@article {18712,
	title = {Rpn1 and Rpn2 coordinate ubiquitin processing factors at the proteasome},
	journal = {Journal of Biological ChemistryJ. Biol. Chem.},
	year = {2012},
	month = {2012/02/08/},
	abstract = {Substrates tagged with (poly)ubiquitin for degradation can be targeted directly to the 26S proteasome where they are proteolysed. Independently, ubiquitin-conjugates may also be delivered by bivalent shuttles. The majority of shuttles attach to the proteasome through a ubiquitin-like domain (UBL) while anchoring cargo at a C-terminal polyubiquitin-binding domain(s). We found that two shuttles of this class, Rad23 and Dsk2, dock at two different receptors embedded within a single subunit of the 19S proteasome regulatory particle (RP), Rpn1. Their association/dissociation constants and affinities for Rpn1 are similar. In contrast, another UBL-containing protein, the deubiquitinase Ubp6, is also anchored by Rpn1, yet dissociates slower, thus behaving as a sometimes proteasome subunit distinct from transiently-associated shuttles. Two neighboring subunits, Rpn10 and Rpn13, show a marked preference for polyubiquitin over UBLs. Rpn10 attaches to the central solenoid portion of Rpn1 although this association is stabilized by the presence of a third subunit, Rpn2. Rpn13 binds directly to the C-terminal portion of Rpn2. These intrinsic polyubiquitin receptors may compete with substrate shuttles for their polyubiquitin-conjugates, thereby aiding release of the emptied shuttles. By binding multiple ubiquitin-processing factors simultaneously, Rpn1 is uniquely suited to coordinate substrate recruitment, deubiquitination, and movement towards the catalytic core. The broad range of affinities for ubiquitin, ubiquitin-like, and non-ubiquitin signals by adjacent yet non-overlapping sites all within the Base illustrates a hub of activity that coordinates the intricate relay of substrates within the proteasome, and consequently influences substrate residency time and commitment to degradation.},
	keywords = {deubiquitination, Proteasome, solenoid, Surface plasmon resonance (SPR), ubiquitin, Ubiquitin-dependent protease},
	isbn = {0021-9258, 1083-351X},
	doi = {10.1074/jbc.M111.316323},
	url = {http://www.jbc.org/content/early/2012/02/08/jbc.M111.316323},
	author = {Rosenzweig,Rina and Bronner,Vered and Zhang,Daoning and Fushman, David and Glickman,Michael H.}
}
@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 {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 {18707,
	title = {Mutations in the Hydrophobic Core of Ubiquitin Differentially Affect Its Recognition by Receptor Proteins},
	journal = {Journal of Molecular Biology},
	volume = {375},
	year = {2008},
	month = {2008/01/25/},
	pages = {979 - 996},
	abstract = {Ubiquitin (Ub) is one of the most highly conserved signaling proteins in eukaryotes. In carrying out its myriad functions, Ub conjugated to substrate proteins interacts with dozens of receptor proteins that link the Ub signal to various biological outcomes. Here we report mutations in conserved residues of Ub{\textquoteright}s hydrophobic core that have surprisingly potent and specific effects on molecular recognition. Mutant Ubs bind tightly to the Ub-associated domain of the receptor proteins Rad23 and hHR23A but fail to bind the Ub-interacting motif present in the receptors Rpn10 and S5a. Moreover, chains assembled on target substrates with mutant Ubs are unable to support substrate degradation by the proteasome in vitro or sustain viability of yeast cells. The mutations have relatively little effect on Ub{\textquoteright}s overall structure but reduce its rigidity and cause a slight displacement of the C-terminal β-sheet, thereby compromising association with Ub-interacting motif but not with Ub-associated domains. These studies emphasize an unexpected role for Ub{\textquoteright}s core in molecular recognition and suggest that the diversity of protein{\textendash}protein interactions in which Ub engages placed enormous constraints on its evolvability.},
	keywords = {hydrophobic core mutation, molecular recognition, proteasomal degradation, ubiquitin, ubiquitin receptors},
	isbn = {0022-2836},
	doi = {10.1016/j.jmb.2007.11.016},
	url = {http://www.sciencedirect.com/science/article/pii/S0022283607014763},
	author = {Haririnia,Aydin and Verma,Rati and Purohit,Nisha and Twarog,Michael Z. and Deshaies,Raymond J. and Bolon,Dan and Fushman, David}
}
@article {18697,
	title = {Crystal Structure and Solution NMR Studies of Lys48-linked Tetraubiquitin at Neutral pH},
	journal = {Journal of Molecular Biology},
	volume = {367},
	year = {2007},
	month = {2007/03/16/},
	pages = {204 - 211},
	abstract = {Ubiquitin modification of proteins is used as a signal in many cellular processes. Lysine side-chains can be modified by a single ubiquitin or by a polyubiquitin chain, which is defined by an isopeptide bond between the C terminus of one ubiquitin and a specific lysine in a neighboring ubiquitin. Polyubiquitin conformations that result from different lysine linkages presumably differentiate their roles and ability to bind specific targets and enzymes. However, conflicting results have been obtained regarding the precise conformation of Lys48-linked tetraubiquitin. We report the crystal structure of Lys48-linked tetraubiquitin at near-neutral pH. The two tetraubiquitin complexes in the asymmetric unit show the complete connectivity of the chain and the molecular details of the interactions. This tetraubiquitin conformation is consistent with our NMR data as well as with previous studies of diubiquitin and tetraubiquitin in solution at neutral pH. The structure provides a basis for understanding Lys48-linked polyubiquitin recognition under physiological conditions.},
	keywords = {crystal structure, Lys48-linked, polyubiquitin chains, tetraubiquitin, ubiquitin},
	isbn = {0022-2836},
	doi = {10.1016/j.jmb.2006.12.065},
	url = {http://www.sciencedirect.com/science/article/pii/S0022283606017554},
	author = {Eddins,Michael J. and Varadan,Ranjani and Fushman, David and Pickart,Cecile M. and Wolberger,Cynthia}
}
@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 {18719,
	title = {Structural Properties of Polyubiquitin Chains in Solution},
	journal = {Journal of Molecular Biology},
	volume = {324},
	year = {2002},
	month = {2002/12/06/},
	pages = {637 - 647},
	abstract = {Because polyubiquitin chain structure modulates Ub-mediated signaling, knowledge of the physiological conformations of chain signals should provide insights into specific recognition. Here, we characterized the solution conformations of K48-linked Ub2 and Ub4 using a combination of NMR techniques, including chemical shift mapping of the interdomain interface, domain orientation measurements on the basis of 15N relaxation and residual dipolar couplings, and the solvent accessibility studies. Our data indicate a switch in the conformation of Ub2, from open to closed, with increasing pH. The closed conformation features a well-defined interface that is related to, but distinguishable from, that observed in the Ub2 crystal structure. This interface is dynamic in solution, such that important hydrophobic residues (L8, I44, V70) that are sequestered at the interface in the closed conformation may be accessible for direct interactions with recognition factors. Our results suggest that the distal two units of Ub4, which is the minimum signal for efficient proteasomal degradation, may adopt the closed Ub2 conformation.},
	keywords = {chemical shift mapping, domain orientation measurements, polyubiquitin chains, spin relaxation, ubiquitin},
	isbn = {0022-2836},
	doi = {10.1016/S0022-2836(02)01198-1},
	url = {http://www.sciencedirect.com/science/article/pii/S0022283602011981},
	author = {Varadan,Ranjani and Walker,Olivier and Pickart,Cecile and Fushman, David}
}
