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Structures of the ATP-fueled ClpXP proteolytic machine bound to protein substrate.

ABSTRACT: ClpXP is an ATP-dependent protease in which the ClpX AAA+ motor binds, unfolds, and translocates specific protein substrates into the degradation chamber of ClpP. We present cryo-EM studies of the E. coli enzyme that show how asymmetric hexameric rings of ClpX bind symmetric heptameric rings of ClpP and interact with protein substrates. Subunits in the ClpX hexamer assume a spiral conformation and interact with two-residue segments of substrate in the axial channel, as observed for other AAA+ proteases and protein-remodeling machines. Strictly sequential models of ATP hydrolysis and a power stroke that moves two residues of the substrate per translocation step have been inferred from these structural features for other AAA+ unfoldases, but biochemical and single-molecule biophysical studies indicate that ClpXP operates by a probabilistic mechanism in which five to eight residues are translocated for each ATP hydrolyzed. We propose structure-based models that could account for the functional results.

SUBMITTER: Fei X

PROVIDER: S-EPMC7112951 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Structures of the ATP-fueled ClpXP proteolytic machine bound to protein substrate.

Fei Xue X Bell Tristan A TA Jenni Simon S Stinson Benjamin M BM Baker Tania A TA Harrison Stephen C SC Sauer Robert T RT

eLife 20200228

ClpXP is an ATP-dependent protease in which the ClpX AAA+ motor binds, unfolds, and translocates specific protein substrates into the degradation chamber of ClpP. We present cryo-EM studies of the <i>E. coli</i> enzyme that show how asymmetric hexameric rings of ClpX bind symmetric heptameric rings of ClpP and interact with protein substrates. Subunits in the ClpX hexamer assume a spiral conformation and interact with two-residue segments of substrate in the axial channel, as observed for other ...[more]

PMID: 32108573

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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

Structures of the ATP-fueled ClpXP proteolytic machine bound to protein substrate.

Publications

Structures of the ATP-fueled ClpXP proteolytic machine bound to protein substrate.

Similar Datasets

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