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1.  Structural mechanism for the regulation of HCN ion channels by the accessory protein TRIP8b 
Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels underlie the cationic Ih current present in many neurons. The direct binding of cAMP to HCN channels increases the rate and extent of channel opening and results in a depolarizing shift in the voltage dependence of activation. TRIP8b is an accessory protein that regulates the cell surface expression and dendritic localization of HCN channels and reduces the cyclic nucleotide dependence of these channels. Here we use electron paramagnetic resonance (EPR) to show that TRIP8b binds to the apo state of the cyclic nucleotide-binding domain (CNBD) of HCN2 channels without changing the overall domain structure. With EPR and nuclear magnetic resonance (NMR), we locate TRIP8b relative to the HCN channel and identify the binding interface on the CNBD. These data provide a structural framework for understanding how TRIP8b regulates the cyclic nucleotide dependence of HCN channels.
PMCID: PMC4394039  PMID: 25800552
2.  E2 enzymes: more than just middle men 
Cell Research  2016;26(4):423-440.
Ubiquitin-conjugating enzymes (E2s) are the central players in the trio of enzymes responsible for the attachment of ubiquitin (Ub) to cellular proteins. Humans have ∼40 E2s that are involved in the transfer of Ub or Ub-like (Ubl) proteins (e.g., SUMO and NEDD8). Although the majority of E2s are only twice the size of Ub, this remarkable family of enzymes performs a variety of functional roles. In this review, we summarize common functional and structural features that define unifying themes among E2s and highlight emerging concepts in the mechanism and regulation of E2s.
PMCID: PMC4822130  PMID: 27002219
E2; ubiquitin-conjugating enzymes; HECT; RING
3.  Structure and Energetics of Allosteric Regulation of HCN2 Ion Channels by Cyclic Nucleotides* 
The Journal of Biological Chemistry  2015;291(1):371-381.
Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels play an important role in regulating electrical activity in the heart and brain. They are gated by the binding of cyclic nucleotides to a conserved, intracellular cyclic nucleotide-binding domain (CNBD), which is connected to the channel pore by a C-linker region. Binding of cyclic nucleotides increases the rate and extent of channel activation and shifts it to less hyperpolarized voltages. We probed the allosteric mechanism of different cyclic nucleotides on the CNBD and on channel gating. Electrophysiology experiments showed that cAMP, cGMP, and cCMP were effective agonists of the channel and produced similar increases in the extent of channel activation. In contrast, electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) on the isolated CNBD indicated that the induced conformational changes and the degrees of stabilization of the active conformation differed for the three cyclic nucleotides. We explain these results with a model where different allosteric mechanisms in the CNBD all converge to have the same effect on the C-linker and render all three cyclic nucleotides similarly potent activators of the channel.
PMCID: PMC4697172  PMID: 26559974
allosteric regulation; cyclic nucleotide; electron paramagnetic resonance (EPR); electrophysiology; fluorescence anisotropy; ion channel; nuclear magnetic resonance (NMR); double electron-electron resonance (DEER)
4.  Intrinsic disorder drives N-terminal ubiquitination by Ube2w 
Nature chemical biology  2014;11(1):83-89.
Ubiquitination of the αN-terminus of protein substrates has been reported sporadically over the past twenty years. However the identity of an enzyme responsible for this unique ubiquitin (Ub) modification has only recently been elucidated. We show the ubiquitin-conjugating enzyme (E2) Ube2w employs a novel mechanism to facilitate the specific ubiquitination of the α-amino group of its substrates that involves recognition of backbone atoms of intrinsically disordered N-termini. We present the NMR-based solution ensemble of full-length Ube2w that reveals a structural architecture unlike any other E2, in which its C-terminus is partly disordered and flexible to accommodate variable substrate N-termini. Flexibility of the substrate is critical for recognition by Ube2w and point mutations in, or removal of, the flexible C-terminus of Ube2w inhibits substrate binding and modification. Mechanistic insights reported here provide guiding principles for future efforts to define the N-terminal-Ubiquitome in cells.
PMCID: PMC4270946  PMID: 25436519
5.  Biochemical and Structural Characterization of the Ubiquitin-Conjugating Enzyme UBE2W Reveals the Formation of a Noncovalent Homodimer 
Cell biochemistry and biophysics  2013;67(1):103-110.
