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

Rodolpho Souza Amado de Carvalho1, Md Shamiul Islam Rasel1, Nitesh K Khandelwal1

  • 1Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, USA.

Life Science Alliance
|August 29, 2024
PubMed

Insights

Phosphorylation activates the regulatory domain of yeast cadmium factor 1 (Ycf1) transporter. Its structure reveals key interactions stabilizing a transport-ready state, crucial for cellular function.

Area of Science:

  • Biochemistry
  • Structural Biology
  • Molecular Biology

Background:

  • ATP-binding cassette transporters are vital membrane proteins involved in transporting various substrates.
  • Regulation of these transporters often involves phosphorylation of intrinsically disordered regions, posing structural elucidation challenges.

Purpose of the Study:

  • To determine the structure of the activated regulatory domain (R-domain) of yeast cadmium factor 1 (Ycf1) transporter.
  • To identify key interactions within the R-domain and with other regions of Ycf1.
  • To understand the role of these interactions in Ycf1 function upon phosphorylation.

Main Methods:

  • Enzymatic enrichment of the phosphorylated state of the Ycf1 R-domain.
  • Cryo-electron microscopy (cryo-EM) to determine the 3.23 Å structure.
  • Systematic mutagenesis by replacing R-domain segments with alanine, glycine, and glutamine.
  • Functional assays under cellular conditions requiring Ycf1 activity.

Main Results:

  • A cryo-EM structure of the activated Ycf1 R-domain revealed four phosphorylated residues and its overall position.
  • Identified critical interactions, including a bridge between nucleotide-binding domains (NBD1 and NBD2) and interaction with the R-insertion region.
  • Mutational analysis confirmed the functional importance of most well-structured segments within the R-domain.

Conclusions:

  • Phosphorylation stabilizes a transport-competent state of Ycf1 by the R-domain.
  • The R-domain likely envelops NBD1 to stabilize this activated conformation.
  • Structural insights into R-domain regulation provide a foundation for understanding ABC transporter mechanisms.

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