@article {18716,
	title = {Structural and biochemical studies of the open state of Lys48-linked diubiquitin},
	journal = {Biochimica et Biophysica Acta (BBA) - Molecular Cell Research},
	year = {2012},
	month = {2012///},
	abstract = {Ubiquitin (Ub) is a small protein highly conserved among eukaryotes and involved in practically all aspects of eukaryotic cell biology. Polymeric chains assembled from covalently-linked Ub monomers function as molecular signals in the regulation of a host of cellular processes. Our previous studies have shown that the predominant state of Lys48-linked di- and tetra-Ub chains at near-physiological conditions is a closed conformation, in which the Ub{\textendash}Ub interface is formed by the hydrophobic surface residues of the adjacent Ub units. Because these very residues are involved in (poly)Ub interactions with the majority of Ub-binding proteins, their sequestration at the Ub{\textendash}Ub interface renders the closed conformation of polyUb binding incompetent. Thus the existence of open conformation(s) and the interdomain motions opening and closing the Ub{\textendash}Ub interface is critical for the recognition of Lys48-linked polyUb by its receptors. Knowledge of the conformational properties of a polyUb signal is essential for our understanding of its specific recognition by various Ub-receptors. Despite their functional importance, open states of Lys48-linked chains are poorly characterized. Here we report a crystal structure of the open state of Lys48-linked di-Ub. Moreover, using NMR, we examined interactions of the open state of this chain (at pH4.5) with a Lys48-linkage-selective receptor, the UBA2 domain of a shuttle protein hHR23a. Our results show that di-Ub binds UBA2 in the same mode and with comparable affinity as the closed state. Our data suggest a mechanism for polyUb signal recognition, whereby Ub-binding proteins select specific conformations out of the available ensemble of polyUb chain conformations. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.},
	keywords = {Lys48-linked diubiquitin, Lysine-48 linkage selectivity, polyubiquitin, ubiquitin, Ubiquitin-associated domain},
	isbn = {0167-4889},
	doi = {10.1016/j.bbamcr.2012.04.003},
	url = {http://www.sciencedirect.com/science/article/pii/S0167488912000948},
	author = {Lai,Ming-Yih and Zhang,Daoning and LaRonde-LeBlanc,Nicole and Fushman, David}
}
@article {18702,
	title = {Exploring the Linkage Dependence of Polyubiquitin Conformations Using Molecular Modeling},
	journal = {Journal of Molecular Biology},
	volume = {395},
	year = {2010},
	month = {2010/01/29/},
	pages = {803 - 814},
	abstract = {Posttranslational modification of proteins by covalent attachment of a small protein ubiquitin (Ub) or a polymeric chain of Ub molecules (called polyubiquitin) is involved in controlling a vast variety of processes in eukaryotic cells. The question of how different polyubiquitin signals are recognized is central to understanding the specificity of various types of polyubiquitination. In polyubiquitin, monomers are linked to each other via an isopeptide bond between the C-terminal glycine of one Ub and a lysine of the other. The functional outcome of polyubiquitination depends on the particular lysine involved in chain formation and appears to rely on linkage-dependent conformation of polyubiquitin. Thus, K48-linked chains, a universal signal for proteasomal degradation, under physiological conditions adopt a closed conformation where functionally important residues L8, I44, and V70 are sequestered at the interface between two adjacent Ub monomers. By contrast, K63-linked chains, which act as a nonproteolytic regulatory signal, adopt an extended conformation that lacks hydrophobic interubiquitin contact. Little is known about the functional roles of the so-called {\textquotedblleft}noncanonical{\textquotedblright} chains (linked via K6, K11, K27, K29, or K33, or linked head-to-tail), and no structural information on these chains is available, except for information on the crystal structure of the head-to-tail-linked diubiquitin (Ub2). In this study, we use molecular modeling to examine whether any of the noncanonical chains can adopt a closed conformation similar to that in K48-linked polyubiquitin. Our results show that the eight possible Ub2 chains can be divided into two groups: chains linked via K6, K11, K27, or K48 are predicted to form a closed conformation, whereas chains linked via K29, K33, or K63, or linked head-to-tail are unable to form such a contact due to steric occlusion. These predictions are validated by the known structures of K48-, K63-, and head-to-tail-linked chains. Our study also predicts structural models for Ub2 chains linked via K6, K11, or K27. The implications of these findings for linkage-selective recognition of noncanonical polyubiquitin signals by various receptors are discussed.},
