Biology subjects

Rheinberger, J.

Publications and source records attributed to Rheinberger, J..

4 recordsLinked to original sources

bioRxiv2022

Inhibited KdpFABC resides in an E1 off-cycle state

KdpFABC is a high-affinity prokaryotic K+ uptake system that forms a functional chimera between a channel-like subunit (KdpA) and a P-type ATPase (KdpB). At high K+ levels, KdpFABC needs to be inhibited to prevent excessive K+ accumulation to the point of toxicity. This is achieved by a phosphorylation of the serine residue in the TGES162 motif in the A domain of the pump subunit KdpB (KdpBS162-P). Here, we explore the structural basis of inhibition by KdpBS162 phosphorylation by determining the conformational landscape of KdpFABC under inhibiting and non-inhibiting conditions. Under turnover conditions, we identified a new inhibited KdpFABC conformation that we termed E1-P tight, which is not part of the canonical Post-Albers transport cycle of P-type ATPases. It likely represents the biochemically described stalled E1-P state adopted by KdpFABC upon KdpBS162 phosphorylation. The E1-P tight state exhibits a compact fold of the three cytoplasmic domains and is likely adopted when the transition from high-energy E1-P states to E2-P states is unsuccessful. This study represents a structural characterization of a biologically relevant off-cycle state in the P-type ATPase family and supports the emerging discussion of P-type ATPase regulation by such conformations.

molecular biology↗

bioRxiv2020

Optimized data acquisition workflow by sample thickness determination

Sample thickness is a known key parameter in cryo-electron microscopy (cryo-EM) and can affect the amount of high-resolution information retained in the image. Yet, common data acquisition approaches in single particle cryo-EM do not take it into account. Here, we demonstrate how the sample thickness can be determined before data acquisition, allowing to identify optimal regions and restrict automated data collection to images with preserved high-resolution details. This quality over quantity approach, almost entirely eliminates the time- and storage-consuming collection of suboptimal images, which are discarded after a recorded session or during early image processing due to lack of high-resolution information. It maximizes data collection efficiency and lowers the electron microscopy time required per dataset. This strategy is especially useful, if the speed of data collection is restricted by the microscope hardware and software, or if microscope access time, data transfer, data storage and computational power are a bottleneck. SynopsisSample thickness is a key parameter in single particle cryo-electron microscopy. Determining sample thickness before data acquisition allows to target optimal areas and maximize data output quality of single particle cryo-electron microscopy sessions. Scripts and optimized workflows for EPU and SerialEM are presented and available as open-source.

biophysics↗

bioRxiv2020

Highly potent bispecific sybodies neutralize SARS-CoV-2

The ongoing COVID-19 pandemic represents an unprecedented global health crisis. Here, we report the identification of a synthetic nanobody (sybody) pair (Sb#15 and Sb#68) that can bind simultaneously to the SARS-CoV-2 spike-RBD and efficiently neutralize pseudotyped and live-viruses by interfering with ACE2 interaction. Two spatially-discrete epitopes identified by cryo-EM translated into the rational design of bispecific and tri-bispecific fusions constructs, exhibiting up to 100- and 1000-fold increase in neutralization potency. Cryo-EM of the sybody-spike complex further revealed a novel up-out RBD conformation. While resistant viruses emerged rapidly in the presence of single binders, no escape variants were observed in presence of the bispecific sybody. The multivalent bispecific constructs further increased the neutralization potency against globally-circulating SARS- CoV-2 variants of concern. Our study illustrates the power of multivalency and biparatopic nanobody fusions for the development of clinically relevant therapeutic strategies that mitigate the emergence of new SARS-CoV-2 escape mutants.

biochemistry↗

bioRxiv2018

Cryo-EM structures reveal bilayer remodeling during Ca2+ activation of a TMEM16 scramblase

The lipid distribution of plasma membranes of eukaryotic cells is asymmetric and phospholipid scramblases disrupt this asymmetry by mediating the rapid nonselective transport of lipids down their concentration gradients. As a result, phosphatidylserine is exposed to the outer leaflet of membrane, an important step in extracellular signaling networks controlling processes such as apoptosis, blood coagulation, membrane fusion and repair. Several members of the TMEM16 family have been identified as Ca2+-activated scramblases but the mechanisms underlying their Ca2+-dependent gating and their effects on the surrounding lipid bilayer remain poorly understood. Here we describe three high-resolution cryo-electron microscopy structures of a fungal scramblase from Aspergillus fumigatus, afTMEM16, reconstituted in lipid nanodiscs, revealing large Ca2+-dependent conformational changes of the protein as well as significant, function dependent membrane reorganization.

biochemistry↗