SUMMARYHistones are ubiquitinated in response to DNA double strand breaks (DSB), promoting recruitment of repair proteins to chromatin1. UBC13 (UBE2N) is an ubiquitin conjugating enzyme (E2) that heterodimerizes with UEV1a2 and synthesizes K63–linked polyubiquitin (K63Ub) chains at DSB sites in concert with the ubiquitin ligase (E3), RNF1683. K63Ub synthesis is regulated in a noncanonical manner by the deubiquitinating enzyme, OTUB1 (OTU domain-containing ubiquitin aldehyde-binding protein 1), which binds preferentially to the UBC13~Ub thiolester4. Residues N-terminal to the OTU domain, which had been implicated in ubiquitin binding5, are required for binding to UBC13~Ub and inhibition of K63Ub synthesis5. Here we describe structural and biochemical studies elucidating how OTUB1 inhibits UBC13 and other E2 enzymes. We unexpectedly find that OTUB1 binding to UBC13~Ub is allosterically regulated by free ubiquitin, which binds to a second site in OTUB1 and increases its affinity for UBC13~Ub, while at the same time disrupting interactions with UEV1a in a manner that depends upon the OTUB1 N-terminus. Crystal structures of an OTUB1-UBC13 complex and of OTUB1 bound to ubiquitin aldehyde and a chemical UBC13~Ub conjugate show that binding of free ubiquitin to OTUB1 triggers conformational changes in the OTU domain and formation of a ubiquitin-binding helix in the N-terminus, thus promoting binding of the conjugated donor ubiquitin in UBC13~Ub to OTUB1. The donor ubiquitin thus cannot interact with the E2 enzyme, which has been shown to be important for ubiquitin transfer6,7. The N-terminal helix of OTUB1 is positioned to interfere with UEV1a binding to UBC13, as well as with attack on the thiolester by an acceptor ubiquitin, thereby inhibiting K63Ub synthesis. OTUB1 binding also occludes the RING E3 binding site on UBC13, thus providing a further component of inhibition. The general features of the inhibition mechanism explain how OTUB1 inhibits other E2 enzymes4 in a non-catalytic manner.

The Kv7 subfamily of voltage-dependent potassium channels, distinct from other subfamilies by dint of its large intracellular COOH terminus, acts to regulate excitability in cardiac and neuronal tissues. KCNQ1 (Kv7.1), the founding subfamily member, encodes a channel subunit directly implicated in genetic disorders, such as the long QT syndrome, a cardiac pathology responsible for arrhythmias. We have used a recombinant protein preparation of the COOH terminus to probe the structure and function of this domain and its individual modules. The COOHterminal proximal half associates with one calmodulin constitutively bound to each subunit where calmodulin is critical for proper folding of the whole intracellular domain. The distal half directs tetramerization, employing tandem coiled-coils. The firstcoiled-coilcomplexisdimericandundergoesconcentrationdependent self-association to form a dimer of dimers. The outer coiled-coil is parallel tetrameric, the details of which have been elucidated based on 2.0 Å crystallographic data. Both coiledcoils act in a coordinate fashion to mediate the formation and stabilization of the tetrameric distal half. Functional studies, including characterization of structure-based and long QT mutants, prove the requirement for both modules and point to complex roles for these modules, including folding, assembly, trafficking, and regulation.

OTUB1 is a Lys48-specific deubiquitinating enzyme that forms a complex in vivo with E2 ubiquitin conjugating enzymes including UBC13 and UBCH5. OTUB1 binds to E2~Ub thioester intermediates and prevent ubiquitin transfer, thereby non-catalytically inhibiting accumulation of polyubiquitin. We report here that a second role of OTUB1-E2 interactions is to stimulate OTUB1 cleavage of Lys48 polyubiquitin, and that this stimulation is regulated by the ratio of charged to uncharged E2 and by the concentration of Lys48-linked polyubiquitin and free ubiquitin. Structural and biochemical studies of human and worm OTUB1 and UBCH5B show that the E2 stimulates binding of the Lys48 polyubiquitin substrate by stabilizing folding of the OTUB1 N-terminal ubiquitin-binding helix. Our results suggest that OTUB1-E2 complexes in the cell are poised to regulate polyubiquitin chain elongation or degradation in response to changing levels of E2 charging and available free ubiquitin.

