A substrate-interacting region of Parkin directs ubiquitination of the mitochondrial GTPase Miro1.

Mutations in the gene encoding for the E3 ubiquitin ligase Parkin have been linked to early-onset Parkinson's disease. Besides many other cellular roles, Parkin is involved in clearance of damaged mitochondria via mitophagy - a process of particular importance in dopaminergic neurons. Upon mitochondrial damage, Parkin accumulates at the outer mitochondrial membrane and is activated, leading to ubiquitination of many mitochondrial substrates and recruitment of mitophagy effectors. While the activation mechanisms of autoinhibited Parkin have been extensively studied, it remains unknown how Parkin recognises its substrates for ubiquitination, and no substrate interaction site in Parkin has been reported. Here, we identify a conserved region in the flexible linker between the Ubl and RING0 domains of Parkin, which is indispensable for Parkin interaction with the mitochondrial GTPase Miro1. Our results explain the preferential targeting and ubiquitination of Miro1 by Parkin and provide a biochemical explanation for the presence of Parkin at the mitochondrial membrane prior to activation induced by mitochondrial damage. Our findings are important for understanding mitochondrial homeostasis and may inspire new therapeutic avenues for Parkinson's disease.

Finding my leadership style.

In this Stories piece, Dr. Helen Walden-Professor and Head of the School of Molecular Biosciences at the University of Glasgow-shares how her past experiences and academic journey have shaped her leadership style.

AlphaFold: Research accelerator and hypothesis generator.

To celebrate the 50th anniversary of Cell Press and the Cell focus issue on structural biology, we discussed with scientists working across diverse fields how AlphaFold has changed their research and brought structural biology to the masses.

Structural and biochemical basis of interdependent FANCI-FANCD2 ubiquitination.

Di-monoubiquitination of the FANCI-FANCD2 (ID2) complex is a central and crucial step for the repair of DNA interstrand crosslinks via the Fanconi anaemia pathway. While FANCD2 ubiquitination precedes FANCI ubiquitination, FANCD2 is also deubiquitinated at a faster rate than FANCI, which can result in a FANCI-ubiquitinated ID2 complex (IUb D2). Here, we present a 4.1 Å cryo-EM structure of IUb D2 complex bound to double-stranded DNA. We show that this complex, like ID2Ub and IUb D2Ub , is also in the closed ID2 conformation and clamps on DNA. The target lysine of FANCD2 (K561) becomes fully exposed in the IUb D2-DNA structure and is thus primed for ubiquitination. Similarly, FANCI's target lysine (K523) is also primed for ubiquitination in the ID2Ub -DNA complex. The IUb D2-DNA complex exhibits deubiquitination resistance, conferred by the presence of DNA and FANCD2. ID2Ub -DNA, on the other hand, can be efficiently deubiquitinated by USP1-UAF1, unless further ubiquitination on FANCI occurs. Therefore, FANCI ubiquitination effectively maintains FANCD2 ubiquitination in two ways: it prevents excessive FANCD2 deubiquitination within an IUb D2Ub -DNA complex, and it enables re-ubiquitination of FANCD2 within a transient, closed-on-DNA, IUb D2 complex.

Cryo-EM reveals a mechanism of USP1 inhibition through a cryptic binding site.

Repair of DNA damage is critical to genomic integrity and frequently disrupted in cancers. Ubiquitin-specific protease 1 (USP1), a nucleus-localized deubiquitinase, lies at the interface of multiple DNA repair pathways and is a promising drug target for certain cancers. Although multiple inhibitors of this enzyme, including one in phase 1 clinical trials, have been established, their binding mode is unknown. Here, we use cryo-electron microscopy to study an assembled enzyme-substrate-inhibitor complex of USP1 and the well-established inhibitor, ML323. Achieving 2.5-Å resolution, with and without ML323, we find an unusual binding mode in which the inhibitor disrupts part of the hydrophobic core of USP1. The consequent conformational changes in the secondary structure lead to subtle rearrangements in the active site that underlie the mechanism of inhibition. These structures provide a platform for structure-based drug design targeting USP1.

Distinct phosphorylation signals drive acceptor versus free ubiquitin chain targeting by parkin.