	keywords = {head-to-tail, isopeptide linkage, modeling, noncanonical linkage, polyubiquitin},
	isbn = {0022-2836},
	doi = {10.1016/j.jmb.2009.10.039},
	url = {http://www.sciencedirect.com/science/article/pii/S0022283609012790},
	author = {Fushman, David and Walker,Olivier}
}
@article {18701,
	title = {Evidence for Bidentate Substrate Binding as the Basis for the K48 Linkage Specificity of Otubain 1},
	journal = {Journal of Molecular Biology},
	volume = {386},
	year = {2009},
	month = {2009/03/06/},
	pages = {1011 - 1023},
	abstract = {Otubain 1 belongs to the ovarian tumor (OTU) domain class of cysteine protease deubiquitinating enzymes. We show here that human otubain 1 (hOtu1) is highly linkage-specific, cleaving Lys48 (K48)-linked polyubiquitin but not K63-, K29-, K6-, or K11-linked polyubiquitin, or linear α-linked polyubiquitin. Cleavage is not limited to either end of a polyubiquitin chain, and both free and substrate-linked polyubiquitin are disassembled. Intriguingly, cleavage of K48-diubiquitin by hOtu1 can be inhibited by diubiquitins of various linkage types, as well as by monoubiquitin. NMR studies and activity assays suggest that both the proximal and distal units of K48-diubiquitin bind to hOtu1. Reaction of Cys23 with ubiquitin-vinylsulfone identified a ubiquitin binding site that is distinct from the active site, which includes Cys91. Occupancy of the active site is needed to enable tight binding to the second site. We propose that distinct binding sites for the ubiquitins on either side of the scissile bond allow hOtu1 to discriminate among different isopeptide linkages in polyubiquitin substrates. Bidentate binding may be a general strategy used to achieve linkage-specific deubiquitination.},
	keywords = {deubiquitination, isopeptide, linkage specificity, otubain, polyubiquitin},
	isbn = {0022-2836},
	doi = {10.1016/j.jmb.2008.12.085},
	url = {http://www.sciencedirect.com/science/article/pii/S0022283608016124},
	author = {Wang,Tao and Yin,Luming and Cooper,Eric M. and Lai,Ming-Yih and Dickey,Seth and Pickart,Cecile M. and Fushman, David and Wilkinson,Keith D. and Cohen,Robert E. and Wolberger,Cynthia}
}
@article {18694,
	title = {Affinity Makes the Difference: Nonselective Interaction of the UBA Domain of Ubiquilin-1 with Monomeric Ubiquitin and Polyubiquitin Chains},
	journal = {Journal of Molecular Biology},
	volume = {377},
	year = {2008},
	month = {2008/03/14/},
	pages = {162 - 180},
	abstract = {Ubiquilin/PLIC proteins belong to the family of UBL{\textendash}UBA proteins implicated in the regulation of the ubiquitin-dependent proteasomal degradation of cellular proteins. A human presenilin-interacting protein, ubiquilin-1, has been suggested as potential therapeutic target for treating Huntington{\textquoteright}s disease. Ubiquilin{\textquoteright}s interactions with mono- and polyubiquitins are mediated by its UBA domain, which is one of the tightest ubiquitin binders among known ubiquitin-binding domains. Here we report the three-dimensional structure of the UBA domain of ubiquilin-1 (UQ1-UBA) free in solution and in complex with ubiquitin. UQ1-UBA forms a compact three-helix bundle structurally similar to other known UBAs, and binds to the hydrophobic patch on ubiquitin with a Kd of 20~μM. To gain structural insights into UQ1-UBA{\textquoteright}s interactions with polyubiquitin chains, we have mapped the binding interface between UQ1-UBA and Lys48- and Lys63-linked di-ubiquitins and characterized the strength of UQ1-UBA binding to these chains. Our NMR data show that UQ1-UBA interacts with the individual ubiquitin units in both chains in a mode similar to its interaction with mono-ubiquitin, although with an improved binding affinity for the chains. Our results indicate that, in contrast to UBA2 of hHR23A that has strong binding preference for Lys48-linked chains, UQ1-UBA shows little or no binding selectivity toward a particular chain linkage or between the two ubiquitin moieties in the same chain. The structural data obtained in this study provide insights into the possible structural reasons for the diversity of polyubiquitin chain recognition by UBA domains.},
	keywords = {polyubiquitin, protein{\textendash}protein interaction, UBA domain, ubiquilin, ubiquitin},
