Cryo-EM reveals a phosphorylated R-domain envelops the NBD1 catalytic domain in an ABC transporter

TL;DR

A cryo-EM structure shows how a phosphorylated R-domain interacts with an ATPase domain in an ABC transporter, regulating its activity.

Contribution

The study presents a 3.23 Å cryo-EM structure of a phosphorylated R-domain and its functional interactions in an ABC transporter.

Findings

The R-domain encircles the ATP catalytic domain in a tetra-phosphorylated state.

Key interactions between the R-domain and NBD1/NBD2 are identified.

Functional scanning confirms the importance of structured R-domain segments.

Abstract

Cryo-EM reveals that the R-domain of an ABCC transporter encircles the ATP catalytic domain-1 in a tetra-phosphorylated manner and regulates the ATPase activity along with cellular transport.

Many ATP-binding cassette transporters are regulated by phosphorylation on long and disordered loops which presents a challenge to visualize with structural methods. We have trapped an activated state of the regulatory domain (R-domain) of yeast cadmium factor 1 (Ycf1) by enzymatically enriching the phosphorylated state. A 3.23 Å cryo-EM structure reveals an R-domain structure with four phosphorylated residues and the position for the entire R-domain. The structure reveals key R-domain interactions including a bridging interaction between NBD1 and NBD2 and an interaction with the R-insertion, another regulatory region. We scanned these interactions by systematically replacing segments along the entire R-domain with scrambled combinations of alanine, glycine, and glutamine and probing function under cellular conditions that require the Ycf1 function. We find a close match with these interactions and interacting regions on our R-domain structure that points to the importance of most well-structured segments for function. We propose a model where the R-domain stabilizes a transport-competent state upon phosphorylation by enveloping NBD1 entirely.

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