The RBR E3 ligase parkin is recruited to the outer mitochondrial membrane (OMM) during oxidative stress where it becomes activated and ubiquitinates numerous proteins. Parkin activation involves binding of a phosphorylated ubiquitin (pUb), followed by phosphorylation of the Ubl domain in parkin, both mediated by the OMM kinase, PINK1. How an OMM protein is selected for ubiquitination is unclear. Parkin targeted OMM proteins have little structural or sequence similarity, with the commonality between substrates being proximity to the OMM. Here, we used chimeric proteins, tagged with ubiquitin (Ub), to evaluate parkin ubiquitination of mitochondrial acceptor proteins pre-ligated to Ub. We find that pUb tethered to the mitochondrial target proteins, Miro1 or CISD1, is necessary for parkin recruitment and essential for target protein ubiquitination. Surprisingly, phosphorylation of parkin is not necessary for the ubiquitination of either Miro1 or CISD1. Thus, parkin lacking its Ubl domain efficiently ubiquitinates a substrate tethered to pUb. Instead, phosphorylated parkin appears to stimulate free Ub chain formation. We also demonstrate that parkin ubiquitination of pUb-tethered substrates occurs on the substrate, rather than the pUb modification. We propose divergent parkin mechanisms whereby parkin-mediated ubiquitination of acceptor proteins is driven by binding to pre-existing pUb on the OMM protein and subsequent parkin phosphorylation triggers free Ub chain formation. This finding accounts for the broad spectrum of OMM proteins ubiquitinated by parkin and has implications on target design for therapeutics.

Mechanism, specificity, and function of FANCD2-FANCI ubiquitination and deubiquitination.

Fanconi anemia (FA) is a rare genetic disorder caused by mutations in any of the currently 22 known FA genes. The products of these genes, along with other FA-associated proteins, participate in a biochemical pathway, known as the FA pathway. This pathway is responsible for the repair of DNA interstrand cross-links (ICL) and the maintenance of genomic stability in response to replication stress. At the center of the pathway is the monoubiquitination of two FA proteins, FANCD2 and FANCI, on two specific lysine residues. This is achieved by the combined action of the UBE2T ubiquitin-conjugating enzyme and a large multicomponent E3 ligase, known as the FA-core complex. This E2-E3 pair specifically targets the FANCI-FANCD2 heterodimer (ID2 complex) for ubiquitination on DNA. Deubiquitination of both FANCD2 and FANCI, which is also critical for ICL repair, is achieved by the USP1-UAF1 complex. Recent work suggests that FANCD2 ubiquitination transforms the ID2 complex into a sliding DNA clamp. Further ubiquitination on FANCI does not alter this closed-on-DNA ID2 conformation. However, the resulting dimonoubiquitinated complex is highly resistant to USP1-UAF1 deubiquitination. This review will provide an update on recent work focusing on how specificity in FANCD2 ubiquitination and deubiquitination is achieved. Recent findings shedding light to the mechanisms, molecular functions, and biological roles of FANCI/FANCD2 ubiquitination and deubiquitination will be also discussed. ENZYMES: UBA1 (6.2.1.45), UBE2T (2.3.2.23), FANCL (2.3.2.27), USP1 (3.4.19.12).

Structural basis of FANCD2 deubiquitination by USP1-UAF1.

Ubiquitin-specific protease 1 (USP1) acts together with the cofactor UAF1 during DNA repair processes to specifically remove monoubiquitin signals. One substrate of the USP1-UAF1 complex is the monoubiquitinated FANCI-FANCD2 heterodimer, which is involved in the repair of DNA interstrand crosslinks via the Fanconi anemia pathway. Here we determine structures of human USP1-UAF1 with and without ubiquitin and bound to monoubiquitinated FANCI-FANCD2. The crystal structures of USP1-UAF1 reveal plasticity in USP1 and key differences to USP12-UAF1 and USP46-UAF1, two related proteases. A cryo-EM reconstruction of USP1-UAF1 in complex with monoubiquitinated FANCI-FANCD2 highlights a highly orchestrated deubiquitination process, with USP1-UAF1 driving conformational changes in the substrate. An extensive interface between UAF1 and FANCI, confirmed by mutagenesis and biochemical assays, provides a molecular explanation for the requirement of both proteins, despite neither being directly involved in catalysis. Overall, our data provide molecular details of USP1-UAF1 regulation and substrate recognition.

