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: 4.33). 08/2011; 413(2):416-29. DOI: 10.1016/j.jmb.2011.08.029
Source: PubMed


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.

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    • "An emerging theme of JAMM domains is that they function by cleaving at the base of the chain, between proximal Ub and substrate (RPN11, CSN5/Jab1), and/or they are highly specific for K63 poly-Ub linkages (RPN11, AMSH, AMSH-LP, BRCC36) [75,79–82]. To date there are 3 crystal structures of human JAMM domains; CSN5/JAB1 [83], AMSH [84], and AMSH-LP in complex with K63 di-Ub, which has yielded insights into K63 poly-Ub specificity [82] "
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    ABSTRACT: The post-translational attachment of one or several ubiquitin molecules to a protein generates a variety of targeting signals that are used in many different ways in the cell. Ubiquitination can alter the activity, localization, protein-protein interactions or stability of the targeted protein. Further, a very large number of proteins are subject to regulation by ubiquitin-dependent processes, meaning that virtually all cellular functions are impacted by these pathways. Nearly a hundred enzymes from five different gene families (the deubiquitinating enzymes or DUBs), reverse this modification by hydrolyzing the (iso)peptide bond tethering ubiquitin to itself or the target protein. Four of these families are thiol proteases and one is a metalloprotease. DUBs of the Ubiquitin C-terminal Hydrolase (UCH) family act on small molecule adducts of ubiquitin, process the ubiquitin proprotein, and trim ubiquitin from the distal end of a polyubiquitin chain. Ubiquitin Specific Proteases (USP) tend to recognize and encounter their substrates by interaction of the variable regions of their sequence with the substrate protein directly, or with scaffolds or substrate adapters in multiprotein complexes. Ovarian Tumor (OTU) domain DUBs show remarkable specificity for different Ub chain linkages and may have evolved to recognize substrates on the basis of those linkages. The Josephin family of DUBs may specialize in distinguishing between polyubiquitin chains of different lengths. Finally, the JAB1/MPN+/MOV34 (JAMM) domain metalloproteases cleave the isopeptide bond near the attachment point of polyubiquitin and substrate, as well as being highly specific for the K63 poly-Ub linkage. These DUBs regulate proteolysis by: directly interacting with and co-regulating E3 ligases; altering the level of substrate ubiquitination; hydrolyzing or remodeling ubiquitinated and poly-ubiquitinated substrates; acting in specific locations in the cell and altering the localization of the target protein; and acting on proteasome bound substrates to facilitate or inhibit proteolysis. Thus, the scope and regulation of the ubiquitin pathway is very similar to that of phosphorylation, with the DUBs serving the same functions as the phosphatase. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System.
    Full-text · Article · Jul 2013 · Biochimica et Biophysica Acta
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    • "Here we used hierarchical clustering to understand the relationships of the amino acid sequences of the 'JAMM' domain proteins of archaea to eukaryotic and bacterial homologues with known or putative functions (Fig. 1). The JAMM domain proteins that function as DUBs or isopeptidases in eukaryotes included in the analysis were Rpn11/Poh1 (Verma et al., 2002; Yao and Cohen, 2002), Csn5/Jab1 (Cope et al., 2002), AMSH and AMSH-LP (McCullough et al., 2004; Sato et al., 2008; Davies et al., 2011), 2A-DUB/MYSM1/KIAA1915 (Zhu et al., 2007), and Brcc36 (Sobhian et al., 2007; Cooper et al., 2009; 2010; Feng et al., 2010; Patterson-Fortin et al., 2010). The cluster analysis also included the bacterial JAMM domain proteins Mycobacterium tuberculosis Mec+ (Rv1334) shown to catalyse the hydrolysis of cysteine from CysOcysteine adduct formed by cysteine synthase M (CysM) (Burns et al., 2005), Pseudomonas fluorescens QbsD thought to remove the cysteine-phenylalanine residues from the C-terminus of the sulphur carrier QbsE (Godert et al., 2007), and Ttc1133 associated with the recently described TtuB-system of protein modification in Thermus thermophilus (Shigi, 2012). "
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    ABSTRACT: Proteins with JAB1/MPN/MOV34 metalloenzyme (JAMM/MPN+) domains are widespread among all domains of life, yet poorly understood. Here we report the purification and characterization of an archaeal JAMM/MPN+ domain protein (HvJAMM1) from Haloferax volcanii that cleaves ubiquitin-like small archaeal modifier proteins (SAMP1/2) from protein conjugates. HvJAMM1 cleaved SAMP1/2 conjugates generated in H. volcanii as well as isopeptide- and linear-linked SAMP1-MoaE in purified form. Cleavage of linear linked SAMP1-MoaE was dependent on the presence of the SAMP domain and the C-terminal VSGG motif of this domain. While HvJAMM1 was inhibited by size exclusion chromatography and metal chelators, its activity could be restored by addition of excess ZnCl(2) . HvJAMM1 residues (Glu31, His88, His90, Ser98 and Asp101) that were conserved with the JAMM/MPN+ active-site motif were required for enzyme activity. Together, these results provide the first example of a JAMM/MPN+ zinc metalloprotease that independently catalyses the cleavage of ubiquitin-like (isopeptide and linear) bonds from target proteins. In archaea, HvJAMM1 likely regulates sampylation and the pools of 'free' SAMP available for protein modification. HvJAMM1-type proteins are thought to release the SAMPs from proteins modified post-translationally as well as those synthesized as domain fusions.
    Full-text · Article · Sep 2012 · Molecular Microbiology
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    • "Several MPN-containing proteins that derived from various species were obtained and their structures were solved by X-ray crystallography [13] [14] [15] [16] [17] [18] [19]. Structural comparison of these proteins and bioinformation analysis of CSN6 MPN domain offer clues to investigate the evolution of JAMM family members and to broaden our understanding of CSN6 function. "
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    ABSTRACT: The COP9 signalosome (CSN) is a multiprotein complex containing eight subunits and is highly conserved from fungi to human. CSN is proposed to widely participate in many physiological processes, including protein degradation, DNA damage response and signal transduction. Among those subunits, only CSN5 and CSN6 belong to JAMM family. CSN5 possesses isopeptidase activity, but CSN6 lacks this ability. Here we report the 2.5Å crystal structure of MPN domain from Drosophila melanogaster CSN6. Structural comparison with other MPN domains, along with bioinformation analysis, suggests that MPN domain from CSN6 may serve as a scaffold instead of a metalloprotease.
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