Structural and thermodynamic comparison of the catalytic domain of AMSH and AMSH-LP: nearly identical fold but different stability.

Brown Laboratory of Chemistry, Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA.
Journal of Molecular Biology (Impact Factor: 3.91). 08/2011; 413(2):416-29. DOI: 10.1016/j.jmb.2011.08.029
Source: PubMed

ABSTRACT AMSH plays a critical role in the ESCRT (endosomal sorting complexes required for transport) machinery, which facilitates the down-regulation and degradation of cell-surface receptors. It displays a high level of specificity toward cleavage of Lys63-linked polyubiquitin chains, the structural basis of which has been understood recently through the crystal structure of a highly related, but ESCRT-independent, protein AMSH-LP (AMSH-like protein). We have determined the X-ray structure of two constructs representing the catalytic domain of AMSH: AMSH244, the JAMM (JAB1/MPN/MOV34)-domain-containing polypeptide segment from residues 244 to 424, and AMSH219(E280A), an active-site mutant, Glu280 to Ala, of the segment from 219 to 424. In addition to confirming the expected zinc coordination in the protein, the structures reveal that the catalytic domains of AMSH and AMSH-LP are nearly identical; however, guanidine-hydrochloride-induced unfolding studies show that the catalytic domain of AMSH is thermodynamically less stable than that of AMSH-LP, indicating that the former is perhaps structurally more plastic. Much to our surprise, in the AMSH219(E280A) structure, the catalytic zinc was still held in place, by the compensatory effect of an aspartate from a nearby loop moving into a position where it could coordinate with the zinc, once again suggesting the plasticity of AMSH. Additionally, a model of AMSH244 bound to Lys63-linked diubiquitin reveals a type of interface for the distal ubiquitin significantly different from that seen in AMSH-LP. Altogether, we believe that our data provide important insight into the structural difference between the two proteins that may translate into the difference in their biological function.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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 spectrometry-based 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.
    Proceedings of the National Academy of Sciences 09/2013; · 9.81 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Polyubiquitin chains target protein substrates to the 26S proteasome, where they are removed by the deubiquitinase Rpn11 to allow efficient substrate degradation. Despite Rpn11's essential function during substrate processing, its detailed structural and biochemical characterization has been hindered by difficulties in purifying the isolated enzyme. Here we report the 2.0-Å crystal structures of Zn(2+)-free and Zn(2+)-bound Saccharomyces cerevisiae Rpn11 in an MPN-domain heterodimer with Rpn8. The Rpn11-Rpn8 interaction occurs via two distinct interfaces that may be conserved in related MPN-domain complexes. Our structural and mutational studies reveal that Rpn11 lacks a conserved surface to bind the ubiquitin Ile44 patch, does not interact with the moiety on the proximal side of the scissile isopeptide bond and exhibits no linkage specificity for ubiquitin cleavage. These findings explain how Rpn11 functions as a promiscuous deubiquitinase for cotranslocational substrate deubiquitination during proteasomal degradation.
    Nature Structural & Molecular Biology 01/2014; · 11.90 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    Proceedings of the National Academy of Sciences 02/2014; · 9.81 Impact Factor


Available from