A mechanistic review of Parkin activation.

Parkin and phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) constitute a feed-forward signalling pathway that mediates autophagic removal of damaged mitochondria (mitophagy). With over 130 mutations identified to date in over 1000 patients with early onset parkinsonism, Parkin is considered a hot spot of signalling pathways involved in PD aetiology. Parkin is an E3 ligase and how its activity is regulated has been extensively studied: inter-domain interactions exert a tight inhibition on Parkin activity; binding to phospho-ubiquitin relieves this auto-inhibition; and phosphorylation of Parkin shifts the equilibrium towards maximal Parkin activation. This review focusses on recent, structural findings on the regulation of Parkin activity. What follows is a mechanistic introduction to the family of E3 ligases that includes Parkin, followed by a brief description of structural elements unique to Parkin that lock the enzyme in an autoinhibited state, contrasted with emerging models that have shed light on possible mechanisms of Parkin activation.

Differential functions of FANCI and FANCD2 ubiquitination stabilize ID2 complex on DNA.

The Fanconi anaemia (FA) pathway is a dedicated pathway for the repair of DNA interstrand crosslinks and is additionally activated in response to other forms of replication stress. A key step in the FA pathway is the monoubiquitination of each of the two subunits (FANCI and FANCD2) of the ID2 complex on specific lysine residues. However, the molecular function of these modifications has been unknown for nearly two decades. Here, we find that ubiquitination of FANCD2 acts to increase ID2's affinity for double-stranded DNA via promoting a large-scale conformational change in the complex. The resulting complex encircles DNA, by forming a secondary "Arm" ID2 interface. Ubiquitination of FANCI, on the other hand, largely protects the ubiquitin on FANCD2 from USP1-UAF1 deubiquitination, with key hydrophobic residues of FANCI's ubiquitin being important for this protection. In effect, both of these post-translational modifications function to stabilize a conformation in which the ID2 complex encircles DNA.

The deubiquitinase USP7 uses a distinct ubiquitin-like domain to deubiquitinate NF-ĸB subunits.

The transcription factor NF-ĸB is a master regulator of the innate immune response and plays a central role in inflammatory diseases by mediating the expression of pro-inflammatory cytokines. Ubiquitination-triggered proteasomal degradation of DNA-bound NF-ĸB strongly limits the expression of its target genes. Conversely, USP7 (deubiquitinase ubiquitin-specific peptidase 7) opposes the activities of E3 ligases, stabilizes DNA-bound NF-ĸB, and thereby promotes NF-ĸB-mediated transcription. Using gene expression and synthetic peptide arrays on membrane support and overlay analyses, we found here that inhibiting USP7 increases NF-ĸB ubiquitination and degradation, prevents Toll-like receptor-induced pro-inflammatory cytokine expression, and represents an effective strategy for controlling inflammation. However, the broad regulatory roles of USP7 in cell death pathways, chromatin, and DNA damage responses limit the use of catalytic inhibitors of USP7 as anti-inflammatory agents. To this end, we identified an NF-ĸB-binding site in USP7, ubiquitin-like domain 2, that selectively mediates interactions of USP7 with NF-ĸB subunits but is dispensable for interactions with other proteins. Moreover, we found that the amino acids 757LDEL760 in USP7 critically contribute to the interaction with the p65 subunit of NF-ĸB. Our findings support the notion that USP7 activity could be potentially targeted in a substrate-selective manner through the development of noncatalytic inhibitors of this deubiquitinase to abrogate NF-ĸB activity.

Characterization of FANCL variants observed in patient cancer cells.

