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

Crystal structure and SAXS analysis of the immune evasive factor GAPDH from Leptospira interrogans

1 p.Background: Immune evasive factors from pathogenic bacteriainclude metabolic enzymes that fulfill housekeeping roles intra-cellularly but which can contribute to infectivity when released as part of the bacterial secretome. The glycolytic enzyme D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been characterized as a moonlighting protein with immune evasive roles in many gram-negative and gram-positive bacteria (1-2). GAPDH from several bacterial pathogens are known to interfere with the C5a-C5aR1 signaling pathway by binding to C5a. There-fore, research to determine the structural features enabling C5a sequestration and the potential for pharmacological intervention represent interesting alternatives to fight bacterial infections.Methods: The gene for Leptospira interrogans (Li)GAPDH wasamplified from gDNA and subcloned into a bacterial expression vector. Expression and purification of LiGAPDH was performed following established procedures. The structure of LiGAPDH wasdetermined by X-ray crystallography and the shape in solution calculated from small-angle X-ray diffraction (SAXS). A crosslinkingassay with BMOE helped to demonstrate the association between LiGAPDH and human C5a. GAPDH activity was measured with standard enzymatic assays and inhibition by two natural products (curcumin and anacardic acid) was evaluated.Results: We have cloned, expressed and purified GAPDH from Leptospira interrogans. We have crystallized it and solved its struc-ture by X-ray crystallography. In addition, we have restored itsshape in solution, validating the crystallographic model. LiGAPDH appears to be able to engage in a transient complex with human C5a, which we have trapped by crosslinking with BMOE, a highlyspecific reagent that crosslinks thiols < 6-8˚A away. We have analyzed the inhibitory potency and inhibition modality of two natural products with antimicrobial properties: anacardic acid and curcumin. Of them, only curcumin showed inhibition potential.Conclusions: The structure of the immune evasive factor GAPDH from Leptospira interrogans has been determined and is now available for experimental and computational drug discovery campaigns. Although weakly, LiGAPDH can interact with human C5a, suggesting that avidity effects caused by the concentration of GAPDH on the bacterium’s cell wall could enhance the C5a sequestration capacity of L. interrogans. Furthermore, curcumin has emerged as an interesting broad-spectrum inhibitor of LiGAPDH.Peer reviewe

Elucidating the Catalytic Reaction Mechanism of Orotate Phosphoribosyltransferase by Means of X-ray Crystallography and Computational Simulations

Orotate phosphoribosyltransferase (OPRTase) catalyzes the reaction between the ribose donor α-d-5-phosphoribosyl-1-pyrophosphate (PRPP) and orotate (OA) in the presence of Mg2+ ion to obtain pyrophosphate and pyrimidine nucleotide orotidine 5′-monophosphate (OMP), a key precursor in de novo biosynthesis of pyrimidine nucleotides. In this work, several structures of the dimeric Escherichia coli OPRTase (EcOPRTase) have been determined at high resolution, and kinetic measurements have been carried out to obtain the catalytic rate and Michaelis constants. Molecular dynamics (MD) simulations have been carried out, and structural analysis from the X-ray and MD simulation structures reveals conformational changes related to the flexible catalytic loop that establishes hydrogen bond interactions with the pyrophosphoryl group of PRPP. It is proposed that the OA substrate can be in equilibrium in its tautomeric forms. Starting from the most stable tautomeric form, all the plausible mechanisms have been explored by means of quantum mechanics/molecular mechanics (QM/MM) MD simulations using the adaptive string method. The most feasible mechanism consists of the proton transfer from the N1 atom of OA to a water molecule and from the water molecule to the α-phosphate O2A atom of PRPP. After that, the nucleophilic attack of the N1 atom of OA to the C1 atom of PRPP proceeds to yield OMP and pyrophosphate. The free energy barrier obtained is in very good agreement with the experimental data reported. Analysis of some relevant distances between key residues and the substrates (OA and PRPP) at the reactant state and transition state (TS) of the rate-limiting step allows us to understand the role of some conserved residues (Lys73, Asp125, Lys103*, Arg99*, and Mg2+ ion) electrostatically stabilizing the TS and preserving the flexible catalytic loop in a closed conformation during the enzymatic reaction

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

15 p.-7 fig.-4 tab.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 a-helical proteins with GAPDH. These findings allow us to propose L. interrogans GAPDH as a potential immune evasive factor targeting the complement system.This work was funded by Spanish Ministerio de Ciencia, Innovación y Universidades-FEDER grants RTI2018-102242-B-I00 (to MCV) and PID2019-104912RB-I00 (to SRdC); and the Spanish Ministerio de Ciencia e Innovación-Recovery, Transformation and Resilience Plan (PRTR) grant PDC2022-133713-I00. It was also funded by Grants S2017/BMD-3673 and S2022/BMD-7278 of the Regional Government of Madrid and the European Commission – NextGenerationEU through CSIC’s Global Health Platform (“PTI Salud Global”) (SGL2103020) (to SRdC and MCV), and the CSIC Special Intramural Grant PIE-201620E064 (to MCV). It was additionally supported by the Research Network on Complement in Health and Disease (RED2022-134750-T). SRdC was also supported by the CIBER de Enfermedades Raras. KdlP was supported by an Industrial PhD grant (IND2018-010094) awarded by the Spanish Ministerio de Economía y Competitividad.Peer reviewe

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

12 p.-7 fig.-2 tab.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 β-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.Spanish Ministry of Economy, Industry and Competitiveness (CTQ2015-66206-C2-2-R, SAF2015-72961-EXP, RTI2018-102242-B-I00), Regional Government of Madrid (S2017/BMD-3673), and CSIC (PIE 20160E064) to M.C.V. All grants were co-funded with European Union ERDF funds (European Regional Development Fund).Peer reviewe

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

16 p.-9 fig.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.This work was funded by Spanish Ministerio de Ciencia, Innovación y Universidades-FEDER grant RTI2018-102242-B-I00 (to M.C.V.) and PID2019-104912RB-I00 (to S.R.C.), and Spanish Ministerio de Economía y Competitividad-FEDER grants SAF2015-72961-EXP (to M.C.V.) and SAF2015-66287-R (to S.R.C.). It was also funded by Grant S2017/BMD-3673 of the Regional Government of Madrid and the European Commission – NextGenerationEU through CSIC’s Global Health Platform (“PTI Salud Global”) (SGL2103020) (to S.R.C. and M.C.V.), and the CSIC Special Intramural Grant PIE-201620E064 (to M.C.V.). It was additionally supported by the Network of Excellence Complement in Health and Disease (SAF2016-81876-REDT). SRC was also supported by the CIBER de Enfermedades Raras. JSL acknowledges the support of the PhD program in Molecular Biosciences of the Universidad Autónoma de Madrid (UAM) and the Ministry of Education, Culture and Sports of Spain (FPU Grant 17/06090). SNY acknowledges the support of the PhD program in Biochemistry, Molecular Biology and Biomedicine of the Universidad Complutense de Madrid (UCM).Peer reviewe