Kylie J. Walters

Ubiquitin-binding domains — from structures to functions

Ubiquitin-binding domains (UBDs) are modular elements that bind non-covalently to the protein modifier ubiquitin. Recent atomic-level resolution structures of ubiquitin–UBD complexes have revealed some of the mechanisms that underlie the versatile functions of ubiquitin in vivo. The preferences of UBDs for ubiquitin chains of specific length and linkage are central to these functions. These preferences originate from multimeric interactions, whereby UBDs synergistically bind multiple ubiquitin molecules, and from contacts with regions that link ubiquitin molecules into a polymer. The sequence context of UBDs and the conformational changes that follow their binding to ubiquitin also contribute to ubiquitin signalling. These new structure-based insights provide strategies for controlling cellular processes by targeting ubiquitin–UBD interfaces.

Proteasome subunit Rpn13 is a novel ubiquitin receptor

Proteasomal receptors that recognize ubiquitin chains attached to substrates are key mediators of selective protein degradation in eukaryotes. Here we report the identification of a new ubiquitin receptor, Rpn13/ARM1, a known component of the proteasome. Rpn13 binds ubiquitin through a conserved amino-terminal region termed the pleckstrin-like receptor for ubiquitin (Pru) domain, which binds K48-linked diubiquitin with an affinity of approximately 90 nM. Like proteasomal ubiquitin receptor Rpn10/S5a, Rpn13 also binds ubiquitin-like (UBL) domains of UBL-ubiquitin-associated (UBA) proteins. In yeast, a synthetic phenotype results when specific mutations of the ubiquitin binding sites of Rpn10 and Rpn13 are combined, indicating functional linkage between these ubiquitin receptors. Because Rpn13 is also the proteasomal receptor for Uch37, a deubiquitinating enzyme, our findings suggest a coupling of chain recognition and disassembly at the proteasome.

Ubiquitin docking at the proteasome through a novel pleckstrin-homology domain interaction

Targeted protein degradation is largely performed by the ubiquitin–proteasome pathway, in which substrate proteins are marked by covalently attached ubiquitin chains that mediate recognition by the proteasome. It is currently unclear how the proteasome recognizes its substrates, as the only established ubiquitin receptor intrinsic to the proteasome is Rpn10/S5a (ref. 1), which is not essential for ubiquitin-mediated protein degradation in budding yeast. In the accompanying manuscript we report that Rpn13 (refs 3–7), a component of the nine-subunit proteasome base, functions as a ubiquitin receptor, complementing its known role in docking de-ubiquitinating enzyme Uch37/UCHL5 (refs 4–6) to the proteasome. Here we merge crystallography and NMR data to describe the ubiquitin-binding mechanism of Rpn13. We determine the structure of Rpn13 alone and complexed with ubiquitin. The co-complex reveals a novel ubiquitin-binding mode in which loops rather than secondary structural elements are used to capture ubiquitin. Further support for the role of Rpn13 as a proteasomal ubiquitin receptor is demonstrated by its ability to bind ubiquitin and proteasome subunit Rpn2/S1 simultaneously. Finally, we provide a model structure of Rpn13 complexed to diubiquitin, which provides insights into how Rpn13 as a ubiquitin receptor is coupled to substrate deubiquitination by Uch37.

DNA-repair protein hHR23a alters its protein structure upon binding proteasomal subunit S5a

The Rad23 family of proteins, including the human homologs hHR23a and hHR23b, stimulates nucleotide excision repair and has been shown to provide a novel link between proteasome-mediated protein degradation and DNA repair. In this work, we illustrate how the proteasomal subunit S5a regulates hHR23a protein structure. By using NMR spectroscopy, we have elucidated the structure and dynamic properties of the 40-kDa hHR23a protein and show it to contain four structured domains connected by flexible linker regions. In addition, we reveal that these domains interact in an intramolecular fashion, and by using residual dipolar coupling data in combination with chemical shift perturbation analysis, we present the hHR23a structure. By itself, hHR23a adopts a closed conformation defined by the interaction of an N-terminal ubiquitin-like domain with two ubiquitin-associated domains. Interestingly, binding of the proteasomal subunit S5a disrupts the hHR23a interdomain interactions and thereby causes it to adopt an opened conformation.

