Bald, D. | Search bioRxiv

bioRxiv2022

Autoinsertion (LAiR): rapid functional reconstitution of integral membrane proteins into lipid bilayers

Functional investigation of purified integral membrane proteins (IMPs) is hampered by the need to insert these hydrophobic proteins from the detergent-solubilized state into liposomal membranes. Here we report reintegration of IMPs into a lipid environment within minutes, an order of magnitude faster than currently used standard techniques. The new approach yielded optimal results for IMPs solubilized in the detergent lauryl-maltose neopentyl glycol (LMNG) and is therefore termed LMNG Auto-insertion Reintegration (LAiR). LAiR displays superior performance to standard methods in terms of protein activity, long-term stability and proton tightness of proteoliposomes. LAiR reconstituted vectorial control of membrane-bound activity by the transmembrane ion motive force, a property particularly important in mitochondrial function, which was undetectable by standard reintegration methods. LAiR also preserved fragile IMP properties that are prone to disruption upon reintegration, including long-term multi-subunit integrity, inhibitor susceptibility, and higher-order oligomeric states. LAiR proved suitable for reintegration into liposomes as well as into surface-tethered membrane bilayers, and was compatible with IMPs and lipids from prokaryotic and eukaryotic sources. We anticipate a broad scope for LAiR as a powerful tool in fundamental research, pharmaceutical applications, and biotechnology.

biochemistry↗

bioRxiv2022

Dissecting the conformational complexity and flipping mechanism of bacterial heme transport.

Iron-bound cyclic tetrapyrroles (hemes) are key redox-active cofactors in membrane-integrated oxygen reductases and other bioenergetic enzymes. However, the mechanisms of heme transport and insertion into respiratory chain complexes remain unclear. Here, we used a combination of cellular, biochemical, structural and computational methods to resolve ongoing controversies around the function of the heterodimeric bacterial ABC transporter CydDC. We provide multi-level evidence that CydDC is a heme transporter required for assembly and functional maturation of cytochrome bd, a pharmaceutically relevant drug target. Our systematic single-particle cryo-EM approach combined with atomistic molecular dynamics simulations provides detailed insight into the conformational landscape of CydDC during substrate binding and occlusion. Our simulations reveal that heme binds laterally from the membrane space to the transmembrane region of CydDC, enabled by a highly asymmetrical inward-facing CydDC conformation. During the binding process, heme propionates interact with positively charged residues on the surface and later in the substrate-binding pocket of the transporter, causing the heme orientation to flip 180 degrees. The membrane-accessible heme entry site of CydDC is primarily controlled by the conformational plasticity of CydD transmembrane helix 4, the extended cytoplasmic segment of which also couples heme confinement to a rotational movement of the CydC nucleotide-binding domain. Our cryo-EM data highlight that this signal transduction mechanism is necessary to drive conformational transitions toward occluded and outward-facing states. One Sentence SummaryThe heterodimeric bacterial ABC transporter CydDC is a heme flippase essential for the functional maturation of cytochrome bd.

microbiology↗