Fanconi Anemia (FA) is a rare genetic disorder characterized by developmental defects, bone marrow failure and high predisposition to cancer. The FA DNA repair pathway is required in humans to coordinate repair of DNA interstrand cross-links. The central event in the activation of the pathway is the monoubiquitination of FANCD2 and FANCI by the E2-E3 pair, Ube2T-FANCL, with the central UBC-RWD (URD) domain of FANCL recognizing the substrates. Whole genome sequencing studies of cancer cells from patients identified point mutations in the FANCL URD domain. We analysed 17 such variants of FANCL, including known substrate binding mutants (W212A, W214A and L248A, F252A, L254A, I265A), a FA mutation (R221C) and 14 cancer-associated mutations (F110S, I136V, L149V, L154S, A192G, E215Q, E217K, R221W, T224K, M247V, F252L, N270K, V287G, E289Q) through recombinant expression analysis, thermal shift assay, interaction with FANCD2, in vitro ubiquitination activity, and cellular sensitivity to an interstrand cross-linking agent. We find that the FANCL mutations I136V, L154S, W212A and L214A, R221W, R221C, and V287G are destabilizing, with N270K and E289Q destabilizing the C-terminal helices of the URD domain. The hydrophobic patch mutant (L248A, F252A, L254A, I265A), along with mutations E217K, T224K, and M247V, cause defects in the catalytic function of FANCL. This highlights the C-terminal lobe of the FANCL URD domain as important for the activity and function of FANCL. These mutations which affect the fold and activity of FANCL may contribute to tumorigenesis in these non-FA cancer patients, and this implicates FA genes in general cancer progression.

Modes of allosteric regulation of the ubiquitination machinery.

Ubiquitination is a post-translational modification crucial for cellular signaling. A diverse range of enzymes constitute the machinery that mediates attachment of ubiquitin onto target proteins. This diversity allows the targeting of various proteins in a highly regulated fashion. Many of the enzymes have multiple domains or subunits that bind allosteric effectors and exhibit large conformational rearrangements to facilitate regulation. Here we consider recent examples of ubiquitin itself as an allosteric effector of RING and RBR E3 ligases, as well as advances in the understanding of allosteric regulatory elements within HECT E3 ligases.

Allosteric mechanism for site-specific ubiquitination of FANCD2.

DNA-damage repair is implemented by proteins that are coordinated by specialized molecular signals. One such signal in the Fanconi anemia (FA) pathway for the repair of DNA interstrand crosslinks is the site-specific monoubiquitination of FANCD2 and FANCI. The signal is mediated by a multiprotein FA core complex (FA-CC) however, the mechanics for precise ubiquitination remain elusive. We show that FANCL, the RING-bearing module in FA-CC, allosterically activates its cognate ubiqutin-conjugating enzyme E2 UBE2T to drive site-specific FANCD2 ubiquitination. Unlike typical RING E3 ligases, FANCL catalyzes ubiquitination by rewiring the intraresidue network of UBE2T to influence the active site. Consequently, a basic triad unique to UBE2T engages a structured acidic patch near the target lysine on FANCD2. This three-dimensional complementarity, between the E2 active site and substrate surface, induced by FANCL is central to site-specific monoubiquitination in the FA pathway. Furthermore, the allosteric network of UBE2T can be engineered to enhance FANCL-catalyzed FANCD2-FANCI di-monoubiquitination without compromising site specificity.

Structural basis of generic versus specific E2-RING E3 interactions in protein ubiquitination.

Protein ubiquitination is a fundamental regulatory component in eukaryotic cell biology, where a cascade of ubiquitin activating (E1), conjugating (E2), and ligating (E3) enzymes assemble distinct ubiquitin signals on target proteins. E2s specify the type of ubiquitin signal generated, while E3s associate with the E2~Ub conjugate and select the substrate for ubiquitination. Thus, producing the right ubiquitin signal on the right target requires the right E2-E3 pair. The question of how over 600 E3s evolved to discriminate between 38 structurally related E2s has therefore been an area of intensive research, and with over 50 E2-E3 complex structures generated to date, the answer is beginning to emerge. The following review discusses the structural basis of generic E2-RING E3 interactions, contrasted with emerging themes that reveal how specificity can be achieved.

Enzymatic preparation of monoubiquitinated FANCD2 and FANCI proteins.

