Willem J. van Dijk

Nicotine and Carbamylcholine Binding to Nicotinic Acetylcholine Receptors as Studied in Achbp Crystal Structures

Nicotinic acetylcholine receptors are prototypes for the pharmaceutically important family of pentameric ligand-gated ion channels. Here we present atomic resolution structures of nicotine and carbamylcholine binding to AChBP, a water-soluble homolog of the ligand binding domain of nicotinic receptors and their family members, GABAA, GABAC, 5HT3 serotonin, and glycine receptors. Ligand binding is driven by enthalpy and is accompanied by conformational changes in the ligand binding site. Residues in the binding site contract around the ligand, with the largest movement in the C loop. As expected, the binding is characterized by substantial aromatic and hydrophobic contributions, but additionally there are close contacts between protein oxygens and positively charged groups in the ligands. The higher affinity of nicotine is due to a main chain hydrogen bond with the B loop and a closer packing of the aromatic groups. These structures will be useful tools for the development of new drugs involving nicotinic acetylcholine receptor-associated diseases.

SUMO modification of the ubiquitin-conjugating enzyme E2-25K

Post-translational modification with small ubiquitin-related modifier (SUMO) alters the function of many proteins, but the molecular mechanisms and consequences of this modification are still poorly defined. During a screen for novel SUMO1 targets, we identified the ubiquitin-conjugating enzyme E2-25K (Hip2). SUMO attachment severely impairs E2-25K ubiquitin thioester and unanchored ubiquitin chain formation in vitro. Crystal structures of E2-25K(1–155) and of the E2-25K(1–155)–SUMO conjugate (E2-25K*SUMO) indicate that SUMO attachment interferes with E1 interaction through its location on the N-terminal helix. The SUMO acceptor site in E2-25K, Lys14, does not conform to the consensus site found in most SUMO targets (ΨKXE), and functions only in the context of an α-helix. In contrast, adjacent SUMO consensus sites are modified only when in unstructured peptides. The demonstration that secondary structure elements are part of SUMO attachment signals could contribute to a better prediction of SUMO targets.

Ubc9 Sumoylation Regulates Sumo Target Discrimination.

Posttranslational modification with small ubiquitin-related modifier, SUMO, is a widespread mechanism for rapid and reversible changes in protein function. Considering the large number of known targets, the number of enzymes involved in modification seems surprisingly low: a single E1, a single E2, and a few distinct E3 ligases. Here we show that autosumoylation of the mammalian E2-conjugating enzyme Ubc9 at Lys14 regulates target discrimination. While not altering its activity toward HDAC4, E2-25K, PML, or TDG, sumoylation of Ubc9 impairs its activity on RanGAP1 and strongly activates sumoylation of the transcriptional regulator Sp100. Enhancement depends on a SUMO-interacting motif (SIM) in Sp100 that creates an additional interface with the SUMO conjugated to the E2, a mechanism distinct from Ubc9 approximately SUMO thioester recruitment. The crystal structure of sumoylated Ubc9 demonstrates how the newly created binding interface can provide a gain in affinity otherwise provided by E3 ligases.

Noncovalent interaction between Ubc9 and SUMO promotes SUMO chain formation

The ubiquitin‐related modifier SUMO regulates a wide range of cellular processes by post‐translational modification with one, or a chain of SUMO molecules. Sumoylation is achieved by the sequential action of several enzymes in which the E2, Ubc9, transfers SUMO from the E1 to the target mostly with the help of an E3 enzyme. In this process, Ubc9 not only forms a thioester bond with SUMO, but also interacts with SUMO noncovalently. Here, we show that this noncovalent interaction promotes the formation of short SUMO chains on targets such as Sp100 and HDAC4. We present a crystal structure of the noncovalent Ubc9–SUMO1 complex, showing that SUMO is located far from the E2 active site and resembles the noncovalent interaction site for ubiquitin on UbcH5c and Mms2. Structural comparison suggests a model for poly‐sumoylation involving a mechanism analogous to Mms2‐Ubc13‐mediated ubiquitin chain formation.

The E3 ligase HOIP specifies linear ubiquitin chain assembly through its RING‐IBR‐RING domain and the unique LDD extension

Activation of the NF‐κB pathway requires the formation of Met1‐linked ‘linear’ ubiquitin chains on NEMO, which is catalysed by the Linear Ubiquitin Chain Assembly Complex (LUBAC) E3 consisting of HOIP, HOIL‐1L and Sharpin. Here, we show that both LUBAC catalytic activity and LUBAC specificity for linear ubiquitin chain formation are embedded within the RING‐IBR‐RING (RBR) ubiquitin ligase subunit HOIP. Linear ubiquitin chain formation by HOIP proceeds via a two‐step mechanism involving both RING and HECT E3‐type activities. RING1‐IBR catalyses the transfer of ubiquitin from the E2 onto RING2, to transiently form a HECT‐like covalent thioester intermediate. Next, the ubiquitin is transferred from HOIP onto the N‐terminus of a target ubiquitin. This transfer is facilitated by a unique region in the C‐terminus of HOIP that we termed ‘Linear ubiquitin chain Determining Domain’ (LDD), which may coordinate the acceptor ubiquitin. Consistent with this mechanism, the RING2‐LDD region was found to be important for NF‐κB activation in cellular assays. These data show how HOIP combines a general RBR ubiquitin ligase mechanism with unique, LDD‐dependent specificity for producing linear ubiquitin chains.

