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17 p.-8 fig. ; Sulfur trafficking in living organisms relies on transpersulfuration reactions consisting in the enzyme-catalyzed transfer of S atoms via activated persulfidic S across protein-protein interfaces. The recent elucidation of the mechanistic basis for transpersulfuration in the CsdA-CsdE model system has paved the way for a better understanding of its role under oxidative stress. Herein we present the crystal structure of the oxidized, inactivated CsdE dimer at 2.4 Å resolution. The structure sheds light into the activation of the Cys61 nucleophile on its way from a solvent-secluded position in free CsdE to a fully extended conformation in the persulfurated CsdA-CsdE complex. Molecular dynamics simulations of available CsdE structures allow to delineate the sequence of conformational changes underwent by CsdE and to pinpoint the key role played by the deprotonation of the Cys61 thiol. The low-energy subunit orientation in the disulfide-bridged CsdE dimer demonstrates the likely physiologic relevance of this oxidative dead-end form of CsdE, suggesting that CsdE could act as a redox sensor in vivo. ; This work was supported by Spanish Instituto de Salud Carlos III (http://www.isciii.es) (PI12/01667 to MCV), Spanish Ministerio de Economía y Competitividad (http://www.mineco.gob.es/portal/site/mineco/) (PET2008_0101, BIO2009-11184, BFU2010- 22260-C02-02, and CTQ2015-66206-C2-2-R to MCV, and CTQ2015-66223-C2-2-P to IT), the Regional Government of Madrid (http://www.madrid.org/) (S2010/BD-2316 to MCV), and the European Commission (Framework Programme 7 (FP7)) (https://ec.europa.eu/research/fp7/index_en.cfm) project ComplexINC (Contract No. 279039) to MCV. ; Peer reviewed

Sulfur trafficking in living organisms relies on transpersulfuration reactions consisting in the enzyme-catalyzed transfer of S atoms via activated persulfidic S across protein-protein interfaces. The recent elucidation of the mechanistic basis for transpersulfuration in the CsdA-CsdE model system has paved the way for a better understanding of its role under oxidative stress. Herein we present the crystal structure of the oxidized, inactivated CsdE dimer at 2.4 Å resolution. The structure sheds light into the activation of the Cys61 nucleophile on its way from a solvent-secluded position in free CsdE to a fully extended conformation in the persulfurated CsdA-CsdE complex. Molecular dynamics simulations of available CsdE structures allow to delineate the sequence of conformational changes underwent by CsdE and to pinpoint the key role played by the deprotonation of the Cys61 thiol. The low-energy subunit orientation in the disulfide-bridged CsdE dimer demonstrates the likely physiologic relevance of this oxidative dead-end form of CsdE, suggesting that CsdE could act as a redox sensor in vivo. ; This work was supported by Spanish Instituto de Salud Carlos III (http://www.isciii.es) (PI12/01667 to MCV), Spanish Ministerio de Economía y Competitividad (http://www.mineco.gob.es/portal/site/mineco/) (PET2008_0101, BIO2009-11184, BFU2010- 22260-C02-02, and CTQ2015-66206-C2-2-R to MCV, and CTQ2015-66223-C2-2-P to IT), the Regional Government of Madrid (http://www.madrid.org/) (S2010/BD-2316 to MCV), and the European Commission (Framework Programme 7 (FP7)) (https://ec.europa.eu/research/fp7/index_en.cfm) project ComplexINC (Contract No. 279039) to MCV. F.J. Fernández (FJF) is currently employed by Abvance Biotech srl and contributed to the work presented here when affiliated to CSIC and, as an employee of Abvance, to writing and reviewing the article. Abvance Biotech srl provided support in the form of salary for author FJF, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. ; Peer reviewed

Medium-resolution cryo-electron microscopy maps, in particular when they include a significant number of α-helices, may allow the building of partial models that are useful for molecular-replacement searches in large crystallo­graphic structures when the structures of homologs are not available and experimental phasing has failed. Here, as an example, the solution of the structure of a bacteriophage portal using a partial 30% model built into a 7.8 Å resolution cryo-EM map is shown. Inspection of the self-rotation function allowed the correct oligomerization state to be determined, and density-modification procedures using rotation matrices and a mask based on the cryo-EM structure were critical for solving the structure. A workflow is described that may be applicable to similar cases and this strategy is compared with direct use of the cryo-EM map for molecular replacement. ; The following funding is acknowledged: Ministry of Science, Innovation and Universities of Spain (grant Nos. BFU2014-53550-P and BFU2017-83720-P to Miquel Coll; grant No. BFU2014-54181 to Jose´ L. Carrascosa; contract No. SEV2013-0347 to Ana Cuervo; contract No. RYC-2011-09071 to Cristina Machon; award No. SEV-2015-0500 to IRB Barcelona; award No. MDM-2014-0435 to IBMB Structural Biology Unit); Catalan Government CERCA Programme (grant to IRB Barcelona).

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 reviewed

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 reviewed