Regulated proteolysis by ATP-dependent proteases is universal in all living cells. Bacterial ClpC, a member of the Clp/Hsp100 family of AAA+ proteins (ATPases associated with diverse cellular activities) with two nucleotide-binding domains (D1 and D2), requires the adaptor protein MecA for activation and substrate targeting. The activated, hexameric MecA-ClpC molecular machine harnesses the energy of ATP binding and hydrolysis to unfold specific substrate proteins and translocate the unfolded polypeptide to the ClpP protease for degradation. Here we report three related crystal structures: a heterodimer between MecA and the amino domain of ClpC, a heterododecamer between MecA and D2-deleted ClpC, and a hexameric complex between MecA and full-length ClpC. In conjunction with biochemical analyses, these structures reveal the organizational principles behind the hexameric MecA-ClpC complex, explain the molecular mechanisms for MecA-mediated ClpC activation and provide mechanistic insights into the function of the MecA-ClpC molecular machine. These findings have implications for related Clp/Hsp100 molecular machines.

The eukaryotic proteasome mediates degradation of polyubiquitinated proteins. Here we report the single-particle cryoelectron microscopy (cryo-EM) structures of the endogenous 26S proteasome from Saccharomyces cerevisiae at 4.6-to 6.3-Å resolution. The fine features of the cryo-EM maps allow modeling of 18 subunits in the regulatory particle and 28 in the core particle. The proteasome exhibits two distinct conformational states, designated M1 and M2, which correspond to those reported previously for the proteasome purified in the presence of ATP-γS and ATP, respectively. These conformations also correspond to those of the proteasome in the presence and absence of exogenous substrate. Structure-guided biochemical analysis reveals enhanced deubiquitylating enzyme activity of Rpn11 upon assembly of the lid. Our structures serve as a molecular basis for mechanistic understanding of proteasome function.protein degradation | proteasome | cryo-EM | structure T he eukaryotic ubiquitin-proteasome system is responsible for the degradation of polyubiquitinated proteins (1). The 26S proteasome consists of one 20S core particle (CP) and two 19S regulatory particles (RPs). The RP is divided into the lid and base assembly intermediates (1). The lid comprises nine Rpn subunits in yeast (Rpn3/5/6/7/8/9/11/12/15) and the base comprises three Rpn subunits (Rpn1/2/13) and six ATPases (Rpt1-6). Rpn10, which consists of an N-terminal von Willebrand factor A (VWA) domain and multiple C-terminal ubiquitin-interacting motifs (UIM), connects the lid and the base. Polyubiquitin (poly-Ub) chains from substrate are recognized by the RP, leading to unfolding of the substrate and its translocation into the CP, where it is degraded.The main function of the lid is to remove poly-Ub chains from the substrate (1). The released Ub chains are recycled via further cleavage into Ub monomers. Six of the nine Rpn subunits in the lid (Rpn3/5/6/7/9/12) contain a solenoid fold followed by a proteasome-CSN-eIF3 (PCI) domain of varying lengths; for Rpn8 and Rpn11, each has an Mpr1-Pad1-N-terminal (MPN) domain. Among all Rpn subunits, Rpn11 is the only deubiquitylating enzyme (DUB); it cleaves the isopeptide bond between the carboxyl terminus of Ub and the e-amino group of Lys in the substrate (2, 3). Except for Rpn15/ Sem1/Dss1, the C-terminal sequences of the other eight Rpn subunits in the lid form a helix bundle, which dictates lid assembly (4, 5).In the base, the six Rpt subunits form a hexameric ring. Powered by ATP hydrolysis, the Rpt ring is responsible for substrate unfolding and translocation of the unfolded substrate through the narrow RP central channel into the CP for degradation (6-8). The barrel-shaped CP consists of two outer α-rings and two inner β-rings, each containing seven subunits (α1-7 or β1-7). X-ray structures of the CP at atomic resolution have been reported for archaeabacteria (9), yeast (10), and mammals (11).Crystallization of the RP or the 26S proteasome is hampered by its dynamic nature. Improvement of cryo-EM technologies has ...

