Overlay of cryo EM maps shows αV24Y T20S has similar conformational changes as ZYA bound T20S and is in the gate-open state

A Overlay of wild-type (WT) T20S (yellow) and αV24Y T20S (red) electron density maps, showing the α and β rings. Whole maps were aligned to one another.

B Overlay of αV24Y T20S (red) and ZYA-T20S (blue) electron density maps, showing the α and β rings. V24Y T20S shows similar but larger conformational changes than does ZYA-T20S.

C Close up of the intersubunit pocket in the WT T20S (yellow) electron density map, highlighted in A in the larger neon green box outline.

D, E Same as C, but for αV24Y T20S (red) and ZYA-T20S (blue). Arrows point to the density corresponding to αV24Y and Z of ZYA, respectively.

F Close up of the WT T20S (yellow) electron density map, corresponding to the YDR motif, the missing density for tyrosine is highlighted by the neon green dotted circle, which is expected in the closed state. The relative position of the close-up is indicated by the smaller neon blue box outline in A.

G, H Same as F, but for αV24Y T20S (red) and ZYA-T20S (blue). Neon green dotted circle highlight the YDR tyrosine side chain density, which is expected to be in this position in the open gate conformation.

I Close up of the WT T20S (yellow) electron density map, corresponding to the IT Switch, highlighted by the neon green dotted circle.

J, K Same as I but for αV24Y T20S (red) and ZYA-T20S (blue). Changes in the conformation of the IT switch indicates switching from the closed to the open state.

Atomic model of αV24Y T20S compared to WT and ZYA-T20S shows it has undergone HbYX-dependent conformational changes associated with 20S gate-opening

A Atomic model overlay of WT T20S (yellow), αV24Y T20S (red), and ZYA-T20S (blue) with bound ZYA (gold), aligned by the β rings to show conformational changes in the α subunits. Top half of sideview of 20S is shown. Conformational changes in the α-ring are clearly visible (black arrows). Helix 4, 5, and 6 are labeled with black asterisks outlined in white.

B Top view of A. Conformational changes noted by (black arrows).

C Closeup view of one of the α intersubunit pockets as in B. αPro17 is shown with a black arrow. Helix 0 is labeled with a black asterisk outlined in white.

D Closeup view of αK66 which is labeled and shown in sticks.

Models in panel b through d are aligned to show intersubunit conformational changes (i.e., aligned by β rings)

αV24Y mutation induces intrasubunit conformational changes in the HbYX hydropbobic pocket, IT switch pocket, and P17 loop

A Overlay of WT T20S (yellow) and αV24Y T20S (red) α subunit models, aligned by the β rings.

B Same as A except the models were aligned according to individual α subunit.

C Close up of the intersubunit pocket in αV24Y T20S (red), showing distances between the α-carbon selected residues (labeled and shown in sticks) to demonstrate conformational changes when compared to the WT T20S (yellow) model in D. Models are aligned by individual α subunit.

D Same as C except it is the WT T20S (yellow) model.

E Close up on the residues of the IT Switch (sticks) and αV24Y (sticks), showing the distance between the α-carbon of αV24Y and αA154 (sticks) to demonstrate conformational changes in the open state, when compared to the WT T20S (yellow) model in F. Models are aligned by individual α subunit.

F Same as E except it is the WT T20S (yellow) model.

G Close up of Helix 0 and selected surrounding residues (shown in sticks) in an overlay of WT T20S (yellow) and αV24Y T20S (red) α subunits, aligned by individual α subunit. Distances are measured between α-carbons and colored according to the model it corresponds to.

H Close up of αV24Y (shown in sticks) in the αV24Y T20S model, demonstrating its side chain’s polar interaction (yellow dotted line) with αD152 (shown in sticks).

αV24Y mutation and ZYA binding both induce similar intrasubunit conformational changes

A Same as 3A except for αV24Y T20S (red) and ZYA-T20S (blue). Models are aligned by the β ring.

B Same as 3B except for αV24Y T20S (red) and ZYA-T20S (blue). Models are aligned by individual α subunit.

C Same as B except showing a closeup of the Helix 4, 5, and 6 bundle.