Abstract-The slow I KS K ϩ channel plays a major role in repolarizing the cardiac action potential and consists of the assembly of KCNQ1 and KCNE1 subunits. Mutations in either KCNQ1 or KCNE1 genes produce the long-QT syndrome, a life-threatening ventricular arrhythmia. Here, we show that long-QT mutations located in the KCNQ1 C terminus impair calmodulin (CaM) binding, which affects both channel gating and assembly. The mutations produce a voltage-dependent macroscopic inactivation and dramatically alter channel assembly. KCNE1 forms a ternary complex with wild-type KCNQ1 and Ca 2ϩ -CaM that prevents inactivation, facilitates channel assembly, and mediates a Ca 2ϩ -sensitive increase of I KS-current, with a considerable Ca 2ϩ -dependent left-shift of the voltage-dependence of activation. Coexpression of KCNQ1 or I KS channels with a Ca 2ϩ -insensitive CaM mutant markedly suppresses the currents and produces a right shift in the voltage-dependence of channel activation. KCNE1 association to KCNQ1 long-QT mutants significantly improves mutant channel expression and prevents macroscopic inactivation. However, the marked right shift in channel activation and the subsequent decrease in current amplitude cannot restore normal levels of I KS channel activity. Our data indicate that in healthy individuals, CaM binding to KCNQ1 is essential for correct channel folding and assembly and for conferring Ca 2ϩ -sensitive I KS -current stimulation, which increases the cardiac repolarization reserve and hence prevents the risk of ventricular arrhythmias. Key Words: KCNQ Ⅲ potassium channels Ⅲ Kv7 Ⅲ calmodulin Ⅲ KCNE Ⅲ long QT K CNQ channels represent a family of voltage-gated K ϩ channels (Kv7) that plays a major role in brain and cardiac excitability. 1,2 Mutations of human KCNQ genes lead to severe cardiovascular and neurological disorders such as the cardiac long-QT syndrome (LQT) and neonatal epilepsy. Coassembly of KCNQ1 with KCNE1 ␤ subunits produces the I KS -current that is crucial for repolarization of the cardiac action potential. [3][4][5] The cytoplasmic KCNQ C-termini were shown to feature 4 ␣ helices. 6 We previously identified the last ␣ helix of the C terminus (helix D, aa.589 -620) as a region important for the tetrameric assembly of KCNQ1 ␣ subunits. 7 This region also binds Yotiao, an A-kinase-anchoring protein that targets PKA on the I KS channel complex. 8 The first 2␣ helices of KCNQ1-5 form a calmodulin-binding domain (CBD), including an IQ motif that mediates Ca 2ϩ -free calmodulin (apoCaM) binding. 6,9 Although KCNQ channels bind calmodulin (CaM), the role of CaM in channel function remains controversial. Recent studies found a role for CaM as a Ca 2ϩ -sensor of KCNQ2/4/5 channels, 10,11 whereas others suggested a role in channel assembly. 9 So far, no information has been available about the interaction of calmodulin with cardiac I KS channels and its pathophysiological impact to KCNQ1-related LQT channnelopathies. Here, we show that LQT mutations located near the IQ motif of KCNQ1 C terminus impair ...

Modification of proteins by ubiquitin or ubiquitin-like proteins (UBLs) is a critical cellular process implicated in a variety of cellular states and outcomes. A prerequisite for target protein modification by a UBL is the activation of the latter by activating enzymes (E1s). Here, we present the crystal structure of the non-canonical homodimeric E1, UBA5, in complex with its cognate UBL, UFM1, and supporting biochemical experiments. We find that UBA5 binds to UFM1 via a trans-binding mechanism in which UFM1 interacts with distinct sites in both subunits of the UBA5 dimer. This binding mechanism requires a region C-terminal to the adenylation domain that brings UFM1 to the active site of the adjacent UBA5 subunit. We also find that transfer of UFM1 from UBA5 to the E2, UFC1, occurs via a trans mechanism, thereby requiring a homodimer of UBA5. These findings explicitly elucidate the role of UBA5 dimerization in UFM1 activation.

It is widely accepted that ubiquitin conjugating enzymes (E2) contain an active site asparagine that serves as an oxyanion hole, thereby stabilizing a negatively charged transition state intermediate and promoting ubiquitin transfer. Using structural and biochemical approaches to study the role of the conserved asparagine to ubiquitin conjugation by Ubc13/Mms2, we conclude that the importance of this residue stems primarily from its structural role in stabilizing an active site loop.

Voltage‐gated K+ channels co‐assemble with auxiliary β subunits to form macromolecular complexes. In heart, assembly of Kv7.1 pore‐forming subunits with KCNE1 β subunits generates the repolarizing K+ current IKS. However, the detailed nature of their interface remains unknown. Mutations in either Kv7.1 or KCNE1 produce the life‐threatening long or short QT syndromes. Here, we studied the interactions and voltage‐dependent motions of IKS channel intracellular domains, using fluorescence resonance energy transfer combined with voltage‐clamp recording and in vitro binding of purified proteins. The results indicate that the KCNE1 distal C‐terminus interacts with the coiled‐coil helix C of the Kv7.1 tetramerization domain. This association is important for IKS channel assembly rules as underscored by Kv7.1 current inhibition produced by a dominant‐negative C‐terminal domain. On channel opening, the C‐termini of Kv7.1 and KCNE1 come close together. Co‐expression of Kv7.1 with the KCNE1 long QT mutant D76N abolished the K+ currents and gated motions. Thus, during channel gating KCNE1 is not static. Instead, the C‐termini of both subunits experience molecular motions, which are disrupted by the D76N causing disease mutation.

Ubiquitylation controls protein function and degradation. Therefore, ubiquitin ligases need to be tightly controlled. We discovered an evolutionarily conserved allosteric restraint mechanism for Nedd4 ligases and demonstrated its function with diverse substrates: the yeast soluble proteins Rpn10 and Rvs167, and the human receptor tyrosine kinase FGFR1 and cardiac I potassium channel. We found that a potential trimerization interface is structurally blocked by the HECT domain α1-helix, which further undergoes ubiquitylation on a conserved lysine residue. Genetic, bioinformatics, biochemical and biophysical data show that attraction between this α1-conjugated ubiquitin and the HECT ubiquitin-binding patch pulls the α1-helix out of the interface, thereby promoting trimerization. Strikingly, trimerization renders the ligase inactive. Arginine substitution of the ubiquitylated lysine impairs this inactivation mechanism and results in unrestrained FGFR1 ubiquitylation in cells. Similarly, electrophysiological data and TIRF microscopy show that NEDD4 unrestrained mutant constitutively downregulates the I channel, thus confirming the functional importance of E3-ligase autoinhibition.