Structure of a Blm10 complex reveals common mechanisms for proteasome binding and gate opening - PubMed (original) (raw)

Structure of a Blm10 complex reveals common mechanisms for proteasome binding and gate opening

Kianoush Sadre-Bazzaz et al. Mol Cell. 2010.

Abstract

The proteasome is an abundant protease that is critically important for numerous cellular pathways. Proteasomes are activated in vitro by three known classes of proteins/complexes, including Blm10/PA200. Here, we report a 3.4 A resolution crystal structure of a proteasome-Blm10 complex, which reveals that Blm10 surrounds the proteasome entry pore in the 1.2 MDa complex to form a largely closed dome that is expected to restrict access of potential substrates. This architecture and the observation that Blm10 induces a disordered proteasome gate structure challenge the assumption that Blm10 functions as an activator of proteolysis in vivo. The Blm10 C terminus binds in the same manner as seen for 11S activators and inferred for 19S/PAN activators and indicates a unified model for gate opening. We also demonstrate that Blm10 acts to maintain mitochondrial function. Consistent with the structural data, the C-terminal residues of Blm10 are needed for this activity.

(c) 2010 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. Structure of the Blm10:proteasome complex

(A) Cartoon of the proteasome-Blm10 complex, side view. Proteasome white (α subunits) and gray (β subunits), Blm10 rainbow from N-terminus (blue) to C-terminus (red). (B) Cutaway view with the molecular surface. (C) Electron density for Blm10. All maps shown in this paper were phased on the proteasome molecular replacement model for which segments approaching Blm10 had been removed. The phases were refined by solvent flattening, histogram shifting, and four-fold non-crystallographic symmetry averaging. (D) Top view, space-filling representation. The opening visible in the center of this view measures only ~6Å between atom centers. The largest opening, which is not visible in this orientation, is indicated with an arrow. (E) Same as panel D, but only showing Blm10 residues that have at least one atom within 6 Å of the proteasome. (F) Close-up view of the largest opening through the Blm10 dome. Orientation as indicated by the arrow in panel D. Asterisks denote the last ordered residues adjacent to disordered segments that have been omitted from the model.

Figure 2

Figure 2. Proteasome conformational changes induced by Blm10

(A) Top view of the proteasome pore region with electron density for the Blm10 complex. The absence of density for the N-terminal residues of proteasome α2, α3, and α4 indicates that they are disordered in the Blm10 complex (white), whereas they are ordered in the closed, unliganded conformation (colors) and in the fully open complex with PA26 (not shown in this panel). (B) Open conformation seen in complexes with PA26 (yellow) and Blm10 (white). The stabilizing cluster residues (Tyr8, Asp9, Pro17, Tyr26; (Forster et al., 2003)) are labeled for the α6/α7 cluster, which is ordered in the unliganded proteasome (Groll et al., 1997) and in both the PA26 and Blm10 complexes shown here. Tyr8 and Asp9 residues are not ordered for α2, α3, or α4 in the Blm10 complex. Residues indicated with an asterisk are ordered in the Blm10 complex but are displaced from the open conformation seen with PA26. A version of this panel that also includes the closed conformation is shown in Figure S2. (C) Contacts that stabilize α5Asp9 away from the open conformation. (D) Contacts that stabilize α7Tyr8 away from the open conformation.

Figure 3

Figure 3. Interactions of the Blm10 C-terminal residues

(A) Side view with Blm10 C-terminus labeled “C”. (B) The electron density map is well defined for the Blm10 penultimate tyrosine (Tyr2142) and surrounding residues. (C) The last three residues of PA26 (green) and Blm10 (red) are shown after overlap of the two complexes on surrounding proteasome residues. Unliganded proteasome (Groll et al., 1997), cyan. Blm10 Tyr2142 stabilizes the open position of α5 by hydrogen bonding with Gly19 O. PA26 stabilizes the same transition by hydrogen bonding interactions of its activation loop residue Glu102.

Figure 4

Figure 4. The C-terminal residues of Blm10 are important for its function

(A) Isogenic strains were constructed in the A364a genetic background with the genotypes indicated (Table S1). Multiple independent colonies of each strain growing on glycerol medium to select for retention of mitochondrial function were used to inoculate rich medium containing glucose. Saturated cultures were diluted and plated on rich glucose medium, then mitochondrial function in clones was assessed using the tetrazolium staining method (Ogur et al., 1957). Results from multiple strains with the same genotype were combined (Table S1, total number indicated as N), with the average percentage yield of petite colonies plotted here. Error bars indicate the standard deviation of the measurements. (B) As in panel A, except isogenic pairs from three other commonly used genetic backgrounds containing or lacking Blm10 were assayed by picking 120 colonies without regard to size, then replica plating to media containing glycerol or glucose to determine the number of petite colonies (Table S1). See also Figures S3.

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