Cu⁺-specific CopB transporter: Revising P_1B-type ATPase classification

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Copper-transporting P1B-ATPases are crucial for cellular copper homeostasis.
• Traditionally, P1B-1-ATPases (CopAs) export Cu+, while P1B-3-ATPases (CopBs) were thought to export Cu2+.

Purpose of the Study:

• To biochemically and spectroscopically characterize Sphaerobacter thermophilus CopB (StCopB).
• To clarify the metal specificity and transport mechanism of P1B-3-ATPases.
• To re-evaluate the classification of copper-transporting P1B-ATPases.

Main Methods:

• Biochemical assays to measure ATPase activity.
• Spectroscopic characterization, including X-ray absorption spectroscopy.
• Bioinformatic analysis and reconsideration of classification schemes.

Main Results:

• StCopB binds Cu2+ but exhibits metal-stimulated ATPase activity only in response to Cu+.
• Cu+ is coordinated by four sulfur ligands in StCopB, not involving the typical transmembrane site.
• The N-terminal region of StCopB is essential for activity but inhibited by divalent metals.
• Both P1B-1- and P1B-3-ATPase subfamilies are identified as Cu+ transporters.

Conclusions:

• The P1B-3-ATPase subfamily, including StCopB, transports Cu+, challenging previous assumptions.
• The classification of P1B-ATPases needs revision, with both major subfamilies transporting Cu+.
• Cellular copper transport mechanisms are consistent with the reducing cytoplasmic environment favoring Cu+.