Structural biochemistry of ATP-driven dimerization and DNA-stimulated activation of SMC ATPases

Structural maintenance of chromosome (SMC) proteins play a central role in higher-order chromosome structure in all kingdoms of life. SMC proteins consist of a long coiled-coil domain that joins an ATP binding cassette (ABC) ATPase domain on one side and a dimerization domain on the other side. SMC proteins require ATP binding or hydrolysis to promote cohesion and condensation, which is suggested to proceed via formation of SMC rings or assemblies. To learn more about the role of ATP in the architecture of SMC proteins, we report crystal structures of nucleotide-free and ATP bound P. furiosus SMC ATPase domains. ATP dimerizes two SMC ATPase domains by binding to opposing Walker A and signature motifs, indicating that ATP binding can directly assemble SMC proteins. DNA stimulates ATP hydrolysis in the engaged SMC ABC domains, suggesting that ATP hydrolysis can be allosterically regulated. Structural and mutagenesis data identify an SMC protein conserved-arginine finger that is required for DNA stimulation of the ATPase activity and directly connects a putative DNA interaction site to ATP. Our results suggest that stimulation of the SMC ATPase activity may be a specific feature to regulate the ATP-driven assembly and disassembly of SMC proteins.     

Chimeras of P-type ATPases and their transcriptional regulators: contributions of a cytosolic amino-terminal domain to metal specificity

Zn²⁺‐responsive repressor ZiaR and Co²⁺‐responsive activator CoaR modulate production of P₁‐type Zn²⁺‐ (ZiaA) and Co²⁺‐ (CoaT) ATPases respectively. What dictates metal selectivity? We show that Δ ziaΔcoa double mutants had similar Zn²⁺ resistance to Δzia single mutants and similar Co²⁺ resistance to Δcoa single mutants. Controlling either ziaA or coaT with opposing regulators restored no resistance to metals sensed by the regulators, but coincident replacement of the deduced cytosolic amino‐terminal domain CoaT_N with ZiaA_N (in ziaR‐_p ziaA‐ziaA_NcoaT) conferred Zn²⁺ resistance to ΔziaΔcoa, Zn²⁺ content was lowered and residual Co²⁺ resistance lost. Metal‐dependent molar absorptivity under anaerobic conditions revealed that purified ZiaA_N binds Co²⁺ in a pseudotetrahedral two‐thiol site, and Co²⁺ was displaced by Zn²⁺. Thus, the amino‐terminal domain of ZiaA inverts the metals exported by zinc‐regulated CoaT from Co²⁺ to Zn²⁺, and this correlates simplistically with metal‐binding preferences; K_ZiaAN Zn²⁺ tighter than Co²⁺. However, Zn²⁺ did not bleach Cu⁺‐ZiaA_N, and only Cu⁺ co‐migrated with ZiaA_N after competitive binding versus Zn²⁺. Bacterial two‐hybrid assays that detected interaction between the Cu⁺‐metallochaperone Atx1 and the amino‐terminal domain of Cu⁺‐transporter PacS_N detected no interaction with the analogous, deduced, ferredoxin‐fold subdomain of ZiaA_N. Provided that there is no freely exchangeable cytosolic Cu⁺, restricted contact with the Cu⁺‐metallochaperone can impose a barrier impairing the formation of otherwise favoured Cu⁺–ZiaA_N complexes.     

Two ATPases

In this article, I reflect on research on two ATPases. The first is F(1)F(0)-ATPase, also known as ATP synthase. It is the terminal enzyme in oxidative phosphorylation and famous as a nanomotor. Early work on mitochondrial enzyme involved purification in large amount, followed by deduction of subunit composition and stoichiometry and determination of molecular sizes of holoenzyme and individual subunits. Later work on Escherichia coli enzyme utilized mutagenesis and optical probes to reveal the molecular mechanism of ATP hydrolysis and detailed facets of catalysis. The second ATPase is P-glycoprotein, which confers multidrug resistance, notably to anticancer drugs, in mammalian cells. Purification of the protein in large quantity allowed detailed characterization of catalysis, formulation of an alternating sites mechanism, and recently, advances in structural characterization.