Structural and Thermodynamic Comparison of the Catalytic Domain of AMSH and AMSH-LP: Nearly Identical Fold but Different Stability

Davies, C.; Paul, Lake N.; Kim, Myungil; Das, Chittaranjan

doi:10.1016/j.jmb.2011.08.029

Cited by 43 publications

(74 citation statements)

References 42 publications

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“…These sidechains, together with a water molecule, form a slightly distorted tetrahedral coordination shell around the metal ion. This core structure is almost identical to that of the DUB AMSH-LP (25,33,34), for which both apo and substrate-bound crystal structures have been characterized at high-resolution ( Fig. 3B and Fig.…”

Section: Resultsmentioning

confidence: 56%

“…The residue Glu48 at the beginning of β-strand βB corresponds to residue Glu292 of AMSH-LP, which is essential for AMSH-LP activity (25,33). Mutating this residue to glutamine abolished Ub 4 cleavage activity in Rpn8-Rpn11 (Fig.…”

Section: Resultsmentioning

confidence: 96%

“…The active site of MPN domain metalloproteases is located between the N-terminal end of helix α3 and the adjacent β-strands βB and βD (31,33,34). Clear density for the catalytic zinc was identified between the sidechains of His109, His111, and Asp122 ( Fig.…”

Section: Resultsmentioning

confidence: 97%

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Crystal structure of the proteasomal deubiquitylation module Rpn8-Rpn11

Pathare

Nagy

Śledź

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

120

131

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The ATP-dependent degradation of polyubiquitylated proteins by the 26S proteasome is essential for the maintenance of proteome stability and the regulation of a plethora of cellular processes. Degradation of substrates is preceded by the removal of polyubiquitin moieties through the isopeptidase activity of the subunit Rpn11. Here we describe three crystal structures of the heterodimer of the Mpr1-Pad1-N-terminal domains of Rpn8 and Rpn11, crystallized as a fusion protein in complex with a nanobody. This fusion protein exhibits modest deubiquitylation activity toward a model substrate. Full activation requires incorporation of Rpn11 into the 26S proteasome and is dependent on ATP hydrolysis, suggesting that substrate processing and polyubiquitin removal are coupled. Based on our structures, we propose that premature activation is prevented by the combined effects of low intrinsic ubiquitin affinity, an insertion segment acting as a physical barrier across the substrate access channel, and a conformationally unstable catalytic loop in Rpn11. The docking of the structure into the proteasome EM density revealed contacts of Rpn11 with ATPase subunits, which likely stabilize the active conformation and boost the affinity for the proximal ubiquitin moiety. The narrow space around the Rpn11 active site at the entrance to the ATPase ring pore is likely to prevent erroneous deubiquitylation of folded proteins.n eukaryotes, the ubiquitin (Ub) proteasome system (UPS) is responsible for the regulated degradation of proteins (1-5). The UPS plays a key role in the maintenance of protein homeostasis by removing misfolded or damaged proteins, which could impair cellular functions, and by removing proteins whose functions are no longer needed. Consequently, the UPS is critically involved in numerous cellular processes, including cell cycle progression, apoptosis, and DNA damage repair, and malfunctions of the system often result in disease.The 26S proteasome executes the degradation of substrates that are marked for destruction by the covalent attachment of polyubiquitin chains. It is a molecular machine of 2.5 MDa comprising two subcomplexes, the 20S core particle (CP) and one or two 19S regulatory particles (RPs), which associate with the ends of the cylinder-shaped CP (6-8). The recognition and recruitment of polyubiquitylated substrates, their deubiquitylation, ATP-dependent unfolding, and translocation into the core particle take place in the RP. The structurally and mechanistically well-characterized CP houses the proteolytic activities and sequesters them from the environment, thereby avoiding collateral damage (9).The RPs attach to the outer α-rings of the CP, which control access to the proteolytic chamber formed by the inner β-subunit rings (10). Recently, the molecular architecture of the 26S holocomplex was established using cryo-EM-based approaches (11,12), and a pseudoatomic model of the holocomplex was put forward (13). The RP is formed by two subcomplexes, known as the base and the lid, which assemble independent...

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Section: Resultsmentioning

confidence: 56%

Section: Resultsmentioning

confidence: 96%

See 1 more Smart Citation

Crystal structure of the proteasomal deubiquitylation module Rpn8-Rpn11

Pathare

Nagy

Śledź

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

120

131

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“…E280 is important for normal coordination of Zn ions in the catalytic center of AMSH. However, D309 has the flexibility to turn in the absence of E280 and bind H 2 O, which results in normal coordination of Zn ions (41). Thus, combination of these two mutations was necessary for inactivating AMSH.…”

