The catalytic sites of 20S proteasomes and their role in subunit maturation: a mutational and crystallographic study

Abstract

We present a biochemical and crystallographic characterization of active site mutants of the yeast 20S proteasome with the aim to characterize substrate cleavage specificity, subunit intermediate processing, and maturation. beta1(Pre3), beta2(Pup1), and beta5(Pre2) are responsible for the postacidic, tryptic, and chymotryptic activity, respectively. The maturation of active subunits is independent of the presence of other active subunits and occurs by intrasubunit autolysis. The propeptides of beta6(Pre7) and beta7(Pre4) are intermediately processed to their final forms by beta2(Pup1) in the wild-type enzyme and by beta5(Pre2) and beta1(Pre3) in the beta2(Pup1) inactive mutants. A role of the propeptide of beta1(Pre3) is to prevent acetylation and thereby inactivation. A gallery of proteasome mutants that contain active site residues in the context of the inactive subunits beta3(Pup3), beta6(Pre7), and beta7(Pre4) show that the presence of Gly-1, Thr1, Asp17, Lys33, Ser129, Asp166, and Ser169 is not sufficient to generate activity.

Figure 1

(a) Topology of the yeast 20S proteasome. The active site threonine 1 residues are located at the inner wall of the cylindrical particle. (b) Scheme of the β-rings with given distances between the active site threonines.

Figure 2

Stereodiagram of the superposition of β1 (green) and N-acetyl-β1 (yellow) around the Thr1 site. The structures match closely.

Figure 3

(a) Stereodiagram of the β1T1A β5K33R double mutant in the vicinity of residue Thr1 in β5. The electron density is calculated with phases from the wild-type β5 model. (b) Stereodiagram of wild-type (green) and β5K33R (white) mutant around Thr1. They superimpose closely except for the site of mutation. β5K33R autolyses and has a free Thr1. (c) Comparison of the wild-type (green) and β5K33A (white) mutant. Loss of the Lys33 side chain leads to a large movement of the backbone of Thr1. The mutant is unable to autolyse and has the propeptide attached.

Figure 4

A gallery of superposition of main chain traces around Thr 1. a and b show the three active subunits β1, β2 and β5. In c and d, β1 is compared with wild-type β3 and β3G1T, respectively, in e and f, β1 is superimposed with wild-type β6 and the 5-fold β6 mutant (β6*), and, in g and h, β1 is compared with β7 and β4.

Figure 5

Stereo diagram of the 5-fold β6 mutant in the vicinity of residue 1. The electron density is calculated with phases from the wild-type β6 model (black bonds). The mutations A129S, G1T, and the rearrangement of K33 are clearly visible (red bonds).