Neuweiler, H. | bioRxiv Science

bioRxiv2025

Bidirectional communication between nucleotide and substrate binding sites in a type IV multidrug ABC transporter

ATP-binding cassette (ABC) transporters use ATP to transport substrates across cellular membranes. In type IV ABC transporters, including many multidrug resistance (MDR) pumps, communication between the nucleotide-binding domains (NBDs) and transmembrane domains (TMDs) occurs via intracellular coupling helices. However, the precise mechanism of interdomain crosstalk and coordination between the ATP and substrate binding sites remain unclear. Combining nuclear magnetic resonance (NMR) spectroscopy, Hydrogen-Deuterium eXchange Mass Spectrometry (HDX-MS), photo-induced electron-transfer fluorescence correlation spectroscopy (PET-FCS) and functional assays, we identified a conserved cluster of residues at the NBD/TMD interface of the bacterial MDR transporter BmrA. This cluster is crucial for transporter function and relays nucleotide and substrate binding between the two domains via coupling helix 2. Mutations impact both local and global transporter dynamics. Our findings reveal a novel interdomain communication pathway in type IV ABC transporters, shedding light on the intricate coupling mechanisms that enable the coordination of these molecular machines.

biophysics↗

bioRxiv2020

Structure, interdomain dynamics and pH-dependent autoactivation of pro-rhodesain, the main lysosomal cysteine protease from African trypanosomes

Rhodesain is the lysosomal cathepsin L-like cysteine protease of T. brucei rhodesiense, the causative agent of Human African Trypanosomiasis. The enzyme is essential for the proliferation and pathogenicity of the parasite as well as its ability to overcome the blood-brain barrier of the host. Lysosomal cathepsins are expressed as zymogens with an inactivating pro-domain that is cleaved under acidic conditions. A structure of the uncleaved maturation intermediate from a trypanosomal cathepsin L-like protease is currently not available. We thus established the heterologous expression of T. brucei rhodesiense pro-rhodesain in E. coli and determined its crystal structure. The trypanosomal pro-domain differs from non-parasitic pro-cathepsins by a unique, extended -helix that blocks the active site and whose interactions resemble that of the antiprotozoal inhibitor K11777. Interdomain dynamics between pro- and core protease domain as observed by photoinduced electron transfer fluorescence correlation spectroscopy increase at low pH, where pro-rhodesain also undergoes autocleavage. Using the crystal structure, molecular dynamics simulations and mutagenesis, we identify a conserved interdomain salt bridge that prevents premature intramolecular cleavage at higher pH values and may thus present a control switch for the observed pH-sensitivity of pro-enzyme cleavage in (trypanosomal) CathL-like proteases.

biochemistry↗