Arylamine N-acetyltransferases: From Structure to Function

Arylamine N-acetyltransferases (NATs) are cytosolic conjugating enzymes which transfer an acetyl group from acetylCoenzyme A to a xenobiotic acceptor substrate. The enzyme has an active site cysteine as part of a catalytic triad with histidine and aspartate. NATs have had an important role in pharmacogenetics. Polymorphism in acetylation (and inactivation) of the anti-tubercular agent isoniazid resides in human NAT2, one of two polymorphic human NATs. In humans there is also a third pseudogene and in rodents there are three isozymes. Comparison of human and rodent NAT enzymes and their genes is aiding our understanding of the roles of the individual isoenzymes. This may have clinical importance since human NAT1 is overexpressed in a sub-population of breast cancers and control of expression of the NAT genes is ripe for investigation. The mammalian NAT enzymes are involved in metabolism of drugs and carcinogens but there is growing evidence, including from transgenic mice, that human NAT1 has an endogenous role in folate degradation. Structural studies and intracellular tracking of polymorphic NAT variants, is contributing to appreciation of how individual mutations result in loss of NAT activity. Genome analyses have identified NAT homologues in bacteria including Mycobacterium tuberculosis, in which the NAT enzyme metabolises inactivation of isoniazid. More intriguingly, deletion of the nat gene in mycobacteria, leads to deficits in cell wall synthesis. Structural comparisons of NATs from prokaryotes and eukaryotes, particularly in relation to CoA binding, provide a platform for understanding how the unique NAT protein fold may lend itself to a wide range of functions.

Gates, Channels, and Switches: Elements of the Proteasome Machine

The proteasome has emerged as an intricate machine that has dynamic mechanisms to regulate the timing of its activity, its selection of substrates, and its processivity. The 19-subunit regulatory particle (RP) recognizes ubiquitinated proteins, removes ubiquitin, and injects the target protein into the proteolytic chamber of the core particle (CP) via a narrow channel. Translocation into the CP requires substrate unfolding, which is achieved through mechanical force applied by a hexameric ATPase ring of the RP. Recent cryoelectron microscopy (cryoEM) studies have defined distinct conformational states of the RP, providing illustrative snapshots of what appear to be progressive steps of substrate engagement. Here, we bring together this new information with molecular analyses to describe the principles of proteasome activity and regulation.

Structure of Proteasome Ubiquitin Receptor hRpn13 and Its Activation by the Scaffolding Protein hRpn2

Rpn13 is a subunit of the proteasome that serves as a receptor for both ubiquitin and Uch37, one of the proteasomes three deubiquitinating enzymes. We have determined the structure of full-length human Rpn13 (hRpn13). Unexpectedly, Rpn13s ubiquitin- and Uch37-binding domains pack against each other when it is not incorporated into the proteasome. This intramolecular interaction reduces hRpn13s affinity for ubiquitin. We find that hRpn13 binding to the proteasome scaffolding protein hRpn2/S1 abrogates its interdomain interactions, thus activating hRpn13 for ubiquitin binding. hRpn13s Uch37-binding domain, a previously unknown fold, contains nine alpha helices. We have mapped its Uch37-binding surface to a region rich in charged amino acids. Altogether, our results provide mechanistic insights into hRpn13s functional activities with Uch37 and ubiquitin and suggest that its role as a ubiquitin receptor is finely tuned for proteasome targeting.

Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome.