In higher eukaryotes, DNA damage repair response pathways are orchestrated by several molecular signals including ubiquitination. In particular the repair of DNA interstrand crosslinks, toxic to transcription and replication processes, involve the activation of the Fanconi anemia repair pathway. At the heart of this pathway lies the monoubiquitination of FANCD2 and FANCI proteins, which triggers the recruitment of DNA repair factors. A major road block in our understanding of this fundamental repair pathway arises from the challenge with generating sufficient quantities of site-specifically monoubiquitinated FANCD2 and FANCI proteins to enable mechanistic and molecular studies. Current in vitro methods rely on the purification of a large (~0.8MDa), multiprotein E3 complex that can only partially monoubiquitinate a FANCD2-FANCI-DNA complex. In this chapter, we describe detailed protocols for the preparation of homogeneously and natively monoubiquitinated FANCD2 and FANCI proteins in isolation. The method relies on the use of a minimal E3 module and an engineered E2 variant that together drive site-specific ubiquitination of the isolated substrates, without the requirement of DNA cofactors. Using the enzymatic approach, we also demonstrate how added functionalities such as a fluorescently labeled ubiquitin can be conjugated on the FANCD2 and FANCI substrates, thus enabling multiple downstream applications.

Specificity for deubiquitination of monoubiquitinated FANCD2 is driven by the N-terminus of USP1.

The Fanconi anemia pathway for DNA interstrand crosslink repair and the translesion synthesis pathway for DNA damage tolerance both require cycles of monoubiquitination and deubiquitination. The ubiquitin-specific protease-1 (USP1), in complex with USP1-associated factor 1, regulates multiple DNA repair pathways by deubiquitinating monoubiquitinated Fanconi anemia group D2 protein (FANCD2), Fanconi anemia group I protein (FANCI), and proliferating cell nuclear antigen (PCNA). Loss of USP1 activity gives rise to chromosomal instability. Whereas many USPs hydrolyse ubiquitin-ubiquitin linkages, USP1 targets ubiquitin-substrate conjugates at specific sites. The molecular basis of USP1's specificity for multiple substrates is poorly understood. Here, we reconstitute deubiquitination of purified monoubiquitinated FANCD2, FANCI, and PCNA and show that molecular determinants for substrate deubiquitination by USP1 reside within the highly conserved and extended N-terminus. We found that the N-terminus of USP1 harbours a FANCD2-specific binding sequence required for deubiquitination of K561 on FANCD2. In contrast, the N-terminus is not required for direct PCNA or FANCI deubiquitination. Furthermore, we show that the N-terminus of USP1 is sufficient to engineer specificity in a more promiscuous USP.

Synergistic recruitment of UbcH7~Ub and phosphorylated Ubl domain triggers parkin activation.

The E3 ligase parkin ubiquitinates outer mitochondrial membrane proteins during oxidative stress and is linked to early-onset Parkinson's disease. Parkin is autoinhibited but is activated by the kinase PINK1 that phosphorylates ubiquitin leading to parkin recruitment, and stimulates phosphorylation of parkin's N-terminal ubiquitin-like (pUbl) domain. How these events alter the structure of parkin to allow recruitment of an E2~Ub conjugate and enhanced ubiquitination is an unresolved question. We present a model of an E2~Ub conjugate bound to the phospho-ubiquitin-loaded C-terminus of parkin, derived from NMR chemical shift perturbation experiments. We show the UbcH7~Ub conjugate binds in the open state whereby conjugated ubiquitin binds to the RING1/IBR interface. Further, NMR and mass spectrometry experiments indicate the RING0/RING2 interface is re-modelled, remote from the E2 binding site, and this alters the reactivity of the RING2(Rcat) catalytic cysteine, needed for ubiquitin transfer. Our experiments provide evidence that parkin phosphorylation and E2~Ub recruitment act synergistically to enhance a weak interaction of the pUbl domain with the RING0 domain and rearrange the location of the RING2(Rcat) domain to drive parkin activity.

RBR ligase-mediated ubiquitin transfer: a tale with many twists and turns.

RBR ligases are an enigmatic class of E3 ubiquitin ligases that combine properties of RING and HECT-type E3s and undergo multilevel regulation through autoinhibition, post-translational modifications, multimerization and interaction with binding partners. Here, we summarize recent progress in RBR structures and function, which has uncovered commonalities in the mechanisms by which different family members transfer ubiquitin through a multistep process. However, these studies have also highlighted clear differences in the activity of different family members, suggesting that each RBR ligase has evolved specific properties to fit the biological process it regulates.