Proteomics

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An inter-subunit protein-protein interface that stabilizes the specific activity and oligomerization of the AAA+ chaperone Reptin


ABSTRACT: Reptin forms multiple inter-subunit protein-protein interaction contacts. Rate-limiting motifs in ligand-dependent oligomer assembly are not defined. We set up a Reptin-self peptide scan assay to identify functional protein-protein interfaces that dominate in Reptin self assembly. The most dominant self-peptide binding mapped to the inter-subunit “rim” of the Reptin oligomer that is formed a tyrosine finger binding into an adjacent hydrophobic region in an adjacent subunit. Reptin binding to the tyrosine finger peptide motif is suppressed by Liddean or ADP suggesting that oligomer formation excludes the self-peptide from binding. HDX-mass spectrometry demonstrated that ATP suppressed deuteration at the dimer interface in both wt and Y340A Reptin. However, the Y340A mutation attenuated deuterium suppression of Reptin within the tyrosine finger in the presence of ligand. The tyrosine finger of Reptin interacts with a more shallow pocket in Pontin. Gel filtration demonstrated that the Y340A Reptin mutant does not form ADP-induced oligomers compared to the D299N mutant or wt- Reptin. The Y340A mutation shows increased AGR2 binding but decreased Pontin or p53 binding. These data indicate that the Y340 tyrosine “finger” plays an important role in stabilizing the Reptin oligomer in the presence of ligand. We propose that the Reptin interactome is regulated by ligand-dependent conversion between monomeric and oligomeric forms that is regulated by a divergent inter-subunit protein-protein interaction motif.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Dominika Coufalová  

LAB HEAD: Ted R. Hupp

PROVIDER: PXD008226 | Pride | 2019-04-23

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
SP061_ReptinY340A_ND.raw Raw
SP061_Reptin_5min.raw Raw
SP061_Reptin_ATP_5min.raw Raw
SP061_Reptin_MAP.msf Msf
SP061_Reptin_MAP.raw Raw
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Publications

An inter-subunit protein-peptide interface that stabilizes the specific activity and oligomerization of the AAA+ chaperone Reptin.

Coufalova Dominika D   Remnant Lucy L   Hernychova Lenka L   Muller Petr P   Healy Alan A   Kannan Srinivasaraghavan S   Westwood Nicholas N   Verma Chandra S CS   Vojtesek Borek B   Hupp Ted R TR   Houston Douglas R DR  

Journal of proteomics 20190309


Reptin is a member of the AAA+ superfamily whose members can exist in equilibrium between monomeric apo forms and ligand bound hexamers. Inter-subunit protein-protein interfaces that stabilize Reptin in its oligomeric state are not well-defined. A self-peptide binding assay identified a protein-peptide interface mapping to an inter-subunit "rim" of the hexamer bridged by Tyrosine-340. A Y340A mutation reduced ADP-dependent oligomer formation using a gel filtration assay, suggesting that Y340 for  ...[more]

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