The yin and yang of proteasomal regulation The ubiquitin-proteasome pathway regulates myriad proteins through their selective proteolysis. The small protein ubiquitin is attached, typically in many copies, to the target protein, which is then recognized and broken down by the proteasome. Shi et al. found a repeat structure in the proteasome for recognizing ubiquitin as well as ubiquitin-like (UBL) proteins. Tandem binding sites allow the proteasome to dock multiple proteins. One of the bound UBL proteins is an enzyme that cleaves ubiquitin-protein conjugates, which antagonizes degradation. Thus, the repetition of related binding sites with distinct specificity achieves a balance of positive and negative regulation of the proteasome. Science, this issue p. 10.1126/science.aad9421 Tandem ligand-binding sites in the proteasome subunit Rpn1 modulate proteasome activity both positively and negatively. INTRODUCTION The ubiquitin-proteasome system comprises hundreds of distinct pathways of degradation, which converge at the step of ubiquitin recognition by the proteasome. Five proteasomal ubiquitin receptors have been identified, two that are intrinsic to the proteasome (Rpn10 and Rpn13) and three reversibly associated proteasomal ubiquitin receptors (Rad23, Dsk2, and Ddi1). RATIONALE We found that the five known proteasomal ubiquitin receptors of yeast are collectively nonessential for ubiquitin recognition by the proteasome. We therefore screened for additional ubiquitin receptors in the proteasome and identified subunit Rpn1 as a candidate. We used nuclear magnetic resonance (NMR) spectroscopy to characterize the structure of the binding site within Rpn1, which we term the T1 site. Mutational analysis of this site showed its functional importance within the context of intact proteasomes. T1 binds both ubiquitin and ubiquitin-like (UBL) proteins, in particular the substrate-delivering shuttle factor Rad23. A second site within the Rpn1 toroid, T2, recognizes the UBL domain of deubiquitinating enzyme Ubp6, as determined by hydrogen-deuterium exchange mass spectrometry analysis and validated by amino acid substitution and functional assays. The Rpn1 toroid thus serves a critical scaffolding role within the proteasome, helping to assemble multiple proteasome cofactors, as well as substrates. RESULTS Our results indicate that proteasome subunit Rpn1 can recognize both ubiquitin and UBL domains of substrate shuttling factors that themselves bind ubiquitin and function as reversibly associated proteasomal ubiquitin receptors. Recognition is mediated by the T1 site within the Rpn1 toroid, which supports proteasome function in vivo. We found that the capacity of T1 to recognize both ubiquitin and UBL shuttling proteins was shared with Rpn10 and Rpn13. The surprising multiplicity of ubiquitin-recognition domains within the proteasome may promote enhanced, multipoint binding of ubiquitin chains. The structures of the T1 site in its free state and in complex with monoubiquitin or lysine 48 (K48)–linked diubiquitin were solved, which revealed that three neighboring outer helices from the T1 toroid engage two ubiquitins. This ubiquitin-binding domain is structurally distinct from those of Rpn10 and Rpn13, despite their common ligands. Moreover, the Rpn1-binding mode leads to a preference for certain ubiquitin chain types, especially K6- and K48-linked chains, in a distinct configuration that can position substrates close to the entry port of the proteasome. The fate of proteasome-docked ubiquitin conjugates is determined by a competition between substrate degradation and deubiquitination; the latter leads to premature release of substrates. Proximal to the T1 site within the Rpn1 toroid is a second UBL-binding site, T2, that does not assist in ubiquitin chain recognition but, rather, in chain disassembly, by binding to the UBL domain of deubiquitinating enzyme Ubp6. Note that the UBL interactors at T1 and T2 are distinct and assign substrate localization to T1 and substrate deubiquitination to T2. CONCLUSION A ligand-binding hotspot was identified in the Rpn1 toroid, consisting of two adjacent receptor sites, referred to as T1 and T2. The Rpn1 toroid represents a distinct class of binding domains for ubiquitin and UBL proteins. The T1 site functions to recruit substrates directly by binding to ubiquitin itself and indirectly by binding to UBL shuttling factors, a feature shared by Rpn10 and Rpn13 despite a lack of structural similarity among these receptors. The T2 site also binds to a UBL domain protein, in this case deubiquitinating enzyme Ubp6. This study thus defines a two-site recognition domain intrinsic to the proteasome that uses distinct ubiquitin-fold ligands to assemble substrates, substrate shuttling factors, and a deubiquitinating enzyme in close proximity. A ligand-binding hotspot in the proteasome for assembling substrates and cofactors. Schematic (top) and model structure (bottom, left) mapping the UBL-binding Rpn1 T1 (indigo) and T2 (orange) sites. (Bottom, right) Enlarged region of the proteasome designed to illustrate Rpn1 T1 and T2 sites bound to a ubiquitinated (yellow) substrate (beige) and deubiquitinating enzyme Ubp6 (green), respectively. Aided by PDB 4CR2, 1WGG, 1VJV, and 2B9R. Hundreds of pathways for degradation converge at ubiquitin recognition by a proteasome. Here, we found that the five known proteasomal ubiquitin receptors in yeast are collectively nonessential for ubiquitin recognition and identified a sixth receptor, Rpn1. A site (T1) in the Rpn1 toroid recognized ubiquitin and ubiquitin-like (UBL) domains of substrate shuttling factors. T1 structures with monoubiquitin or lysine 48 diubiquitin show three neighboring outer helices engaging two ubiquitins. T1 contributes a distinct substrate-binding pathway with preference for lysine 48–linked chains. Proximal to T1 within the Rpn1 toroid is a second UBL-binding site (T2) that assists in ubiquitin chain disassembly, by binding the UBL of deubiquitinating enzyme Ubp6. Thus, a two-site recognition domain intrinsic to the proteasome uses distinct ubiquitin-fold ligands to assemble substrates, shuttling factors, and a deubiquitinating enzyme.

