Search

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).

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.

1 p., Background: Communication between the humoral and cellu-lar branches and the innate and the adaptive branches of vertebrateimmunity is essential for mounting an effective immune response,for surveying the presence of pathogens, immune complexes andapoptotic cell debris and for maintaining homeostasis (Merle et al.,2015). Complement activation on cell surfaces results in theiropsonization and subsequent presentation to immune cells bearingcomplement receptors. The complex formed by the complement’siC3b opsonin and the integrin complement receptor type 3 (CR3)is thought to mediate this communication and stimulate adaptiveimmune cells (Xu et al., 2017). Despite the centrality of the iC3b-CR3 complex to immune signaling, a high-resolution structure ofthe complex between the complete opsonin and the I domain orthe entire headpiece of CR3 is still lacking. Furthermore, the poten-tial involvement of regions of iC3b other than the TED domain inthe interaction remains unclear., Methods: The iC3b-CR3 I domain complex was purifiedby size-exclusion chromatography and subjected to crystalliza-tion screenings. Diffraction-quality crystals were obtained, whichyielded a complete diffraction data set to 3.4˚A resolution on thePX2A beamline at the SOLEIL synchrotron (Paris, France). Small-angle X-ray scattering (SAXS) profiles of the complex were collectedon the BioSAXS beamline at the ESRF (Grenoble, France). KD valueswere measured by surface plasmon resonance (SPR). MD simula-tions were carried out with GROMACS, Results: Here we present the high-resolution (3.4˚A) crystallo-graphic structure of human iC3b and the von Willebrand A inserted domain of the CR3 ( I) receptor and a model built from crystal-lographic, biochemical, biophysical and SAXS data. In addition tothe known interaction between the thioester-domain (TED) of iC3band the I domain, the crystal structure reveals the involvement ofnovel interaction surfaces on the C3c macroglobulin ring in secur-ing and orientating the complex., Conclusions: Based on available data and novel insights wehave built an integrative structural biology model that reconciles previously known information about the iC3b-CR3 interaction,and which allows us to predict the dynamics of the iC3b-CR3interaction across biological cell surfaces.

15 p.-8 fig.-2 tab.1 graph. abst.+ 8 fig. supl.-1 tab. supl., 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., This work was supported by the Spanish Ministerio de Economı́a y Competitividad (2015) and Ministerio de Ciencia,Innovación y Universidades (2018) through grants cofinanced with European Union ERDF funds PGC2018-094852-B-C22 to IT and CTQ2015-66206-C2-2-R and RTI2018-1018-102242-B-I00 to M.C.V., by Generalitat Valenciana AICO/2018/238 project and by Universitat Jaume I UJI-B2016-28project to IT and M.R., respectively, and CSIC intramuralgrant 2016E064 to M.C.V. M.R. thanks Ministerio deEconomía y Competitividad for a“Ramón y Cajal”contract(RYC-2014-16592)

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).