Cryo-EM Reveals a Phosphorylated R-domain Envelops the NBD1 Catalytic Domain in an ABC Transporter

Overview

Journal Life Sci Alliance

Specialties Biochemistry
Biology
Cell Biology
Molecular Biology

Date 2024 Aug 29

PMID 39209537

Authors

Rodolpho Souza Amado de Carvalho

Md Shamiul Islam Rasel

Nitesh K Khandelwal

Thomas M Tomasiak

Affiliations

Soon will be listed here.

Abstract

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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