reliability of the data. Here, we define a "good-quality" sample as a protein preparation that fulfills three criteria: (1) the preparation contains a protein that is pure and soluble and exhibits structural and functional integrity, (2) the protein must be structurally homogeneous, and (3) the preparation must be reproducible. To ensure effective quality control (QC) of all these parameters, we suggest to follow a simple workflow involving the use of gel electrophoresis, light scattering, and spectroscopic experiments. We describe the techniques used in every step of this workflow and provide easy-to-use standard protocols for each step.