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Funding Information: We thank L?gia S. Nobre and Joana M. Baptista for contribution at the initial stage of the work, and Cl?udia S. Freitas for technical support. We also thank Professor Miguel Teixeira of ITQB-NOVA for critical reading of the manuscript. We thank the XALOC staff and floor coordinators at the synchrotron ALBA for the YtfEM data collection. We acknowledge the ESRF for provision of synchrotron radiation facilities and we would like to thank Gianluca Santoni for assistance using the beamline ID30A-3 for the YtfEM-E159L data collection. We also thank Diamond Light Source for beamtime and the staff of beamline I04 for assistance with crystal testing and data collection of YtfEM-E125L. Funding. This work was financially supported by Funda??o para a Ci?ncia e Tecnologia (Portugal) through fellowship SFRH/BD/118545/2016 (LOS) and R&D unit LISBOA-01-0145-FEDER007660 (MostMicro) co-funded by FCT/MCTES and FEDER funds under the PT2020 Partnership Agreement. This work was partially supported by PPBI ? Portuguese Platform of BioImaging (PPBI-POCI-01-0145-FEDER-022122) co-funded by national funds from OE ? ?Or?amento de Estado? and by European funds from FEDER ? ?Fundo Europeu de Desenvolvimento Regional.? We also acknowledge funding from the European Union?s Horizon 2020 Research and Innovation Program under grant agreement no. 810856. Publisher Copyright: © Copyright © 2021 Silva, Matias, Romão and Saraiva. Copyright: Copyright 2021 Elsevier B.V., All rights reserved. ; Escherichia coli YtfE is a di-iron protein of the widespread Repair of Iron Centers proteins (RIC) family that has the capacity to donate iron, which is a crucial component of the biogenesis of the ubiquitous family of iron-sulfur proteins. In this work we identify in E. coli a previously unrecognized link between the YtfE protein and the major bacterial system for iron-sulfur cluster (ISC) assembly. We show that YtfE establishes protein-protein interactions with the scaffold IscU, where the transient cluster is formed, and the cysteine desulfurase IscS. Moreover, we found that promotion by YtfE of the formation of an Fe-S cluster in IscU requires two glutamates, E125 and E159 in YtfE. Both glutamates form part of the entrance of a protein channel in YtfE that links the di-iron center to the surface. In particular, E125 is crucial for the exit of iron, as a single mutation to leucine closes the channel rendering YtfE inactive for the build-up of Fe-S clusters. Hence, we provide evidence for the key role of RICs as bacterial iron donor proteins involved in the biogenesis of Fe-S clusters. ; publishersversion ; published

Pontin and Reptin are related partner proteins belonging to the AAA+ (ATPases Associated with various cellular Activities) family. They are implicated in multiple and seemingly unrelated processes encompassing the regulation of gene transcription, the remodeling of chromatin, DNA damage sensing and repair, and the assembly of protein and ribonucleoprotein complexes, among others. The 2nd International Workshop on Pontin and Reptin took place at the Instituto de Tecnologia Química e Biológica António Xavier in Oeiras, Portugal on October 10–12, 2014, and reported significant new advances on the mechanisms of action of these two AAA+ ATPases. The major points under discussion were related to the mechanisms through which these proteins regulate gene transcription, their roles as co-chaperones, and their involvement in pathophysiology, especially in cancer and ciliary biology and disease. Finally, they may become anticancer drug targets since small chemical inhibitors were shown to produce anti-tumor effects in animal models. ; This work was supported by Fundação para a Ciência e Tecnologia (Portugal) grants PTDC/BBB-BEP/1724/2012 to PM and TB, and PEst-OE/EQB/LA0004/2011 to PM; Creative Research Initiatives Program (Research Center for Chromatin Dynamics, 2009-0081563) to SB; Ministry of Economy and Competitiveness of the Spanish Government grants SAF2011-22988 and SAF2014-52301-R to OL; NIH grants R01 GM084465 and CA166054 to AD; The Canadian Institutes of Health Research grant (MOP-93778) to WAH; Equipe Labélisée Ligue Contre le Cancer and Institut National du Cancer grant PLBIO10-155 to JR. ; Peer reviewed ; Peer Reviewed

