A Conserved Motif in Intracellular Loop 1 Stabilizes the Outward-Facing Conformation of TmrAB

Millan, Cinthia; Francis, Martina; Khandelwal, Nitesh; Thompson, Valery F.; Thaker, Tarjani; Tomasiak, Thomas

doi:10.1016/j.jmb.2021.166834

Cited by 7 publications

(7 citation statements)

References 68 publications

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“…Asp919 and Lys935 form H-bonds with Asn1051 and Glu1241, respectively, while Ile923 and Val1238 make nonpolar interactions. Finally, the interactions in the substrate cavity accompany dephosphorylated R-domain contacts with a critical allosteric junction, the "GRD" motif of ICL-1, which couples with the X-loop of NBD1 to communicate ATP binding 23 .…”

Section: Dephosphorylation Leads To R-domain Occlusion Of the Substra...mentioning

confidence: 99%

Structural Basis for Autoinhibition by the Dephosphorylated Regulatory Domain of Ycf1

Khandelwal

Tomasiak

2023

Preprint

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Yeast Cadmium Factor 1 (Ycf1) sequesters glutathione and glutathione-heavy metal con-jugates into yeast vacuoles as a cellular detoxification mechanism. Ycf1 belongs to the C sub-family of ATP Binding Cassette (ABC) transporters characterized by long flexible linkers, notably the regulatory domain (R-domain). R-domain phosphorylation is necessary for activity, whereas dephosphorylation induces autoinhibition through an undefined mechanism. Because of its tran-sient and dynamic nature, no structure of the dephosphorylated Ycf1 exists, limiting understand-ing of this R-domain regulation. Here, we capture the dephosphorylated Ycf1 using cryo-EM and show that the unphosphorylated R-domain indeed forms an ordered structure with an unex-pected helix-strand hairpin topology bound within the Ycf1 substrate cavity. This architecture and binding mode resemble that of a viral peptide inhibitor of an ABC transporter and the secreted bacterial WXG peptide toxins. We further reveal the subset of phosphorylation sites within the hairpin turn that drive the reorganization of the R-domain conformation, suggesting a mechanism for Ycf1 activation by phosphorylation-dependent release of R-domain mediated autoinhibition.

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Section: Dephosphorylation Leads To R-domain Occlusion Of the Substra...mentioning

confidence: 99%

Structural Basis for Autoinhibition by the Dephosphorylated Regulatory Domain of Ycf1

Khandelwal

Tomasiak

2023

Preprint

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“…Finally, the interactions in the substrate cavity accompany dephosphorylated R-domain residue Asp920 contacts with a critical allosteric junction residue R1058 of the “GRD” motif of ICL-1 (Fig. 3D ), which couples with the X-loop of NBD1 to communicate ATP binding 25 .…”

Section: Resultsmentioning

confidence: 99%

“…In this state, phosphorylation of several adjacent residues along the R-domain helix, specifically S903, S908, and T911, is likely the master switch that controls the transition of the R-domain from an autoinhibitor to activator conformation. In this context, a few mechanisms by which autoinhibition in Ycf1 occurs are likely: 1) the R-domain occludes substrate binding directly to prevent transport, 2) the R-domain physically blocks the assembly of the TMDs and, consequently, NBD dimerization forming the power stroke of the ABC transporter conformational switch, 3) the R-domain blocks engagement of the GRD motif, a key motif required for allosteric coupling between NBDs and TMDs 25 , and lastly 4) R-domain sequesters away from the allosteric activation site on NBD1, which our previous phosphorylated Ycf1 structure 5 showed requires R-domain docking for Ycf1 stimulation. Scenarios 1-3 suggest a direct inhibition of function by the dephosphorylated R-domain and suggest phosphorylation plays a permissive role in transport, whereas scenario 4 represents the loss of a stimulatory function upon losing the R-domain phosphosites when dephosphorylated and suggests a stimulatory role.…”

Section: Discussionmentioning

confidence: 99%

Structural basis for autoinhibition by the dephosphorylated regulatory domain of Ycf1

Khandelwal,

Tomasiak

2024

Nat Commun

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Yeast Cadmium Factor 1 (Ycf1) sequesters glutathione and glutathione-heavy metal conjugates into yeast vacuoles as a cellular detoxification mechanism. Ycf1 belongs to the C subfamily of ATP Binding Cassette (ABC) transporters characterized by long flexible linkers, notably the regulatory domain (R-domain). R-domain phosphorylation is necessary for activity, whereas dephosphorylation induces autoinhibition through an undefined mechanism. Because of its transient and dynamic nature, no structure of the dephosphorylated Ycf1 exists, limiting understanding of this R-domain regulation. Here, we capture the dephosphorylated Ycf1 using cryo-EM and show that the unphosphorylated R-domain indeed forms an ordered structure with an unexpected hairpin topology bound within the Ycf1 substrate cavity. This architecture and binding mode resemble that of a viral peptide inhibitor of an ABC transporter and the secreted bacterial WXG peptide toxins. We further reveal the subset of phosphorylation sites within the hairpin turn that drive the reorganization of the R-domain conformation, suggesting a mechanism for Ycf1 activation by phosphorylation-dependent release of R-domain mediated autoinhibition.

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“…S3C) which was insufficiently detailed to enable model building. Concurrently, several coupling elements that connect the NBDs and TMDs, including the NBD1 X-loop and GRD motif 32 , are brought into contact upon transition to IFnarrow (Fig. 3E).…”

Section: The Two Structures Of Ycf1 Show Different Nbd Orientation With a Single-occluded Atp Site In Ifnarrowmentioning

confidence: 99%

The structural basis for regulation of the glutathione transporter Ycf1 by regulatory domain phosphorylation

et al. 2021

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Yeast Cadmium Factor-1 (Ycf1) sequesters heavy metals and glutathione into the vacuole to counter cell stress. Ycf1 belongs to the ATP binding cassette C-subfamily (ABCC) of transporters, many of which are regulated by phosphorylation on intrinsically disordered domains. The regulatory mechanism of phosphorylation is still poorly understood. Here, we report two cryo-EM structures of Ycf1 at 3.4Å and 4.0Å in distinct inward-facing open conformations capturing a previously unobserved ordered state of the intrinsically disordered regulatory domain (R-domain). R-domain phosphorylation is clearly evident and induces a topology promoting electrostatic and hydrophobic interactions with Nucleotide Binding Domain 1 (NBD1) and the lasso domain. These interactions stay constant between the structures and are related by rigid body movements of the NBD1/R-domain complex. Biochemical data further show R-domain phosphorylation reorganizes the Ycf1 architecture and is required for maximal ATPase activity. Together, we provide long-sought after insights into how R-domains control ABCC transporter activity.

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A Conserved Motif in Intracellular Loop 1 Stabilizes the Outward-Facing Conformation of TmrAB

Cited by 7 publications

References 68 publications

Structural Basis for Autoinhibition by the Dephosphorylated Regulatory Domain of Ycf1

Structural Basis for Autoinhibition by the Dephosphorylated Regulatory Domain of Ycf1

Structural basis for autoinhibition by the dephosphorylated regulatory domain of Ycf1

The structural basis for regulation of the glutathione transporter Ycf1 by regulatory domain phosphorylation

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