Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2011 Oct 21;413(2):416-29.
doi: 10.1016/j.jmb.2011.08.029. Epub 2011 Aug 24.

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

Christopher W Davies  1 , Lake N Paul, Myung-Il Kim, Chittaranjan Das
Affiliations
Comparative Study

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

Christopher W Davies et al. J Mol Biol. .

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.

PubMed Disclaimer

Figures

Figure 1
Figure 1
c(s) distribution plots of the DUB domains of AMSH244 and AMSH-LP as determined by AUC. (a) c(s) distribution plot of AMSH244 using interference optics shows that the majority discrete species at 2.1S is a globular, monomeric protein. The protein concentrations used were 30µM (large dashes), 60µM (solid line), and 120µM (small dashes). (b) c(s) distribution plot of AMSH-LP using absorbance at 280nm shows that the majority discrete species at 2.03S is a globular, monomeric protein. The protein concentrations used were 25µM (large dashes), 47µM (solid line), 94µM (small dashes).
Figure 2
Figure 2
The structure of the catalytic domain of AMSH. (a) Ribbon representation of the X-ray structure of AMSH244. (b) Expanded view of the active-site zinc coordination. (c) Expanded view of the second zinc coordination site. The coordinating residues are shown as sticks, with carbon shown in green, oxygen in red, nitrogen in blue and sulfur in yellow. Zinc is shown as grey sphere.
Figure 3
Figure 3
Superposition of the active sites of AMSH244 and AMSH219E280A. AMSH244 is shown in green and AMSH219E280A in pink. The zinc-coordinating residues in AMSH219E280A are outlined in electron density (2Fo−Fc) at 1σ.
Figure 4
Figure 4
Structural comparison of the catalytic domains of AMSH and AMSH-LP. (a) Backbone superposition of AMSH (shown in green) and AMSH-LP (shown in cyan). (b) Expanded view of the zinc-coordinating residues in the active site. (c) Expanded view of the residues coordinating the second zinc. The coordinating residues are shown as sticks, with carbon shown in green and cyan, oxygen in red, nitrogen in blue and sulfur in yellow. Zinc is shown as grey sphere.
Figure 5
Figure 5
A model of AMSH244 bound to Lys63-linked ubiquitin dimer. Backbone superposition of AMSH244 (green), the DUB domain of AMSH-LP (cyan), and the DUB domain of AMSH-LP bound to Lys63-linked diubiquitin (gray). The proximal ubiquitin and distal ubiquitin are shown in magenta and yellow ribbon, respectively.
Figure 6
Figure 6
Sequence alignment of AMSH from different species including AMSH-LP and RPN11. (a) Sequence alignment comparing the residues implicated in the proximal ubiquitin binding. (b) Sequence alignment comparing the residues implicated in the distal ubiquitin binding.
Figure 7
Figure 7
A view of the residues involved in the proximal ubiquitin recognition. Superposition of AMSH244 (green ribbon), the DUB domain of AMSH-LP (cyan ribbon) and the DUB domain of AMSH-LP bound to Lys63-linked diubiquitin (gray ribbon). Residues from the proximal ubiquitin making contact at the proximal site are shown in magenta.
Figure 8
Figure 8
A view of the residues involved in the distal ubiquitin recognition. Superposition of AMSH244 (green ribbon) and the DUB domain of AMSH-LP bound to Lys63-linked diubiquitin (gray ribbon). The distal ubiquitin is shown in yellow. Hydrogen bonding interactions are shown as black dashes and van der Waals interactions are shown as blue dashes.
Figure 9
Figure 9
Fraction unfolded curves comparing the stability of the catalytic domains of AMSH and AMSH-LP with increasing concentrations of GdHCl followed by circular dichroism spectroscopy (CD) at 220nm. Data for AMSH244 is shown in filled circles and that for the DUB domain of AMSH-LP is shown in open circles.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Maytal-Kivity V, Reis N, Hofmann K, Glickman MH. MPN+, a putative catalytic motif found in a subset of MPN domain proteins from eukaryotes and prokaryotes, is critical for Rpn11 function. BMC Biochem. 2002;3:28. - PMC - PubMed
    1. Nijman SM, Luna-Vargas MP, Velds A, Brummelkamp TR, Dirac AM, Sixma TK, Bernards R. A genomic and functional inventory of deubiquitinating enzymes. Cell. 2005;123:773–786. - PubMed
    1. Komander D, Clague MJ, Urbe S. Breaking the chains: structure and function of the deubiquitinases. Nat Rev Mol Cell Biol. 2009;10:550–563. - PubMed
    1. Wilkinson KD. DUBs at a glance. J Cell Sci. 2009;122:2325–2329. - PMC - PubMed
    1. Komander D. Mechanism, specificity and structure of the deubiquitinases. Subcell Biochem. 2010;54:69–87. - PubMed

Publication types

MeSH terms

LinkOut - more resources