Occupancy of the HbYX hydrophobic pocket is sufficient to induce gate opening in the archaeal 20S proteasomes

Reviewer #1 (Public review):

Summary:

In this manuscript, Chua, Daugherty, and Smith analyze a new set of archaeal 20S proteasomes obtained by cryo-EM that illustrate how the occupancy of the HbYX binding pocket induces gate opening. They do so primarily through a V24Y mutation in the α-subunit. These results are supported by a limited set of mutations in K66 in the α subunit, bringing new emphasis to this unit.

Strengths:

The new structure's analysis is comprehensive, occupying the entire manuscript. As such, the scope of this manuscript is very narrow, but the strength of the data is solid, and they offer an interesting and important new piece to the gate-opening literature.

Weaknesses:

Major Concerns

(1) This manuscript rests on one new cryo-EM structure, leading to a single (albeit convincing) experiment demonstrating the importance of occupying the pocket and moving K66. Could a corresponding bulky mutation at K66 not activate the 20S proteasome?

(2) To emphasize the importance of this work, the authors highlight the importance of gate-opening to human 20S proteasomes. However, the key distinctions between these proteasomes are not given sufficient weight.
(a) As the authors note, the six distinct Rpt C-termini can occupy seven different pickets. However, how these differences would impact activation is not thoroughly discussed.
(b) With those other sites, the relative importance of various pockets, such as the one controlling the α3 N-terminus, should be discussed more thoroughly as a potential critical difference.
(c) These differences can lead to eukaryote 20S gates shifting between closed and open and having a partially opened state. This becomes relevant if the goal is to lead to an activated 20S. It would have been interesting to have archaea 20S with a mix of WT and V24Y α-subunits. However, one might imagine the subclassification problem would be challenging and require an extraordinary number of particles.
(d) Furthermore, the conservation of the amino acids around the binding pocket was not addressed. This seems particularly important in the relative contribution of a residue analogous to K66 or V24.

Reviewer #2 (Public review):

Summary:

The manuscript by Chuah et al. reports the experimental results that suggest the occupancy of the HbYX pockets suffices for proteasome gate opening. The authors conducted cryo-EM reconstructions of two mutant archaeal proteasomes. The work is technically sound and may be of special interest in the field of structural biology of the proteasomes.

Strengths:

Overall, the work incrementally deepens our understanding of the proteasome activation and expands the structural foundation for therapeutic intervention of proteasome function. The evidence presented appears to be well aligned with the existing literature, which adds confidence in the presentation.

Weaknesses:

The paper may benefit from some minor revision by making improvements on the figures and necessary quantitative comparative studies.