Structural mechanism for cargo recognition by the retromer complex

Retromer is a multi-protein complex that recycles transmembrane cargo from endosomes to the trans-Golgi network and the plasma membrane. Defects in retromer impair various cellular processes and underlie some forms of Alzheimer's disease and Parkinson's disease. Although retromer was discovered over 15 years ago, the mechanisms for cargo recognition and recruitment to endosomes have remained elusive. Here, we present an X-ray crystallographic analysis of a four-component complex comprising the VPS26 and VPS35 subunits of retromer, the sorting nexin SNX3, and a recycling signal from the divalent cation transporter DMT1-II. This analysis identifies a binding site for canonical recycling signals at the interface between VPS26 and SNX3. In addition, the structure highlights a network of cooperative interactions among the VPS subunits, SNX3, and cargo that couple signal-recognition to membrane recruitment.We thank Alberto Marina (CIC bioGUNE) for technical assistance. This work was supported by the Carlos III Health Institute grant PI11/00121, the Basque Government grant PI2011-26, the Spanish Ministry of Economy and Competitiveness Grant BFU2014-59759-R (to A.H.), and the intramural program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH (ZIA HD001607) (to J.S.B.). This study made use of the Diamond Light Source (Oxfordshire, UK), synchrotron SOLEIL (Gif-sur Yvette, France), the European Synchrotron Radiation Facility (ESRF, Grenoble, France) and ALBA synchrotron beamline BL13-XALOC, funded in part by the European Community’s Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement N°283570). We thank all the staff from these facilities, and in particular to Andrew Thomson from SOLEIL, for assistance with X-ray data collection and processing, and Robert Rambo from Diamond for assistance with SAXS data collection. We also thank Peter Cullen, Carol R. Haft and Mitsuaki Tabuchi for kind gifts of reagents, and Philip McCoy (NHLBI, NIH) for cell sorting

Targeting the Spike: Repurposing Mithramycin and Dihydroergotamine to Block SARS-CoV-2 Infection

: The urgency to find complementary therapies to current SARS-CoV-2 vaccines, whose effectiveness is preserved over time and not compromised by the emergence of new and emerging variants, has become a critical health challenge. We investigate the possibility of jamming the opening of the Receptor Binding Domain (RBD) of the spike protein of SARS-CoV-2 with small compounds. Through in silico screening, we identified two potential candidates that would lock the Receptor Binding Domain (RBD) in a closed configuration, preventing the virus from infecting the host cells. We show that two drugs already approved by the FDA, mithramycin and dihydroergotamine, can block infection using concentrations in the μM range in cell-based assays. Further STD-NMR experiments support dihydroergotamine's direct interaction with the spike protein. Overall, our results indicate that repurposing of these compounds might lead to potential clinical drug candidates for the treatment of SARS-CoV-2 infection

Architecture of the ESCPE-1 membrane coat

Recycling of membrane proteins enables the reuse of receptors, ion channels and transporters. A key component of the recycling machinery is the endosomal sorting complex for promoting exit 1 (ESCPE-1), which rescues transmembrane proteins from the endolysosomal pathway for transport to the trans-Golgi network and the plasma membrane. This rescue entails the formation of recycling tubules through ESCPE-1 recruitment, cargo capture, coat assembly and membrane sculpting by mechanisms that remain largely unknown. Herein, we show that ESCPE-1 has a single-layer coat organization and suggest how synergistic interactions between ESCPE-1 protomers, phosphoinositides and cargo molecules result in a global arrangement of amphipathic helices to drive tubule formation. Our results thus define a key process of tubule-based endosomal sorting.This work was funded by MCIN/AEI/10.13039/501100011033 (PID2020- 119132GB-I00, CEX2021-001136-S) (to A.H.), the Intramural Program of NICHD, NIH (ZIA HD001607 to J.S.B.), the Swiss National Science Foundation grant 205321 179041 (to D.C.-D.), the Human Frontiers Science Program grant RGP0017/2020 (to D.C.-D.) and the PID2021- 127309NB-I00 funded by AEI/10.13039/501100011033/ FEDER, UE (to D.C.-D.). This study made use of the Diamond Light Source proposal MX20113, ALBA synchrotron beamline BL13-XALOC, the cryo-EM facilities at the UK Electron Bio-Imaging Centre, proposals EM17171- 6 and EM17171, and the Midlands Regional Cryo-EM Facility at the Leicester Institute of Structural and Chemical Biology (LISCB). We thank C. Savva (LISCB, University of Leicester) for his help in cryo-EM data collection. With funding from the Spanish government through the Severo Ochoa Centre of Excellence’ accreditation (CEX2021-001136-S

Structural insight into the membrane targeting domain of the Legionella deAMPylase SidD

AMPylation, the post-translational modification with adenosine monophosphate (AMP), is catalyzed by effector proteins from a variety of pathogens. Legionella pneumophila is thus far the only known pathogen that, in addition to encoding an AMPylase (SidM/DrrA), also encodes a deAMPylase, called SidD, that reverses SidM-mediated AMPylation of the vesicle transport GTPase Rab1. DeAMPylation is catalyzed by the N-terminal phosphatase-like domain of SidD. Here, we determined the crystal structure of full length SidD including the uncharacterized C-terminal domain (CTD). A flexible loop rich in aromatic residues within the CTD was required to target SidD to model membranes in vitro and to the Golgi apparatus within mammalian cells. Deletion of the loop (??loop) or substitution of its aromatic phenylalanine residues rendered SidD cytosolic, showing that the hydrophobic loop is the primary membrane-targeting determinant of SidD. Notably, deletion of the two terminal alpha helices resulted in a CTD variant incapable of discriminating between membranes of different composition. Moreover, a L. pneumophila strain producing SidD??loop phenocopied a L. pneumophila ??sidD strain during growth in mouse macrophages and displayed prolonged co-localization of AMPylated Rab1 with LCVs, thus revealing that membrane targeting of SidD via its CTD is a critical prerequisite for its ability to catalyze Rab1 deAMPylation during L. pneumophila infection

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