Cryo-EM reveals the conformation of a substrate analogue in the human 20S proteasome core

Abstract

The proteasome is a highly regulated protease complex fundamental for cell homeostasis and controlled cell cycle progression. It functions by removing a wide range of specifically tagged proteins, including key cellular regulators. Here we present the structure of the human 20S proteasome core bound to a substrate analogue inhibitor molecule, determined by electron cryo-microscopy (cryo-EM) and single-particle analysis at a resolution of around 3.5 Å. Our map allows the building of protein coordinates as well as defining the location and conformation of the inhibitor at the different active sites. These results open new prospects to tackle the proteasome functional mechanisms. Moreover, they also further demonstrate that cryo-EM is emerging as a realistic approach for general structural studies of protein-ligand interactions.

Figure 1. Cryo-EM map of the human 20S–AdaAhx₃L₃VS complex.

(a,b) Individual sections 1 Å thick of the 3D map, as determined by the 3D reconstruction algorithm, are represented as grey-scale. Regions showing the pattern of α-helical secondary structure (a) and the separation of sheet forming β strands (b) are boxed. (c) Surface representation and (d) sections of the map surface rendered and colour coded according to local resolution as determined using ResMap.

Figure 2. Mesh representation of the map of the human 20S–AdaAhx₃L₃VS complex, Fourier low-pass filtered to 3.4 Å, with built protein coordinates.

(a) Section of the map showing the global agreement between the map densities and the coordinates of the 20S proteasome complex. Close-up representations of an α-helix (b) and a β strand (c) are shown, illustrating substantial recovery of side-chain information.

Figure 3. The ligand-binding pockets at the three 20S proteasome core active sites.

(a) Structural formula of the inhibitor AdaAhx₃L₃VS. The map of the human 20S–AdaAhx₃L₃VS complex, Fourier low-pass filtered to 3.4 Å, is shown as a mesh (b–e). Clear densities are seen extending from the N-terminal Thr residues of the β5 (b,c), β2 (d) and β1 (e) subunits that are consistent with the -L₃VS moiety of the AdaAhx₃L₃VS molecule, with the vinyl sulfone group and the side chains of the three leucine residues shown clearly resolved at the β5 site (c).

Figure 4. Conformations of the -L₃VS moiety of AdaAhx₃L₃VS and ZL₃VS bound to the 20S proteasome core.

(a) Structural formulae of AdaAhx₃L₃VS and ZL₃VS with their -L₃VS moiety boxed. (b) The -L₃VS moiety of the AdaAhx₃L₃VS molecule (in magenta) at the human 20S proteasome core β5 subunit active site is shown with superimposed coordinates for ZL₃VS (grey), as found in a yeast 20S proteasome core crystallographic structure.