Dissecting the conformational complexity and flipping mechanism of a prokaryotic heme transporter

Abstract

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 highly specific heme transporter required for assembly and 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 translocation. We found 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 degree. 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 open states.

Structural and mechanistic insights into human choline and ethanolamine transport

“…5b). Of note, neither the dimensions, nor the residues lining the central cavities of both FLVCRs indicate a structural adaptation for accommodating a heme molecule as observed in other heme transporters 30–32 .…”

Profiling the Heme‐Binding Proteomes of Bacteria Using Chemical Proteomics

“…Gratifyingly, heme-regulated transport efflux pump HrtBA [54] (EF_0792 and EF_0793), heme chaperone HemW (EF_1305) and cytochrome bd subunit CydA were clearly induced in the presence of either heme or heme probe 1. Expression of heme transporter proteins CydDC [55,56] (EF_2058 and EF_2059) and noncanonical heme chaperones AhpC and Gap-2 [57] was unaffected by heme treatment and likewise for heme probe 1, while neither CydB nor newly reported heme transport regulator FhtR [54] were detected by mass spectrometry. However, expression of the heme-dependent catalase KatA [53] was induced only in the presence of heme and not heme probe 1.…”

Untersuchung des Häm‐bindenden bakteriellen Proteoms mittels chemischer Proteomik

“…Erfreulicherweise wurde die Expression der Häm-regulierten Transport-Efflux-Pumpe HrtBA [54] (EF 0792 und EF 0793), des Häm-Chaperon HemW (EF 1305) und der Cytochrom bd-Untereinheit CydA in Gegenwart von Häm oder der Häm-Sonde 1 deutlich induziert. Die Expression der Häm-Transporter-Proteine CydDC [55,56] (EF 2058 und EF 2059) und der nicht-kanonischen Häm-Chaperone AhpC und Gap-2 [57] wurde weder durch die Behandlung mit Häm noch durch die Häm-Sonde 1 beeinflusst, während weder CydB noch der neu publizierte Häm-Transport-Regulator FhtR [54] durch Massenspektrometrie nachgewiesen werden konnten. Die Expression der Häm-abhängigen Katalase KatA [53] wurde jedoch nur in Gegenwart von Häm und nicht von Häm-Sonde 1 induziert.…”