The mitogen-activated protein (MAP) kinases are essential signaling molecules that mediate many cellular effects of growth factors, cytokines, and stress stimuli. Full activation of the MAP kinases requires dual phosphorylation of the Thr and Tyr residues in the TXY motif of the activation loop by MAP kinase kinases. Down-regulation of MAP kinase activity can be initiated by multiple serine/threonine phosphatases, tyrosine-specific phosphatases, and dual specificity phosphatases (MAP kinase phosphatases). This would inevitably lead to the formation of monophosphorylated MAP kinases. However, the biological functions of these monophosphorylated MAP kinases are currently not clear. In this study, we have prepared MAP kinase p38␣, a member of the MAP kinase family, in all phosphorylated forms and characterized their biochemical properties. Our results indicated the following: (i) p38␣ phosphorylated at both Thr-180 and Tyr-182 was 10 -20-fold more active than p38␣ phosphorylated at Thr-180 only, whereas p38␣ phosphorylated at Tyr-182 alone was inactive; (ii) the dual-specific MKP5, the tyrosine-specific hematopoietic protein-tyrosine phosphatase, and the serine/ threonine-specific PP2C␣ are all highly specific for the dephosphorylation of p38␣, and the dephosphorylation rates were significantly affected by different phosphorylated states of p38␣; (iii) the N-terminal domain of MPK5 has no effect on enzyme catalysis, whereas deletion of the MAP kinase-binding domain in MKP5 leads to a 370-fold decrease in k cat /K m for the dephosphorylation of p38␣. This study has thus revealed the quantitative contributions of phosphorylation of Thr, Tyr, or both to the activation of p38␣ and to the substrate specificity for various phosphatases. Mitogen-activated protein kinases (MAPKs)3 play a pivotal role in controlling numerous cellular processes, including differentiation, mitogenesis, oncogenesis, and apoptosis (1-6). A typical MAPK cascade consists of three tiers of sequentially activating protein kinases, which commonly are referred to as MAPK, MAPK kinase (MAPKK), and MAPK kinase kinase (MAPKKK). An activated MAPKKK phosphorylates and activates a specific MAPKK, which then activates a specific MAPK. The three best characterized MAPK cascades are the extracellular signal-regulated kinase (ERK) pathway, the c-Jun N-terminal kinase (JNK) pathway, and the p38 kinase pathway. ERKs are activated by a range of stimuli, including growth factors, cell adhesion, tumor-promoting phorbol esters, and oncogenes, whereas JNK and p38 are preferentially activated by proinflammatory cytokines, and a variety of environmental stresses such as UV and osmotic stress. After activation, each MAPK phosphorylates a distinct spectrum of substrates, which include key regulatory enzymes, cytoskeletal proteins, nuclear receptors, regulators of apoptosis, and many transcription factors.Like many protein kinases, the activity of MAPKs is regulated by phosphorylation in an activation loop located near their active sites (7). The hallmark of the MAPKs is ...

The ubiquitin system is important for drug discovery, and the discovery of selective small-molecule inhibitors of deubiquitinating enzymes (DUBs) remains an active yet extremely challenging task. With a few exceptions, previously developed inhibitors have been found to bind the evolutionarily conserved catalytic centers of DUBs, resulting in poor selectivity. The small molecule IU1 was the first-ever specific inhibitor identified and exhibited surprisingly excellent selectivity for USP14 over other DUBs. However, the molecular mechanism for this selectivity was elusive. Herein, we report the high-resolution co-crystal structures of the catalytic domain of USP14 bound to IU1 and three IU1 derivatives. All the structures of these complexes indicate that IU1 and its analogs bind to a previously unknown steric binding site in USP14, thus blocking the access of the C-terminus of ubiquitin to the active site of USP14 and abrogating USP14 activity. Importantly, this steric site in USP14 is very unique, as suggested by structural alignments of USP14 with several known DUB X-ray structures. These results, in conjunction with biochemical characterization, indicate a coherent steric blockade mechanism for USP14 inhibition by compounds of the IU series. In light of the recent report of steric blockade of USP7 by FT671, this work suggests a potential generally applicable allosteric mechanism for the regulation of DUBs via steric blockade, as showcased by our discovery of IU1-248 which is 10-fold more potent than IU1.

Chemical ubiquitination is an effective approach for accessing structurally defined, atypical ubiquitin (Ub) chains that are difficult to prepare by other techniques. Herein, we describe a strategy that uses a readily accessible premade isopeptide-linked 76-mer (isoUb), which has an N-terminal Cys and a C-terminal hydrazide, as the key building block to assemble atypical Ub chains in a modular fashion. This method avoids the use of auxiliary-modified Lys and instead employs the canonical and therefore more robust Cys-based native chemical ligation technique. The efficiency and capacity of this isoUb-based strategy is exemplified by the cost-effective synthesis of several linkage- and length-defined atypical Ub chains, including K27-linked tetra-Ub and K11/K48-branched tri-, tetra-, penta-, and hexa-Ubs.

Regulated proteolysis by ATP-dependent proteases is universal in all living cells. In Bacillus subtilis, the degradation of the competence transcription factor ComK is mediated by a ternary complex involving the adaptor protein MecA and the ATP-dependent protease ClpCP. Here we demonstrate that a C-terminal, 98-amino acid domain of MecA (residues 121-218) serves as a non-recycling, degradation tag and targets a variety of fusion proteins to the ClpCP protease for degradation. MecA-(121-218) facilitates productive oligomerization of ClpC, stimulates the ATPase activity of ClpC, and allows the activated ClpC complex to stably associate with ClpP. Importantly, the ClpCP protease undergoes dynamic cycles of assembly and disassembly, which are triggered by association with MecA and the degradation of MecA, respectively.

New synthetic strategies that exploited the strengths of both chemoselective ligation and recombinant protein expression were developed to prepare K27 di-ubiquitins (diUb), which enabled mechanistic studies on the molecular recognition of K27-linked Ubs by single-molecule Fçrster resonance energy transfer (smFRET) and X-ray crystallography.The results revealed that free K27 diUb adopted acompact conformation, whereas upon binding to UCHL3, K27 diUb was remodeled to an open conformation. The K27 isopeptide bond remained rigidly buried inside the diUb moiety during binding,a ni nteresting unique structural feature that may explain the distinctive biological function of K27 Ub chains.

Background:The structure of the type II AAA ϩ hexameric molecular machine is highly dynamic.