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X-ray and cryo-EM structures of inhibitor-bound cytochrome bc₁ complexes for structure-based drug discovery.

ABSTRACT: Cytochrome bc₁, a dimeric multi-subunit electron-transport protein embedded in the inner mitochondrial membrane, is a major drug target for the treatment and prevention of malaria and toxoplasmosis. Structural studies of cytochrome bc₁ from mammalian homologues co-crystallized with lead compounds have underpinned structure-based drug design to develop compounds with higher potency and selectivity. However, owing to the limited amount of cytochrome bc₁ that may be available from parasites, all efforts have been focused on homologous cytochrome bc₁ complexes from mammalian species, which has resulted in the failure of some drug candidates owing to toxicity in the host. Crystallographic studies of the native parasite proteins are not feasible owing to limited availability of the proteins. Here, it is demonstrated that cytochrome bc₁ is highly amenable to single-particle cryo-EM (which uses significantly less protein) by solving the apo and two inhibitor-bound structures to ?4.1?Å resolution, revealing clear inhibitor density at the binding site. Therefore, cryo-EM is proposed as a viable alternative method for structure-based drug discovery using both host and parasite enzymes.

SUBMITTER: Amporndanai K

PROVIDER: S-EPMC5947725 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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X-ray and cryo-EM structures of inhibitor-bound cytochrome bc1 complexes for structure-based drug discovery.

Amporndanai Kangsa K Johnson Rachel M RM O'Neill Paul M PM Fishwick Colin W G CWG Jamson Alexander H AH Rawson Shaun S Muench Stephen P SP Hasnain S Samar SS Antonyuk Svetlana V SV

IUCrJ 20180220 Pt 2

Cytochrome bc1, a dimeric multi-subunit electron-transport protein embedded in the inner mitochondrial membrane, is a major drug target for the treatment and prevention of malaria and toxoplasmosis. Structural studies of cytochrome bc1 from mammalian homologues co-crystallized with lead compounds have underpinned structure-based drug design to develop compounds with higher potency and selectivity. However, owing to the limited amount of cytochrome bc<su ...[more]

PMID: 29765610

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Project description:ABCG2 is a transporter protein of the ATP-binding-cassette (ABC) family that is expressed in the plasma membrane in cells of various tissues and tissue barriers, including the blood-brain, blood-testis and maternal-fetal barriers1-4. Powered by ATP, it translocates endogenous substrates, affects the pharmacokinetics of many drugs and protects against a wide array of xenobiotics, including anti-cancer drugs5-12. Previous studies have revealed the architecture of ABCG2 and the structural basis of its inhibition by small molecules and antibodies13,14. However, the mechanisms of substrate recognition and ATP-driven transport are unknown. Here we present high-resolution cryo-electron microscopy (cryo-EM) structures of human ABCG2 in a substrate-bound pre-translocation state and an ATP-bound post-translocation state. For both structures, we used a mutant containing a glutamine replacing the catalytic glutamate (ABCG2EQ), which resulted in reduced ATPase and transport rates and facilitated conformational trapping for structural studies. In the substrate-bound state, a single molecule of estrone-3-sulfate (E1S) is bound in a central, hydrophobic and cytoplasm-facing cavity about halfway across the membrane. Only one molecule of E1S can bind in the observed binding mode. In the ATP-bound state, the substrate-binding cavity has collapsed while an external cavity has opened to the extracellular side of the membrane. The ATP-induced conformational changes include rigid-body shifts of the transmembrane domains, pivoting of the nucleotide-binding domains (NBDs), and a change in the relative orientation of the NBD subdomains. Mutagenesis and in vitro characterization of transport and ATPase activities demonstrate the roles of specific residues in substrate recognition, including a leucine residue that forms a 'plug' between the two cavities. Our results show how ABCG2 harnesses the energy of ATP binding to extrude E1S and other substrates, and suggest that the size and binding affinity of compounds are important for distinguishing substrates from inhibitors.

Dataset Information

X-ray and cryo-EM structures of inhibitor-bound cytochrome bc₁ complexes for structure-based drug discovery.

Publications

X-ray and cryo-EM structures of inhibitor-bound cytochrome <i>bc</i><sub>1</sub> complexes for structure-based drug discovery.

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