Tracing the substrate translocation mechanism in P-glycoprotein.

P-glycoprotein (Pgp) is a prototypical ATP-binding cassette (ABC) transporter of great biological and clinical significance.Pgp confers cancer multidrug resistance and mediates the bioavailability and pharmacokinetics of many drugs (Juliano and Ling, 1976; Ueda et al., 1986; Sharom, 2011). Decades of structural and biochemical studies have provided insights into how Pgp binds diverse compounds (Loo and Clarke, 2000; Loo et al., 2009; Aller et al., 2009; Alam et al., 2019; Nosol et al., 2020; Chufan et al., 2015), but how they are translocated through the membrane has remained elusive. Here, we covalently attached a cyclic substrate to discrete sites of Pgp and determined multiple complex structures in inward- and outward-facing states by cryoEM. In conjunction with molecular dynamics simulations, our structures trace the substrate passage across the membrane and identify conformational changes in transmembrane helix 1 (TM1) as regulators of substrate transport. In mid-transport conformations, TM1 breaks at glycine 72. Mutation of this residue significantly impairs drug transport of Pgp in vivo, corroborating the importance of its regulatory role. Importantly, our data suggest that the cyclic substrate can exit Pgp without the requirement of a wide-open outward-facing conformation, diverting from the common efflux model for Pgp and other ABC exporters. The substrate transport mechanism of Pgp revealed here pinpoints critical targets for future drug discovery studies of this medically relevant system.

Cotranslational assembly of membrane protein/nanoparticles in cell-free systems.

Nanoparticles composed of amphiphilic scaffold proteins and small lipid bilayers are valuable tools for reconstitution and subsequent functional and structural characterization of membrane proteins. In combination with cell-free protein production systems, nanoparticles can be used to cotranslationally and translocon independently insert membrane proteins into tailored lipid environments. This strategy enables rapid generation of protein/nanoparticle complexes by avoiding detergent contact of nascent membrane proteins. Frequently in use are nanoparticles assembled with engineered derivatives of either the membrane scaffold protein (MSP) or the Saposin A (SapA) scaffold. Furthermore, several strategies for the formation of membrane protein/nanoparticle complexes in cell-free reactions exist. However, it is unknown how these strategies affect functional folding, oligomeric assembly and membrane insertion efficiency of cell-free synthesized membrane proteins. We systematically studied membrane protein insertion efficiency and sample quality of cell-free synthesized proteorhodopsin (PR) which was cotranslationally inserted in MSP and SapA based nanoparticles. Three possible PR/nanoparticle formation strategies were analyzed: (i) PR integration into supplied preassembled nanoparticles, (ii) coassembly of nanoparticles from supplied scaffold proteins and lipids upon PR expression, and (iii) coexpression of scaffold proteins together with PR in presence of supplied lipids. Yield, homogeneity as well as the formation of higher PR oligomeric complexes from samples generated by the three strategies were analyzed. Conditions found optimal for PR were applied for the synthesis of a G-protein coupled receptor. The study gives a comprehensive guideline for the rapid synthesis of membrane protein/nanoparticle samples by different processes and identifies key parameters to modulate sample yield and quality.

Know your detergents: A case study on detergent background in negative stain electron microscopy.

Electron cryo-microscopy (cryo-EM) of purified macromolecular complexes is now providing 3D-structures at near-atomic resolution (Kühlbrandt, 2014). Cryo-EM can tolerate heterogeneous specimens, however, high-resolution efforts demand highly optimized samples. Therefore, significant pre-screening and evaluation is essential before a final dataset can be obtained. While cryo-EM is comparably slow and requires access to expensive high-end electron microscopes, room temperature negative stain EM is fast, inexpensive and provides immediate feedback. This has made it a popular approach for sample quality control in the early phases of a project. Optimization in negative stain can be critical not only for cryo-EM, but also for X-ray crystallography, as highlighted for example by studies on GPCR complexes (Kang et al., 2015; Rasmussen et al., 2012). However, when not done carefully and interpreted correctly, negative stain can be prone to artifacts. A typical problem, which is often overlooked in the interpretation of EM data of small membrane proteins, is the background, caused by empty detergent micelles, as it can be easily confused with detergent embedded protein samples. To counteract this ubiquitous problem, we present a case study on commonly used detergents.We show that most detergents produce significant background in negative stain EM, even below nominal critical micelle concentration (CMC). Unawareness of such artefacts can lead to misinterpretation of sample quality and homogeneity. We hope that this study can serve as a template to evaluate images in the early phases of a project.