Mechanism of UCH-L5 Activation and Inhibition by DEUBAD Domains in RPN13 and INO80G

Summary Deubiquitinating enzymes (DUBs) control vital processes in eukaryotes by hydrolyzing ubiquitin adducts. Their activities are tightly regulated, but the mechanisms remain elusive. In particular, the DUB UCH-L5 can be either activated or inhibited by conserved regulatory proteins RPN13 and INO80G, respectively. Here we show how the DEUBAD domain in RPN13 activates UCH-L5 by positioning its C-terminal ULD domain and crossover loop to promote substrate binding and catalysis. The related DEUBAD domain in INO80G inhibits UCH-L5 by exploiting similar structural elements in UCH-L5 to promote a radically different conformation, and employs molecular mimicry to block ubiquitin docking. In this process, large conformational changes create small but highly specific interfaces that mediate activity modulation of UCH-L5 by altering the affinity for substrates. Our results establish how related domains can exploit enzyme conformational plasticity to allosterically regulate DUB activity. These allosteric sites may present novel insights for pharmaceutical intervention in DUB activity.

The nucleosome acidic patch plays a critical role in RNF168-dependent ubiquitination of histone H2A

During DNA damage response, the RING E3 ligase RNF168 ubiquitinates nucleosomal H2A at K13–15. Here we show that the ubiquitination reaction is regulated by its substrate. We define a region on the RING domain important for target recognition and identify the H2A/H2B dimer as the minimal substrate to confer lysine specificity to the RNF168 reaction. Importantly, we find an active role for the substrate in the reaction. H2A/H2B dimers and nucleosomes enhance the E3-mediated discharge of ubiquitin from the E2 and redirect the reaction towards the relevant target, in a process that depends on an intact acidic patch. This active contribution of a region distal from the target lysine provides regulation of the specific K13–15 ubiquitination reaction during the complex signalling process at DNA damage sites.

Enabling high-throughput ligation-independent cloning and protein expression for the family of ubiquitin specific proteases

High-throughput methods to produce a large number of soluble recombinant protein variants are particularly important in the process of determining the three-dimensional structure of proteins and their complexes. Here, we describe a collection of protein expression vectors for ligation-independent cloning, which allow co-expression strategies by implementing different affinity tags and antibiotic resistances. Since the same PCR product can be inserted in all but one of the vectors, this allows efficiency in versatility while screening for optimal expression strategies. We first demonstrate the use of these vectors for protein expression in Escherichia coli, on a set of proteins belonging to the ubiquitin specific protease (USP) Family. We have selected 35 USPs, created 145 different expression constructs into the pETNKI-His-3C-LIC-kan vector, and obtained 38 soluble recombinant proteins for 21 different USPs. Finally, we exemplify the use of our vectors for bacterial co-expression and for expression in insect cells, with USP4 and USP7 respectively. We conclude that our ligation-independent cloning strategy allows for high-throughput screening for the expression of soluble proteins in a variety of vectors in E. coli and in insect cells. In addition, the same vectors can be used for co-expression studies, at least for simple binary complexes. Application in the family of ubiquitin specific proteases led to a number of soluble USPs that are used for functional and crystallization studies.

BAP1/ASXL1 recruitment and activation for H2A deubiquitination

The deubiquitinating enzyme BAP1 is an important tumor suppressor that has drawn attention in the clinic since its loss leads to a variety of cancers. BAP1 is activated by ASXL1 to deubiquitinate mono-ubiquitinated H2A at K119 in Polycomb gene repression, but the mechanism of this reaction remains poorly defined. Here we show that the BAP1 C-terminal extension is important for H2A deubiquitination by auto-recruiting BAP1 to nucleosomes in a process that does not require the nucleosome acidic patch. This initial encounter-like complex is unproductive and needs to be activated by the DEUBAD domains of ASXL1, ASXL2 or ASXL3 to increase BAP1s affinity for ubiquitin on H2A, to drive the deubiquitination reaction. The reaction is specific for Polycomb modifications of H2A as the complex cannot deubiquitinate the DNA damage-dependent ubiquitination at H2A K13/15. Our results contribute to the molecular understanding of this important tumor suppressor.

Ubiquitin-specific protease 4 is inhibited by its ubiquitin-like domain

USP4 is a member of the ubiquitin‐specific protease (USP) family of deubiquitinating enzymes that has a role in spliceosome regulation. Here, we show that the crystal structure of the minimal catalytic domain of USP4 has the conserved USP‐like fold with its typical ubiquitin‐binding site. A ubiquitin‐like (Ubl) domain inserted into the catalytic domain has autoregulatory function. This Ubl domain can bind to the catalytic domain and compete with the ubiquitin substrate, partially inhibiting USP4 activity against different substrates. Interestingly, other USPs, such as USP39, could relieve this inhibition.