The biochemical and structural characterization of ubiquitin-conjugating enzymes (E2s) over the past 30 years has fostered important insights into ubiquitin transfer mechanisms. Although many of these enzymes share high sequence and structural conservation, their functional roles in the cell are decidedly diverse. Here, we report that the mono-ubiquitinating E2 UBE2W forms a homodimer using two distinct protein surfaces. Dimerization is primarily driven by residues in the ß-sheet region and Loops 4 and 7 of the catalytic domain. Mutation of two residues in the catalytic domain of UBE2W is capable of disrupting UBE2W homodimer formation, however, we find that dimerization of this E2 is not required for its ubiquitin transfer activity. In addition, residues in the C-terminal region, although not compulsory for the dimerization of UBE2W, play an ancillary role in the dimer interface. In all current E2 structures, the C-terminal helix of the UBC domain is at least 15Å away from the primary dimerization surface shown here for UBE2W. This leads to the proposal that the C-terminal region of UBE2W adopts a noncanonical position that places it closer to the UBC ß-sheet, providing the first indication that at least some E2s adopt C-terminal conformations different from the canonical structures observed to date.
PMCID: PMC3758794  PMID: 23709311
Ubiquitin; Ubiquitin-conjugating enzyme; UBE2W; Dimerization; NMR
6.  Structure of an E3:E2~Ub complex reveals an allosteric mechanism shared among RING/U-box ligases 
Molecular cell  2012;47(6):933-942.
Despite the widespread importance of RING/U-box E3 ubiquitin ligases in ubiquitin (Ub) signaling, the mechanism by which this class of enzymes facilitates Ub transfer remains enigmatic. Here we present a structural model for a RING/U-box E3:E2~Ub complex poised for Ub transfer. The model and additional analyses reveal that E3 binding biases dynamic E2~Ub ensembles toward closed conformations with enhanced reactivity for substrate lysines. We identify a key hydrogen bond between a highly conserved E3 sidechain and an E2 backbone carbonyl, observed in all structures of active RING/U-Box E3/E2 pairs, as the linchpin for allosteric activation of E2~Ub. The conformational biasing mechanism is generalizable across diverse E2s and RING/U-box E3s, but is not shared by HECT-type E3s. The results provide a structural model for a RING/U-box E3:E2~Ub ligase complex and identify the long sought-after source of allostery for RING/U-Box activation of E2~Ub conjugates.
PMCID: PMC3462262  PMID: 22885007
7.  OTUB1 co-opts Lys48-linked ubiquitin recognition to suppress E2 enzyme function 
Molecular Cell  2012;45(3):384-397.
Ubiquitylation entails the concerted action of E1, E2 and E3 enzymes. We recently reported that OTUB1, a deubiquitylase, inhibits the DNA damage response independently of its isopeptidase activity. OTUB1 does so by blocking ubiquitin transfer by UBC13, the cognate E2 enzyme for RNF168. OTUB1 also inhibits E2s of the UBE2D and UBE2E families. Here we elucidate the structural mechanism by which OTUB1 binds E2s to inhibit ubiquitin transfer. OTUB1 recognizes ubiquitin-charged E2s through contacts with both donor ubiquitin and the E2 enzyme. Surprisingly, free ubiquitin associates with the canonical distal ubiquitin-binding site on OTUB1 to promote formation of the inhibited E2 complex. Lys48 of donor ubiquitin lies near the OTUB1 catalytic site and the C-terminus of free ubiquitin, a configuration that mimics the products of Lys48-linked ubiquitin chain cleavage. OTUB1 therefore co-opts Lys48-linked ubiquitin chain recognition to suppress ubiquitin conjugation and the DNA damage response.
PMCID: PMC3306812  PMID: 22325355
8.  The acidic transcription activator Gcn4 binds the Mediator subunit Gal11/Med15 using a simple protein interface forming a fuzzy complex 
Molecular cell  2011;44(6):942-953.
The structural basis for binding of the acidic transcription activator Gcn4 and one activator-binding domain of the Mediator subunit Gal11/Med15 was examined by NMR. Gal11 activator-binding domain 1 has a four-helix fold with a small shallow hydrophobic cleft at its center. In the bound complex, eight residues of Gcn4 adopt a helical conformation allowing three Gcn4 aromatic/aliphatic residues to insert into the Gal11 cleft. The protein-protein interface is dynamic and surprisingly simple, involving only hydrophobic interactions. This allows Gcn4 to bind Gal11 in multiple conformations and orientations, an example of a “fuzzy complex” where the Gcn4-Gal11 interface cannot be described by a single conformation. Gcn4 uses a similar mechanism to bind two other unrelated activator-binding domains. Functional studies in yeast show the importance of residues at the protein interface, define the minimal requirements for a functional activator, and suggest a mechanism by which activators bind to multiple unrelated targets.
PMCID: PMC3246216  PMID: 22195967
transcription; mediator complex; Gcn4; Gal11/Med15; acidic activator; activation domain; intrinsically disordered; NMR
9.  UbcH7 reactivity profile reveals Parkin and HHARI to be RING/HECT hybrids 
Nature  2011;474(7349):105-108.
