The Y\u03a6 motif defines the structure-activity relationships of human 20S proteasome activators

Kwadwo A Opoku-Nsiah,Nikita Chopra,Jason E Gestwicki,Andres H De La Pena,Sarah K Williams,Andrej Sali,Gabriel C Lander

doi:10.1038/s41467-022-28864-x

Abstract

The 20S proteasome (20S) facilitates turnover of most eukaryotic proteins. Substrate entry into the 20S first requires opening of gating loops through binding of HbYX motifs that are present at the C-termini of certain proteasome activators (PAs). The HbYX motif has been predominantly characterized in the archaeal 20S, whereas little is known about the sequence preferences of the human 20S (h20S). Here, we synthesize and screen ~120 HbYX-like peptides, revealing unexpected differences from the archaeal system and defining the h20S recognition sequence as the Y-F/Y (YФ) motif. To gain further insight, we create a functional chimera of the optimized sequence, NLSYYT, fused to the model activator, PA26E102A. A cryo-EM structure of PA26E102A-h20S is used to identify key interactions, including non-canonical contacts and gate-opening mechanisms. Finally, we demonstrate that the YФ sequence preferences are tuned by valency, allowing multivalent PAs to sample greater sequence space. These results expand the model for termini-mediated gating and provide a template for the design of h20S activators.

Highlights

1,2, Nikita Chopra[1,4,5], The 20S proteasome (20S) facilitates turnover of most eukaryotic proteins
The canonical HbYX model is defined as having a hydrophobic residue at P3, a preference for Tyr at P2, and any residue at P1
Another key part of the HbYX motif is that it contains the terminal carboxylate at the P1 position, which forms a critical salt bridge with a conserved cationic side chain at the base of the αpocket; for example αLys[66] of the Thermoplasma acidophilum 20S (Fig. 1a)[9]

Summary

Introduction

1,2, Nikita Chopra[1,4,5], The 20S proteasome (20S) facilitates turnover of most eukaryotic proteins. Substrate entry into the 20S first requires opening of gating loops through binding of HbYX motifs that are present at the C-termini of certain proteasome activators (PAs). N-terminal extensions of the α-subunits gate entry into the pore, limiting the degradation of bystander proteins[2] This barrier creates a key regulatory role for the proteasome activators (PAs)[3–5], large particles that bind the 20S, open the gates, and facilitate substrate selection and entry. 20S proteasome (h20S) is a hetero-oligomer, containing seven distinct α-pockets for binding to the HbYX motif It is not clear whether the same structure-activity relationships (SAR) that govern the recognition of the HbYX motif in the archaeal system are conserved in the h20S. Beyond the important insights into the molecular mechanisms of h20S gate opening, such investigations might be expected to inform the design of small molecules that bind the αpockets[23]

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Results

Conclusion