Abstract
Three receptors (Rpn1/S2/PSMD2, Rpn10/S5a, Rpn13/Adrm1) in the proteasome bind substrates by interacting with conjugated ubiquitin chains and/or shuttle factors (Rad23/HR23, Dsk2/PLIC/ubiquilin, Ddi1) that carry ubiquitinated substrates to proteasomes. We solved the structure of two such receptors with their preferred shuttle factor, namely hRpn13(Pru):hPLIC2(UBL) and scRpn1 T1:scRad23(UBL). We find that ubiquitin folds in Rad23 and Dsk2 are fine-tuned by residue substitutions to achieve high affinity for Rpn1 and Rpn13, respectively. A single substitution in hPLIC2 yields enhanced interactions with the Rpn13 ubiquitin contact surface and sterically blocks hRpn13 binding to its preferred ubiquitin chain type, K48-linked chains. Rpn1 T1 binds two ubiquitins in tandem and we find that Rad23 binds exclusively to the higher-affinity Helix28/Helix30 site. Rad23 contacts at Helix28/Helix30 are optimized compared to ubiquitin by multiple conservative amino acid substitutions. Thus, shuttle factors deliver substrates to proteasomes through fine-tuned ubiquitin-like surfaces.
Keywords:
Dsk2; Rad23; Rpn1; Rpn13; proteasome; ubiquitin.
Published by Elsevier Ltd.
MeSH terms
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Adaptor Proteins, Signal Transducing
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Amino Acid Sequence
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Amino Acid Substitution
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Autophagy-Related Proteins
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Binding Sites
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Cell Cycle Proteins / chemistry*
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism
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Crystallography, X-Ray
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DNA-Binding Proteins / chemistry*
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / metabolism
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Gene Expression
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Humans
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Intracellular Signaling Peptides and Proteins
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Membrane Glycoproteins / chemistry*
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Membrane Glycoproteins / genetics
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Membrane Glycoproteins / metabolism
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Molecular Docking Simulation
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Molecular Dynamics Simulation
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Mutation
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Proteasome Endopeptidase Complex / chemistry*
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Proteasome Endopeptidase Complex / genetics
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Proteasome Endopeptidase Complex / metabolism
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Protein Binding
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Protein Conformation, alpha-Helical
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Protein Conformation, beta-Strand
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Protein Interaction Domains and Motifs
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Saccharomyces cerevisiae / chemistry*
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins / chemistry*
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / metabolism
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Sequence Alignment
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Sequence Homology, Amino Acid
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Substrate Specificity
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Thermodynamics
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Ubiquitins / chemistry*
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Ubiquitins / genetics
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Ubiquitins / metabolism
Substances
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ADRM1 protein, human
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Adaptor Proteins, Signal Transducing
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Autophagy-Related Proteins
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Cell Cycle Proteins
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DNA-Binding Proteins
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Intracellular Signaling Peptides and Proteins
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Membrane Glycoproteins
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RAD23 protein, S cerevisiae
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RPN1 protein, S cerevisiae
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Recombinant Proteins
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Saccharomyces cerevisiae Proteins
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UBQLN2 protein, human
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Ubiquitins
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Proteasome Endopeptidase Complex