	isbn = {0022-2836},
	doi = {10.1016/j.jmb.2007.12.029},
	url = {http://www.sciencedirect.com/science/article/pii/S0022283607016452},
	author = {Zhang,Daoning and Raasi,Shahri and Fushman, David}
}
@article {18706,
	title = {Mapping the Interactions between Lys48 and Lys63-Linked Di-ubiquitins and a Ubiquitin-Interacting Motif of S5a},
	journal = {Journal of Molecular Biology},
	volume = {368},
	year = {2007},
	month = {2007/05/04/},
	pages = {753 - 766},
	abstract = {Numerous cellular processes are regulated by (poly)ubiquitin-mediated signaling events, which involve a covalent modification of the substrate protein by a single ubiquitin or a chain of ubiquitin molecules linked via a specific lysine. Remarkably, the outcome of polyubiquitination is linkage-dependent. For example, Lys48-linked chains are the principal signal for proteasomal degradation, while Lys63-linked chains act as nonproteolytic signals. Despite significant progress in characterization of various cellular pathways involving ubiquitin, understanding of the structural details of polyubiquitin chain recognition by downstream cellular effectors is missing. Here we use NMR to study the interaction of a ubiquitin-interacting motif (UIM) of the proteasomal subunit S5a with di-ubiquitin, the simplest model for polyubiquitin chain, to gain insights into the mechanism of polyubiquitin recognition by the proteasome. We have mapped the binding interface and characterized the stoichiometry and the process of UIM binding to Lys48- and Lys63-linked di-ubiquitin chains. Our data provide the first direct evidence that UIM binding involves a conformational transition in Lys48-linked di-ubiquitin, which opens the hydrophobic interdomain interface. This allows UIM to enter the interface and bind directly to the same ubiquitin hydrophobic-patch surface as utilized in UIM:monoubiquitin complexes. The results indicate that up to two UIM molecules can bind di-ubiquitin, and the binding interface between UIM and ubiquitin units in di-ubiquitin is essentially the same for both Lys48- and Lys63-linked chains. Our data suggest possible structural models for the binding of UIM and of full-length S5a to di-ubiquitin.},
	keywords = {chemical shift perturbation mapping, polyubiquitin, S5a, ubiquitin, ubiquitin-interacting motif},
	isbn = {0022-2836},
	doi = {10.1016/j.jmb.2007.02.037},
	url = {http://www.sciencedirect.com/science/article/pii/S0022283607002185},
	author = {Haririnia,Aydin and D{\textquoteright}Onofrio,Mariapina and Fushman, David}
}
@article {18705,
	title = {Interdomain mobility in di-ubiquitin revealed by NMR},
	journal = {Proteins: Structure, Function, and Bioinformatics},
	volume = {63},
	year = {2006},
	month = {2006///},
	pages = {787 - 796},
	abstract = {Domain orientation and dynamics can play an essential role in the function of multidomain proteins. Lys48-linked polyubiquitin chains, the principal signal for proteasomal protein degradation, adopt a closed conformation at physiological conditions, in which the functionally important residues Leu8, Ile44, and Val70 are sequestered at the interdomain interface. This interface must open in order for these groups to become available for interactions with various chain-recognition factors. Knowledge of the mechanism of domain motion leading to the opening of the interdomain interface in polyubiqutin is, therefore, essential for the understanding of the processes controlling molecular recognition events in polyubiquitin signaling. Here we use NMR to characterize the interdomain dynamics that open the interface in a di-ubiquitin chain. This process occurs via domain reorientations on a 10-ns time scale and with the amplitudes that are sufficient for making functionally important hydrophobic residues in polyubiquitin available for direct interactions with various ubiquitin-binding factors. The analysis revealed the structures of the interconverting conformational states of di-ubiquitin and the rates and amplitudes of this process at near-physiological and acidic pH. The proposed mechanism of domain reorientation is quite general and could serve as a paradigm of interdomain mobility in other multidomain systems. Proteins 2006. {\textcopyright} 2006 Wiley-Liss, Inc.},
	keywords = {anisotropic diffusion, domain motion, interdomain orientation, polyubiquitin, spin relaxation},
	isbn = {1097-0134},
	doi = {10.1002/prot.20917},
	url = {http://onlinelibrary.wiley.com/doi/10.1002/prot.20917/abstract},
	author = {Ryabov,Yaroslav and Fushman, David}
}
