9AYC

Tetra-phosphorylated, E1435Q Ycf1 mutant in inward-facing wide conformation

Summary for 9AYC

• Entry DOI: 10.2210/pdb9ayc/pdb
• EMDB information: 43985
• Descriptor: Metal resistance protein YCF1 (1 entity in total)
• Functional Keywords: abc-transporter, ycf1, r-domain, phosphorylation, heavy metal, membrane protein
• Biological source: Saccharomyces cerevisiae (brewer's yeast)
• Total number of polymer chains: 1
• Total formula weight: 176263.23

Authors

Carvalho, R.S.A.,Rasel, M.S.I.,Khandelwal, N.K.,Tomasiak, T.M. (deposition date: 2024-03-07, release date: 2024-08-21, Last modification date: 2025-09-03)

Primary citation

Souza Amado de Carvalho, R.,Rasel, M.S.I.,Khandelwal, N.K.,Tomasiak, T.M.
Cryo-EM reveals a phosphorylated R-domain envelops the NBD1 catalytic domain in an ABC transporter.
Life Sci Alliance, 7:-, 2024

PubMed 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.

PubMed: 39209537
DOI: 10.26508/lsa.202402779

Experimental method

ELECTRON MICROSCOPY (3.23 Å)