Structural Role of the First Four Transmembrane Helices in ZntA, a P_1B-Type ATPase from Escherichia coli

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• ZntA from Escherichia coli is a P1B-ATPase transporter conferring resistance to toxic heavy metals like Pb²⁺, Zn²⁺, and Cd²⁺.
• P1B-type ATPases, including human ATP7A and ATP7B, typically possess an N-terminal metal-binding domain and eight transmembrane helices.
• A splice variant of ATP7B lacking the N-terminal domain and first four transmembrane helices exhibits altered expression patterns.

Purpose of the Study:

• To investigate the functional and structural consequences of truncating ZntA, specifically removing the N-terminal metal-binding domain and first four transmembrane helices (Δ231-ZntA).
• To elucidate the role of the N-terminal domain and transmembrane helices in ZntA's metal binding affinity, specificity, and overall structure.

Main Methods:

• Creation and characterization of a truncated ZntA mutant (Δ231-ZntA).
• Assessment of in vitro and in vivo metal transport activity.
• Determination of metal ion binding affinities and specificities.
• Site-specific mutagenesis of key metal-binding residues (Cys392, Cys394, Asp714).
• Construction of three-dimensional homology models of ZntA and Δ231-ZntA dimers.

Main Results:

• Δ231-ZntA exhibited significantly reduced in vitro and no detectable in vivo activity.
• The truncated mutant bound metal ions with 15-19000-fold higher affinity at the transmembrane site, indicating substantial structural changes.
• Metal ion specificity shifted: Cd²⁺ showed the highest affinity for Δ231-ZntA, whereas Pb²⁺ had the highest affinity for wild-type ZntA.
• Mutagenesis revealed flexibility in metal binding, with different residue combinations involved in Zn²⁺/Pb²⁺ binding compared to Cd²⁺ binding.
• Homology models demonstrated dramatic differences in dimer structures between ZntA and Δ231-ZntA.

Conclusions:

• The N-terminal domain and the first four transmembrane helices of ZntA are essential for its proper function and dimer structure.
• Truncation of these regions leads to altered metal binding affinity and specificity, highlighting their critical role in P1B-ATPase architecture.
• The findings underscore the importance of the transmembrane domain in maintaining the structural integrity and metal transport capabilities of P1B-type ATPases.