D αV24Y T20S and ZYA-T20S are aligned by β subunits and αV24Y side chain and carboxybenzyl group (Z) of ZYA are both shown in space-fill model to appreciate the level of spatial overlap between the αV24Y tyrosine ring (red) and the Z ring (gold) of ZYA, demonstrating they each occupy the same HbYX hydrophobic pocket in the T20S.

E Close up of the αV24Y T20S (red) α intersubunit pocket showing measurements between the αV24Y tyrosine and labeled α carbons (shown in sticks). Comparison against F indirectly demonstrates the position of αV24Y’s side chain relative to the carboxybenzyl group of ZYA in the ZYA-T20S model.

F Measurements of distances between ZYA’s carboxybenzyl group (gold) and the α-carbon of surrounding residues (shown in sticks) in the ZYA-T20S model (blue).

G The HbYX hydrophobic pocket measurement: αT124 to αL21 (main chain shown in sticks) distance in WT T20S (yellow), αV24Y T20S (red), and ZYA-T20S (blue).

H Same as 3E & F, except with alignment of ZYA-T20S (blue) and αV24Y T20S (red) α subunit models. Distances noted are colored according to the models they correspond to representing intrasubunit differences.

αK66 plays a key role in stabilizing the open state of the T20S, even in the absence of proteasome activator binding

A Overlay of WT T20S (yellow) and αV24Y T20S (red), aligned by individual α subunits, showing a close-up of the αK66 (shown in sticks) back loop. Distance measured demonstrates the extent of intrasubunit conformational changes in the “back loop” and K66 α carbon.

B Close up of αV24Y T20S (red) model, showing the polar interactions (yellow dotted lines) between αK66 and surrounding residues (shown in sticks). Distances are measured between the exact atoms involved in the polar interactions.

C Same as B except it is the WT T20S (yellow) model.

D Same as B except it is the ZYA-T20S (blue) model and ZYA (gold).

E WT T20S and mutants were measured for activity based on LFP degradation rate (rfu/min), normalized to the WT T20S as a control. Data (means) are representative of three or more independent experiments, each performed in triplicate. Error bars represent ± standard deviation.

The interunit pocket of the αV24Y T20S is less populated with waters, relative to ZYA T20S and WT T20S

A Intersubunit pocket of WT T20S model (yellow) showing waters (spheres) and their interactions (yellow dotted lines) with the residues (shown in sticks, listed in table) in the intersubunit pocket. Spheres are colored individually and match the colors on the table (right) to identify residues it interacts with.

B, C Same as A except αV24Y T20S model (red) and ZYA-T20S model (blue).

A large portion of the N-terminal domain of the αV24F T20S mutant is unstructured

A Angled view of αV24F T20S 2.2Å electron density map (green), showing the α and β rings.

B Overlay of αV24F T20S map with αV24Y T20S map (red; transparent surface; left) and WT T20S map (yellow; transparent surface; right).

C Same as B except αV24Y T20S model (red, cartoon, left) and WT T20S model (yellow; cartoon; right).

E Difference map generated by subtracting αV24F T20S map from αV24Y T20S map, using ChimeraX.

F Same as E except WT T20S map instead of αV24Y T20S map.

Model for induction of gate opening by binding of the Hb group binding to the HbYX hydrophobic site in the 20S intersubunit pockets

Binding of the Hb group of the HbYX motif, or tyrosine of αV24Y, is expected to initiate an allosteric chain of events that leads to 20S gate opening. Hb group binding causes:

1) Increase the size of the Hb pocket

2) Affects IT switch Hb pocket promoting IT switching to open state

3) IT switching lengthens P17 loop (increases loop slack)

4) E25 H-bond rearrangement further lengthens P17 loop

5) Combine effects on P17 loop slack promotes rigid body rotations against the neighboring Helix 0.

6) Rigid body rotations effect back loop (not shown), which repositions K66 to further stabilize open state.

αV24Y T20S Processing Scheme

A Cryo-EM workflow for alphaV24Y T20S. All steps were performed in Cryosparc.

B Final 2D class averages after 2 rounds of 2D Classification shown with a mixture of top and size views.

C Homogenous refinement of class of particles that showed the open gate conformation.

αV24Y T20S Validation

A Micrograph representative showing αV24Y T20S. Scale bar represents 5nm.

