Biochemical and structural characterization of a complex involved in chaperone mediated proteasomal degradation

Abstract

Eukaryotic cells have evolved numerous strategies to maintain proteostasis, the delicate balance of synthesis, folding, trafficking and degradation of intra-and extracellular proteins. Molecular chaperones are at the heart of proteostasis as they play essential roles in both protein folding and degradation pathways. The change in the role of the chaperone is dictated by the interaction with different cochaperones which can either favor the folding of a substrate or its degradation through two established and specific protein degradation mechanisms, the ubiquitin-proteasome system (UPS) and the autophagy system. One of those cochaperones is Bag1 (Bcl-2-associated athanogene 1), a nucleotide exchange factor of the chaperone Hsp70 that contains a ubiquitin like (UBL) domain shown to mediate the interaction with the proteasome. In this work, we aim to characterize in detail the interaction between Hsp70 and the proteasome mediated by Bag1. For that, the ability of Bag1 to interact with different proteasomal subunits has been tested in vitro. It was found that Bag1 specifically interacts with the subunit Rpn1, and that Bag1 can simultaneously bind both Rpn1 and Hsp70. Binary (Rpn1:Bag1) and ternary (Rpn1:Bag1:Hsp70) complexes have been isolated and characterized by a combination of biochemical and biophysical techniques. Using isothermal titration calorimetry (ITC) experiments, it has been calculated the affinity of Bag1 for Rpn1 (KD = 500 nM) and for Hsp70 (KD = 50 nM), observing that both interactions are independent. Additionally, a stable quaternary complex formed by Rpn1, Bag1, Hsp70 and a model substrate of Hsp70 has been isolated. This evidence suggests the existence of a substrate transference mechanism from the chaperone to the proteasome facilitated by the interaction of Bag1 and Rpn1 subunit. To gain further insight into the interactions among these proteins, the purified complexes have been structurally characterized using cryogenic electron microscopy (cryoEM) and three-dimensional processing. The obtained maps, complemented with cross-linking and mass spectrometry information, have allowed to propose a model in which the UBL of Bag1 is contacting the Rpn1 and its Bag domain (BD) is attached to the nucleotide binding domain (NBD) of Hsp70. The intrinsic flexibility of these complexes, necessary to accommodate different substrates, has prevented obtaining high resolution maps. Finally, a high-resolution map of the complex between the proteasome and Bag1 has been obtained. This map shows the binding of the UBL domain of Bag1 to the toroidal region of Rpn1 in the so called T2 site. This interaction would allow the positioning of Hsp70 in the proximity of the proteasomal gate, favoring the transference of the substrate