Article

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.

0 Bookmarks
 · 
90 Views
  • 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; DOI:10.1073/pnas.1400546111
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: AMSH, a conserved zinc metallo deubiquitinase (DUB), controls down-regulation and degradation of cell-surface receptors mediated by the endosomal sorting complexes required for transport (ESCRT) machinery. It displays high specificity toward Lys63-linked polyubiquitin chain, which is used as a signal for ESCRT-mediated endosomal-lysosomal sorting of receptors. Herein, we report the crystal structures of the catalytic domain of AMSH orthologue Sst2 from fission yeast, its ubiquitin (product)-bound form, and its Lys63-linked diubiquitin (substrate)-bound form at 1.45 Å, 1.7 Å, and 2.3 Å, respectively. The structures reveal that the P-side product fragment maintains nearly all the contacts with the enzyme as seen with the P portion (distal ubiquitin) of the Lys63-linked diubiquitin substrate, with additional coordination of Gly76 carboxylate group of the product with the active-site Zn2+. One of the product-bound structures described herein is the result of an attempt to co-crystallize diubiquitin substrate bound to an active site mutant presumed to render the enzyme inactive, instead yielding a co-crystal structure of the enzyme bound to the P-side ubiquitin fragment of the substrate (distal ubiquitin). This fragment was generated in situ from residual activity of the mutant enzyme. In this structure, the catalytic water is seen placed between the active-site Zn2+ and the carboxylate group of Gly76 of ubiquitin, providing what appears to be a snapshot of the active site when the product is about to depart. Comparison of this structure with that of the substrate-bound form suggests the importance of dynamics of a flexible flap near the active site in catalysis. The crystal structure of the Thr319Ile mutant of the catalytic domain of Sst2 provides insight into structural basis of microcephaly-capillary malformation (MIC-CAP) syndrome. Isothermal titration calorimetry (ITC) yields a dissociation constant (KD) of 10.2 ± 0.6 μM for ubiquitin binding to the enzyme, a value comparable to the KM of the enzyme catalyzing hydrolysis of Lys63-linked diubiquitin substrate (∼20 μM). These results, together with the previously reported observation that the intracellular concentration of free ubiquitin (~ 20 μM) exceeds that of Lys63-linked polyubiquitin chains, imply that the free, cytosolic form of the enzyme remains inhibited by being tightly bound to free ubiquitin. We propose that when AMSH associates with endosomes, inhibition would be relieved due to ubiquitin binding domains (UBDs) present on its endosomal-binding partners that would shift the balance toward better recognition of polyubiquitin chains through avidity effect.
    Biochemistry 05/2014; 53(19). DOI:10.1021/bi5003162
  • [Show abstract] [Hide abstract]
    ABSTRACT: Recent research in wireless power transfer (WPT) using resonant inductive coupling has demonstrated very high efficiencies (above 40%) at large distances compared to the transmitting element dimensions, thereby exponentially increasing the number of potential applications of WPT. Since resonant inductive coupling is a very multidisciplinary field, different approaches have been proposed to predict the behaviour of these systems from the physical theory of resonators (coupled-mode theory), reflected load theory and circuit theory. Also, there is in this field a heterogeneous definition of metrics without a clear optimization process. In this article we unify the different metrics and demonstrate how to maximize the power transfer efficiency in a non-radiative resonant wireless power transfer link from a circuit-centric point of view providing design guidelines in terms of optimal load impedance, optimal source impedance and optimal distance between coils.
    Circuits and Systems (ISCAS), 2013 IEEE International Symposium on; 01/2013

Preview

Download
0 Downloads
Available from