Although the functional interaction between ubiquitin conjugating enzymes (E2s) and ubiquitin ligases (E3s) is essential in ubiquitin (Ub) signaling, the criteria that define an active E2–E3 pair are not well-established. The human E2 UbcH7 (Ube2L3) shows broad specificity for HECT-type E3s1, but often fails to function with RING E3s in vitro despite forming specific complexes2–4. Structural comparisons of inactive UbcH7/RING complexes with active UbcH5/RING complexes reveal no defining differences3,4, highlighting a gap in our understanding of Ub transfer. We show that, unlike many E2s that transfer Ub with RINGs, UbcH7 lacks intrinsic, E3-independent reactivity with lysine, explaining its preference for HECTs. Despite lacking lysine reactivity, UbcH7 exhibits activity with the RING-In Between-RING (RBR) family of E3s that includes Parkin and human homologue of ariadne (HHARI)5,6. Found in all eukaryotes7, RBRs regulate processes such as translation8 and immune signaling9. RBRs contain a canonical C3HC4-type RING, followed by two conserved Cys/His-rich Zn2+-binding domains, In-Between-RING (IBR) and RING2 domains, which together define this E3 family7. Here we show that RBRs function like RING/HECT hybrids: they bind E2s via a RING domain, but transfer Ub through an obligate thioester-linked Ub (denoted ‘~Ub’), requiring a conserved cysteine residue in RING2. Our results define the functional cadre of E3s for UbcH7, an E2 involved in cell proliferation10 and immune function11, and suggest a novel mechanism for an entire class of E3s.
PMCID: PMC3444301  PMID: 21532592
10.  Ubiquitin in Motion: Structural studies of the E2~Ub conjugate 
Biochemistry  2011;50(10):1624-1633.
Ubiquitination of proteins provides a powerful and versatile post-translational signal in the eukaryotic cell. The formation of a thioester bond between ubiquitin (Ub) and the active site of a ubiquitin-conjugating enzyme (E2) is critical for Ub transfer to substrates. Assembly of a functional ubiquitin ligase (E3) complex poised for Ub transfer involves recognition and binding of an E2~Ub conjugate. Therefore, full characterization of the structure and dynamics of E2~Ub conjugates is required for further mechanistic understanding of Ub transfer reactions. Here we present characterization of the dynamic behavior of E2~Ub conjugates of two human enzymes, UbcH5c~Ub and Ubc13~Ub, in solution as determined by NMR and SAXS. Within each conjugate, Ub retains great flexibility with respect to the E2, indicative of highly dynamic species that adopt manifold orientations. The population distribution of Ub conformations is dictated by the identity of the E2: UbcH5c~Ub populates an array of extended conformations and the population of Ubc13~Ub conjugates favors a closed conformation in which the hydrophobic surface of Ub faces Helix 2 of Ubc13. We propose that the varied conformations adopted by Ub represent available binding modes of the E2~Ub species and thus provide insight into the diverse E2~Ub protein interactome, particularly regarding interaction with Ub ligases.
PMCID: PMC3056393  PMID: 21226485
ubiquitin; ubiquitin conjugating enzyme; ubiquitination; UbcH5; Ubc13; NMR; spin label; SAXS
11.  Structural and functional characterization of the monomeric U-box domain from E4B† 
Biochemistry  2010;49(2):347-355.
Substantial evidence has accumulated indicating a significant role for oligomerization in the function of E3 ubiquitin ligases. Among the many characterized E3 ligases, the yeast U-box protein Ufd2 and its mammalian homolog E4B appear to be unique in functioning as monomers. An E4B U-box domain construct (E4BU) has been sub-cloned, over-expressed in E. Coli and purified, which enabled determination of a high resolution NMR solution structure and detailed biophysical analysis. E4BU is a stable monomeric protein that folds into the same structure observed for other structurally characterized U-box domains, all of which are homodimers. Multiple sequence alignment combined with comparative structural analysis reveals substitutions in the sequence that inhibit dimerization. The interaction between E4BU and the E2 conjugating enzyme UbcH5c has been mapped using NMR and this data has been used to generate a structural model for the complex. The E2 binding site is found to be similar to that observed for dimeric U-box and RING domain E3 ligases. Despite the inability to dimerize, E4BU was found to be active in a standard autoubiquitination assay. The structure of E4BU and its ability to function as a monomer are discussed in light of the ubiquitous observation of U-box and RING domain oligomerization.
PMCID: PMC2806929  PMID: 20017557

Results 1-11 (11)