This work was supported by the European Union's Horizon 2020 research and innovation program under Grant 810856, National Funds Grant ERA-MBT/ 0003/2014, Ph.D. fellowship PD/BD/135187/2017 (to I. B. T.), and postdoctoral fellowships SFRH/BPD/94050/2013 and SFRH/BPD/93164/2013 (to E. M. and B. F. M.), through the FCT-Fundação para a Ciência e Tecnologia. The authors declare that they have no conflicts of interest with the contents of this article. Acknowledgments—We thank Isabel Pacheco for help in the purification of SfSIP and Dr. Américo Duarte for providing ferredoxin. We also thank all members of the Inorganic Biochemistry and NMR Laboratory for discussions and comments and feedback regarding the preparation of the manuscript. The NMR experiments were performed at CERMAX (Centro de Ressonância Magnetica António Xavier) and was financially supported by Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE2020-Programa Opera-cional Competitividade e Internacionalização (POCI), the European Community's Seventh Framework Program (FP7/2007–2013)) under Grant Agreement 283570 (BioStruct-X). Beamtime at I04 at the Diamond Light Source and assistance from the beamline staff during the synchrotron data collections are gratefully acknowledged. ; Siderophores make iron accessible under iron-limited conditions and play a crucial role in the survival of microorganisms. Because of their remarkable metal-scavenging properties and ease in crossing cellular envelopes, siderophores hold great potential in biotechnological applications, raising the need for a deeper knowledge of the molecular mechanisms underpinning the siderophore pathway. Here, we report the structural and functional characterization of a siderophore-interacting protein from the marine bacterium Shewanella frigidimarina NCIBM400 (SfSIP). SfSIP is a flavin-containing ferric-siderophore reductase with FAD- and NAD(P)H-binding domains that have high homology with other characterized SIPs. However, we found here that it mechanistically departs from what has been described for this family of proteins. Unlike other FAD-containing SIPs, SfSIP did not discriminate between NADH and NADPH. Furthermore, SfSIP required the presence of the Fe 2+ -scavenger, ferrozine, to use NAD(P)H to drive the reduction of Shewanella-produced hydroxamate ferric-siderophores. Additionally, this is the first SIP reported that also uses a ferredoxin as electron donor, and in contrast to NAD(P)H, its utilization did not require the mediation of ferrozine, and electron transfer occurred at fast rates. Finally, FAD oxidation was thermodynamically coupled to deprotonation at physiological pH values, enhancing the solubility of ferrous iron. On the basis of these results and the location of the SfSIP gene downstream of a sequence for putative binding of aerobic respiration control protein A (ArcA), we propose that SfSIP contributes an additional layer of regulation that maintains cellular iron homeostasis according to environmental cues of oxygen availability and cellular iron demand. ; publishersversion ; published

This journal is © The Royal Society of Chemistry 2020. ; The bone marrow tyrosine kinase in chromosome X (BMX) is pursued as a drug target because of its role in various pathophysiological processes. We designed BMX covalent inhibitors with single-digit nanomolar potency with unexploited topological pharmacophore patterns. Importantly, we reveal the first X-ray crystal structure of covalently inhibited BMX at Cys496, which displays key interactions with Lys445, responsible for hampering ATP catalysis and the DFG-out-like motif, typical of an inactive conformation. Molecular dynamic simulations also showed this interaction for two ligand/BMX complexes. Kinome selectivity profiling showed that the most potent compound is the strongest binder, displays intracellular target engagement in BMX-transfected cells with two-digit nanomolar inhibitory potency, and leads to BMX degradation PC3 in cells. The new inhibitors displayed anti-proliferative effects in androgen-receptor positive prostate cancer cells that where further increased when combined with known inhibitors of related signaling pathways, such as PI3K, AKT and Androgen Receptor. We expect these findings to guide development of new selective BMX therapeutic approaches. ; Funded under the Royal Society (URF to G. J. L. B., URF\R\180019), FCT Portugal (iFCT to G. J. L. B., IF/00624/2015, Postdoctoral Fellowship SFRH/BPD/95253/2013 to J. D. S., project PTDC/MED-QUI/28764/2017 to J. D. S., 02/SAICT/2017 grant 28333 to T. R., doctoral studentship SFRH/BD/143583/2019 to B. B. S., and DL 57/2016/CP1451/CT0025 to J. D. S.). This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreements No. 807281 and 702428. The authors acknowledge the Wellcome Trust (104633/Z/14/Z, to D. W. and C. V. R.), Ministerio de Economía y Competitividad (project RTI2018-099592-B-C21 to F. C.), the Royal G. and Mae H. Westaway Family Memorial Fund (A. N. K.), National Science Foundation Graduate Research Fellowship (Grant No ...