Abstract
Hsp104 is a hexameric AAA+ yeast disaggregase capable of solubilizing disordered aggregates and amyloid. Hsp104 couples ATP hydrolysis to polypeptide translocation through its central channel. Substrate binding by Hsp104 is mediated primarily by two conserved tyrosine residues in nucleotide binding domain (NBD) 1 and NBD2. Recent structural studies have revealed that an additional tyrosine residue (Y650) located in NBD2 appears to contact substrate and may play an important role in Hsp104 function. Here, we functionally analyze the properties of this proposed Hsp104 –substrate interaction. We find that Y650 is not essential for Hsp104 to confer thermotolerance. Supporting these findings, in a potentiated Hsp104 variant background, the Y650A mutation does not abolish potentiation. However, modulation of this site does have subtle effects on the activity of this potentiated Hsp104 variant. We therefore suggest that while Y650 is not essential for Hsp104 function, its modulation may be useful for fine-tuning Hsp104 properties.
Highlights
Protein disaggregases engage and dissolve proteins that aggregate and form amyloid
A loop region centered around Y650 projects into the central channel of Hsp104
While extensive studies have supported the functional role of Y257 and Y662, prior to our work, there has been no demonstration of the functional role of this third putative pore-loop of Hsp104
Summary
Protein aggregation is implicated in a range of diseases, and so it is important to understand the structure and mechanism of disaggregases. Hsp104 assembles into a ring structure with a central channel through which substrate is threaded[1,2,3]. ATP hydrolysis powers a ratchet-like mechanism that advances substrate through the central channel of Hsp104[4, 5]. Hsp104 confers stress resilience by solubilizing diverse substrates thereby allowing their refolding to native structure and function[6,7,8]. Yeast harbor beneficial prions that are thought to promote adaptation, and Hsp104 plays an important role in remodeling these species[9]
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