Section: Resultsmentioning

confidence: 99%

Lysine 63-linked polyubiquitination is required for EGF receptor degradation

Huang

Zeng

Kim

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

111

100

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Ubiquitination mediates endocytosis and endosomal sorting of various signaling receptors, transporters, and channels. However, the relative importance of mono-versus polyubiquitination and the role of specific types of polyubiquitin linkages in endocytic trafficking remain controversial. We used mass spectrometrybased targeted proteomics to show that activated epidermal growth factor receptor (EGFR) is ubiquitinated by one to two short (two to three ubiquitins) polyubiquitin chains mainly linked via lysine 63 (K63) or conjugated with a single monoubiquitin. Multimonoubiquitinated EGFR species were not found. To directly test whether K63 polyubiquitination is necessary for endocytosis and post-endocytic sorting of EGFR, a chimeric protein, in which the K63 linkage-specific deubiquitination enzyme AMSH [associated molecule with the Src homology 3 domain of signal transducing adaptor molecule (STAM)] was fused to the carboxyl terminus of EGFR, was generated. MS analysis of EGFR-AMSH ubiquitination demonstrated that the fraction of K63 linkages was substantially reduced, whereas relative amounts of monoubiquitin and K48 linkages increased, compared with that of wild-type EGFR. EGFR-AMSH was efficiently internalized into early endosomes, but, importantly, the rates of ligand-induced sorting to late endosomes and degradation of EGFR-AMSH were dramatically decreased. The slow degradation of EGFR-AMSH resulted in the sustained signaling activity of this chimeric receptor. Ubiquitination patterns, rate of endosomal sorting, and signaling kinetics of EGFR fused with the catalytically inactive mutant of AMSH were reversed to normal. Altogether, the data are consistent with the model whereby short K63-linked polyubiquitin chains but not multimonoubiquitin provide an increased avidity for EGFR interactions with ubiquitin adaptors, thus allowing rapid sorting of activated EGFR to the lysosomal degradation pathway.

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“…Structural Model for the AMSH⅐STAM⅐Tri-Ub ComplexThe structural basis for the preference of AMSH-STAM for longer ubiquitin chains was not clear given the current structures of AMSH, STAM, and associated complexes (26,40,41). We modeled the structure of STAM bound to AMSH using the existing structures of AMSH-like protease bound to di-ubiquitin and the known structures of domains of STAM and homology modeling of the UIM of STAM to provide structural support for our biochemical findings.…”

mentioning

confidence: 95%

The Vps27/Hrs/STAM (VHS) Domain of the Signal-transducing Adaptor Molecule (STAM) Directs Associated Molecule with the SH3 Domain of STAM (AMSH) Specificity to Longer Ubiquitin Chains and Dictates the Position of Cleavage

Baiady¹,

Padala²,

Mashahreh³

et al. 2016

Journal of Biological Chemistry

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The deubiquitinating enzyme associated molecule with the SH3 domain of STAM (AMSH) is crucial for the removal of ubiquitin molecules during receptor-mediated endocytosis and lysosomal receptor sorting. AMSH interacts with signal transducing adapter molecule (STAM) 1 or 2, which enhances the activity of AMSH through an unknown mechanism. This stimulation is dependent on the ubiquitin-interacting motif of STAM. Here we investigate the specific mechanism of AMSH stimulation by STAM proteins and the role of the STAM Vps27/ Hrs/STAM domain. We show that, in the presence of STAM, the length of the ubiquitin chains affects the apparent cleavage rate. Through measurement of the chain cleavage kinetics, we found that, although the k cat of Lys 63 -linked ubiquitin chain cleavage was comparable for di-and tri-ubiquitin, the K m value was lower for tri-ubiquitin. This increased affinity for longer chains was dependent on the Vps27/Hrs/STAM domain of STAM and required that the substrate ubiquitin chain contain homogenous Lys 63 -linkages. In addition, STAM directed AMSH cleavage toward the distal isopeptide bond in tri-ubiquitin chains. Finally, we generated a structural model of AMSH-STAM to show how the complex binds Lys 63 -linked ubiquitin chains and cleaves at the distal end. These data show how a deubiquitinating enzyme-interacting protein dictates the efficiency and specificity of substrate cleavage.The ubiquitin system plays a role in a wide range of cellular processes, including protein degradation, cell signaling, transcription regulation, and DNA damage response (1). The regulation of numerous cellular processes by ubiquitin is ascribed to the ability of ubiquitin to form a large spectrum of distinct modifications, from monoubiquitination of a target protein to polyubiquitin chains (2). Polyubiquitin chains are formed by connecting one of seven lysines or the N-terminal ␣-amine within one ubiquitin to Gly 76 of another ubiquitin (3, 4). These linkages are described by the lysine within ubiquitin that donates the amine (e.g. Lys 63 or Lys 48 ). Similar to other posttranslational modifications, ubiquitination is a reversible modification, and the removal of ubiquitin from substrates or the reduction of the polyubiquitin chain length, called "trimming," is catalyzed by a group of enzymes called deubiquitinating enzymes (DUBs).3 About 100 DUBs are encoded by the human genome. They are divided into six families on the basis of their structure and catalytic mechanism (5). Five of the families are cysteine proteases, including ubiquitin-specific proteases, ubiquitin C-terminal hydrolases, ovarian tumor domain DUBs, Machado-Joseph disease proteases, and monocyte chemotactic protein-induced protein DUBs, whereas the sixth family, JAB1/ MPN/MOV34 domain DUBs, are metalloproteases (5). DUBs that cleave polyubiquitin chains exhibit different cleavage specificities depending on the ubiquitin chain linkage. Some DUBs only cleave chains of a single linkage, whereas others cleave several linkage types and still others exhibit l...

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Structural and Thermodynamic Comparison of the Catalytic Domain of AMSH and AMSH-LP: Nearly Identical Fold but Different Stability

Cited by 43 publications

References 42 publications

Crystal structure of the proteasomal deubiquitylation module Rpn8-Rpn11

Crystal structure of the proteasomal deubiquitylation module Rpn8-Rpn11

Lysine 63-linked polyubiquitination is required for EGF receptor degradation

The Vps27/Hrs/STAM (VHS) Domain of the Signal-transducing Adaptor Molecule (STAM) Directs Associated Molecule with the SH3 Domain of STAM (AMSH) Specificity to Longer Ubiquitin Chains and Dictates the Position of Cleavage

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