Targeted protein degradation is largely performed by the ubiquitin-proteasome pathway, in which substrate proteins are marked by covalently attached ubiquitin chains that mediate recognition by the proteasome. It is currently unclear how the proteasome recognizes its substrates, as the only established ubiquitin receptor intrinsic to the proteasome is Rpn10/S5a 1 , which is not essential for ubiquitin-mediated protein degradation in budding yeast 2 . In the accompanying manuscript we report that Rpn13 3-7 , a component of the 9-subunit proteasome base, functions as a ubiquitin receptor 8 , complementing its known role in docking deubiquitinating enzyme Uch37/UCHL5 [4][5][6] to the proteasome. Here, we merge crystallography and NMR data to describe Rpn13's ubiquitin binding mechanism. We determined the structure of Rpn13 alone and complexed with ubiquitin. The co-complex reveals a novel ubiquitin binding mode in which loops rather than secondary structural elements are used to capture ubiquitin. Further support for Rpn13's role as a proteasomal ubiquitin receptor is demonstrated by its ability to bind ubiquitin and proteasome subunit Rpn2/S1 simultaneously. Finally, we provide a model structure of Rpn13 complexed to diubiquitin, which provides insights into how Rpn13's role as a ubiquitin receptor is coupled to substrate deubiquitination by Uch37. The structure of murine Rpn13 (mRpn13) (1-150) was determined at 1.7 Å resolution by Xray crystallography, and found to contain a Pleckstrin Homology (PH) domain fold ( Figure 1a and 1b) (Structure determination and refinement statistics are provided in the Supplement). In particular, whereas the first 21 N-and last 20 C-terminal amino acids are unstructured, residues 22-130 form a PH domain fold. This result was surprising, as primary sequence alignment did not identify Rpn13 to be homologous to previously characterized proteins. This finding coupled with its ubiquitin receptor properties 8 prompted us to name the N-terminal domain of Rpn13 Pleckstrin-like receptor for ubiquitin (Pru).Though very divergent at their sequence level, all PH domains have a common β-sandwich fold. The PH domain of Rpn13 is composed of a 4-stranded twisted antiparallel β-sheet (β 1-4 : residues 22-34, 45-52, 56-62, 71-74) that packs almost orthogonally against a second triple stranded β-sheet (β 5-7 : residues 80-85, 92-98, 103-110) (Supplementary Figure 1). Similar to other PH domains, Rpn13 Pru forms a hydrophobic core containing conserved hydrophobic residues (F26, V47, I49, F59, F82, Y94, L96, F107 and M109), which are located within β-sheets. One end of the β-sandwich is capped by a long Cterminal amphipathic α-helix (residues 117-128), which is stabilized by interactions between V124 and L128, whereas the other corner of the hydrophobic core is closed by three loops formed by residues located between strands S1/S2, S3/S4 and S6/S7 ( Figure 1a and Supplementary Figure 1).Despite much effort, we were unable to crystallize the Rpn13 Pru:ubiquitin complex; however, we were able to...

Cytochrome c release from mitochondria is a key event in apoptosis signaling that is regulated by Bcl-2 family proteins. Cleavage of the BH3-only protein Bid by multiple proteases leads to the formation of truncated Bid (tBid), which, in turn, promotes the oligomerization/insertion of Bax into the mitochondrial outer membrane and the resultant release of proteins residing in the intermembrane space. Bax, a monomeric protein in the cytosol, is targeted by a yet unknown mechanism to the mitochondria. Several hypotheses have been put forward to explain this targeting specificity. Using mitochondria isolated from different mutants of the yeast Saccharomyces cerevisiae and recombinant proteins, we have now investigated components of the mitochondrial outer membrane that might be required for tBid/Bax-induced cytochrome c release. Here, we show that the protein translocase of the outer mitochondrial membrane is required for Bax insertion and cytochrome c release.The release of cytochrome c and other pro-apoptotic proteins from the mitochondrial intermembrane space into the cytosol is a key event in the activation of apoptosis. Once in the cytosol, cytochrome c interacts with APAF-1, in the presence of dATP, to form the apoptosome and trigger the activation of pro-caspase-9 (1). Caspase-9 then cleaves and activates procaspase-3, the main executioner caspase. Although cytochrome c-mediated activation of the caspases is regarded as the main outcome of mitochondrial membrane permeabilization, other intermembrane space proteins play additional, independent roles in cell death signaling (2). During the early phase of apoptosis, only the mitochondrial outer membrane is permeabilized, whereas other intracellular membranes as well as the plasma membrane remain intact.The mechanism(s) by which the pro-apoptotic Bcl-2 family proteins permeabilize the mitochondrial outer membrane have been studied extensively during recent years. A major pathway involves the soluble cytoplasmic proteins Bid and Bax. Upon cleavage of Bid by proteases, e.g. caspase-8, a truncated 16-kDa C-terminal fragment (tBid) 4 is formed, which subsequently can promote the oligomerization/insertion of Bax into the outer mitochondrial membrane (3, 4). The oligomeric form of Bax is believed to form a pore that allows the extrusion of intermembrane space proteins into the cytosol (5). Oligomerization results in a change in Bax structure with exposure of an N-terminal domain, which is normally not exposed to the cytosol, and the insertion of the C-terminal domain and of the central pore-forming ␣ helices 5 and 6 into the mitochondrial outer membrane (6, 7). The C terminus is of critical importance for the pro-apoptotic function of Bax, and it shows some similarity to the signals that normally direct tail-anchored proteins to mitochondria (8).Several hypotheses have been elaborated to explain the specificity by which Bax targets the mitochondria. One of them involves an interaction of Bax with the mitochondrial porin, also called voltage-dependent anion channel, a...

A complex of RON(M1254T) with AMP-PNP and Mg(2+) reveals a substratelike positioning of Tyr1238 as well as likely catalysis-competent placement of the AMP-PNP and Mg(2+) components and indicates a tendency for cis phosphorylation. The structure shows how the oncogenic mutation may cause the constitutive activation and suggests a mechanistic hypothesis for the autophosphorylation of receptor tyrosine kinases.

Crystal structures of the MAP kinase ERK5 in complex with XMD8-92 and four novel inhibitors reveal an allosteric binding site between the kinase P-loop and αC helix. Binding at this site displaces the P-loop into the ATP-binding site and was shown to be ATP-competitive.