B. Standard FSC-0.143 graph showing corrected 2.38Å resolution.

C View of particle angle distribution showing a mixture of side and top view particles.

D Particles from selected ab-initio model were refined with homogenous refinement and D7 symmetry applied. D7 symmetry was confirmed as appropriate by homogenous refinement with C1 symmetry; the resulting C1 map was copied then flipped 180° around the 2-fold axis then rotated 180° about the 7-fold axis. The flipped and rotated map was essentially identical to the original, confirming D7 symmetry of the complex. Final 2.38Å D7 map was colored in Chimera to show range of resolution of 3D reconstructed map.

αV24Y T20S differ from WT T20S

A Close up of αV24Y T20S sharpened map and model, demonstrating quality of map and model fit in the shown region.

B. Overlay of CCP-EM generated confidence maps for αV24Y T20S (yellow) and WT T20S (cyan), indicating statistically significant conformational changes. Maps were set at a threshold of 0.01 FDR. View is similar to Figure 4H showing clear conformational changes in A154 relative to helix 0. Arrows point out the distance differences between the same two residues in the WT or the V24Y maps. Gaussian smoothing was applied to both maps.

Additional measurements in the intersubunit pocket in V24Y T20S

Complementary to Figure 3. A Measurement between P17 and neighboring Helix 0 for β ring-aligned WT and αV24Y T20S structures representing absence of intersubunit conformational differences in this distance.

B Measurement between P17 and its own Helix 0 (E25) show intrasubunit changes to this distance.

C Close up of intrasubunit conformational change in the IT switch and αP17 loop providing flexibility to allow for intersubunit rigid body rotation. Distance shown are intrasubunit conformational change in the αV24Y T20S model and do not represent the global conformational changes, as the P17 packing against the neighboring Helix 0 is similar in open and closed states (SFig. 3a)

V24Y mutation and ZYA binding both alter E25’s H-bonding partner

E25 hydrogen bonding is affected by hydrophobic group occupancy in the HbYX hydrophobic pocket as it is positioned at the top of this pocket.

A E25 is H-bonded to R28 in the WT T20S.

B Distance between E25 and R20 in the WT T20S.

C E25 H-bonds with R20 in the V24Y T20S.

D Distance between E25 and R20 in the V24Y T20S

E E25 H-bonds with R20 in ZYA-T20S structure.

F Distance between E25 and R20 in the ZYA-T20S.

Comparison of HbYX hydrophobic pocket in V24Y T20S and ZYA-T20S structures

A Distances between the α-carbon of selected residues (shown in sticks) to be compared against B to demonstrate the similarities and minimal level of intrasbunit conformational changes between these structures. B Same as A except the ZYA-T20S model (blue) was used.

αV24F T20S Processing Scheme

A Cryo-EM workflow for αV24F T20S. All steps were performed in Cryosparc.

B Final 2D class averages after 2 rounds of 2D Classification shown with a mixture of top and size views.

C Homogenous refinement of class of particles that showed the open gate conformation.

αV24Y T20S Validation

A Micrograph representative showing αV24F T20S. Scale bar represents 5nm.

B Standard FSC-0.143 graph showing corrected 2.20Å resolution.

C View of particle angle distribution showing a mixture of side and top view particles.

D Particles from selected ab-initio model were refined with homogenous refinement and D7 symmetry applied (D7 symmetry was appropriately confirmed as discussed in SFig. 2d). Final 2.20Å D7 map was colored in Chimera to show range of resolution of 3D reconstructed map.

αV24Y T20S Validation

A Alignment showing sequences in the first 30 positions of the α subunits of T20S and H20S (listed according to gene names). Alignment was performed using ClustalΩ. Conversation and figure was generated using Jalview 2.11.2.5. Alignments are colored using the Clustal color scheme and color intensity indicates conservation (darkest: highly conserved). The bar graph in yellow indicates the conservation score, accounting for size, charge, hydrophobicity, and polarity. Red arrow points to αL21 and the corresponding residues in the alignment.

B Same as A except the alignment shows sequences in the positions 145 to 176 of the α subunits of T20S and H20S (listed according to gene names). The red arrow points to αA154 and the corresponding residues in the alignment.