Journal: bioRxiv

Article Title: Site-specific phosphorylation affects the structure and interactions of the Ycf1p R region

doi: 10.64898/2026.01.07.698183

Figure Lengend Snippet: (A) Schematic diagram of Ycf1p showing the transmembrane domains (TMD0, TMD1, TMD2) and L0 linker in grey, nucleotide binding domain 1 (NBD1) in blue, NBD2 in green, and the intrinsically disordered regulatory (R) region in red. Coupling helices (CH1-CH4) link the cytoplasmic extensions of transmembrane helices in TMD1 and TMD2 and interact with the NBDs. The two half transporters of the ABC core in Ycf1p are comprised of TMD1-NBD1 and TMD2-NBD2. Phosphorylation sites in the L0 linker (S251) and in the R region (S903, S908, T911, and S914) ( , ) are indicated with a ‘P’ circled in red. The Pep4 protease cleavage site is denoted by a pink asterisk (*). (B) Schematic representation of proteins used in the present study. As in other studies, NBD1 consists of residues D610-N859 ( , ), whereas NBD2 is comprised of residues E1265-N1515 . NBD1-R consists of residues D610-E930, and includes NBD1 and the R region as a single polypeptide ( , ), leaving the R region to be defined as N860-E930. Residues S903, S908, T911, and S914 are highlighted in Supplementary Fig. 1.

Article Snippet: Briefly, the His 6 -SUMO Ycf1p proteins were expressed in E. coli LOBSTR-BL21 (DE3) RIL (Kerafast Inc) using a pET26b-derived expression vector ( ).

Techniques: Binding Assay, Phospho-proteomics

Journal: bioRxiv

Article Title: Site-specific phosphorylation affects the structure and interactions of the Ycf1p R region

doi: 10.64898/2026.01.07.698183

Figure Lengend Snippet: (A) In the dephosphorylated Ycf1p cryo-EM structure, R region occupies the substrate binding cavity in a helix-strand hairpin conformation . In this state, the phosphorylation sites are in the substrate binding cavity and would need to be dislodged in order for phosphorylation to occur. Trp fluorescence quenching data show that non-phosphorylated R region can also interact with NBD1 near the ATP binding site ( , ), consistent with NMR data presented here. Thus, segments of the R region not observed in the cryo-EM structure of the auto-inhibited Ycf1p are shown with their possible NBD1 interactions and residual 2° structures. (B) NMR data in this paper also show that R region residues comprising α3- and α4-helices interact with NBD2 and can represent a state to allow for R region phosphorylation. Dislodging of the R region from the substrate cavity may occur either stochastically or be induced by substrate binding, as in other ABCC proteins ( – , ). The latter case may result in basal levels of transport, provided that the non-phosphorylated R region adopts a conformation that allows for nucleotide binding and dimerization of NBD1 and NBD2, as seen in our NMR data. The ‘fuzziness’ around the entire R region represents the limited interactions observed for the R region with the nucleotide-bound and dimerized NBDs from the NMR data. (C) Upon phosphorylation, the R region relocates to the periphery of NBD1 opposite the ATP and NBD2 binding sites. Different cryo-EM structures show that different residues of phosphorylated R region can interact with NBD1 ( , , , ), and is depicted with the two models. Further, parts of the R region are also missing in these structures. Thus, the phosphorylated R region can engage in different interactions with NBD1, leading to a fuzzy NBD1/R region complex. Potential interactions of non-observed R region residues (at the N-terminal end and comprising the phosphorylation sites) are shown as fuzzy elements. (D) Regardless, movement of the R region to the peripheral side of NBD1 allows for substrate binding and formation of an NBD1/NBD2 MgATP sandwich dimer. (E) Formation of the outward facing conformation follows, leading to transport. Note that all of the phosphorylated R region is shown as a fuzzy curve to highlight the fact that NBD1/R region interactions are disrupted when NBD1 binds MgATP and dimerizes with NBD2. As with other ABC transporters , MgATP hydrolysis at the NBD2 composite site resets that transporter for another transport cycle. The model presented here shows possible interactions of the R region at different stages of the ATP hydrolysis and transport cycles. Note that due to the dynamic nature of the disordered R region, interactions of R region with the substrate-binding cavity and with the NBDs are likely transient.

Article Snippet: Briefly, the His 6 -SUMO Ycf1p proteins were expressed in E. coli LOBSTR-BL21 (DE3) RIL (Kerafast Inc) using a pET26b-derived expression vector ( ).

Techniques: Cryo-EM Sample Prep, Binding Assay, Phospho-proteomics, Fluorescence