A bis-Benzylidine Piperidone Targeting Proteasome Ubiquitin Receptor RPN13/ADRM1 as a therapy for cancer

The bis-benzylidine piperidone RA190 covalently binds to cysteine 88 of ubiquitin receptor RPN13 in the 19S regulatory particle and inhibits proteasome function, triggering rapid accumulation of polyubiquitinated proteins. Multiple myeloma (MM) lines, even those resistant to bortezomib, were sensitive to RA190 via endoplasmic reticulum stress-related apoptosis. RA190 stabilized targets of human papillomavirus (HPV) E6 oncoprotein, and preferentially killed HPV-transformed cells. After oral or intraperitoneal dosing of mice, RA190 distributed to plasma and major organs except the brain and inhibited proteasome function in skin and muscle. RA190 administration profoundly reduced growth of MM and ovarian cancer xenografts, and oral RA190 treatment retarded HPV16(+) syngeneic mouse tumor growth, without affecting spontaneous HPV-specific CD8(+) T cell responses, suggesting its therapeutic potential.

Structure and mobility of the PUT3 dimer.

The solution structure and backbone dynamics of the transcriptional activator PUTS (31–100) has been characterized using NMR spectroscopy. PUT3 (31–100) contains three distinct domains: a cysteine zinc cluster, linker, and dimerization domain. The cysteine zinc cluster of PUT3 closely resembles the solution structure of GAL4, while the dimerization domain forms a long coiled-coil similar to that observed in the crystal structures of GAL4 and PPR1. However, the residues at the N-terminal end of the coiled-coil behave very differently in each of these proteins. A comparison of the structural elements within this region provides a model for the DMA binding specificity of these proteins. Furthermore, we have characterized the dynamics of PUT3 to find that the zinc cluster and dimerization domains have very diverse dynamics in solution. The dimerization domain behaves as a large protein, while the peripheral cysteine zinc clusters have dynamic properties similar to small proteins.