β-Sheet Augmentation Is a Conserved Mechanism of Priming HECT E3 Ligases for Ubiquitin Ligation

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Highlights

  • The Huwe1 ligase synthesizes K6- and K48-linked ubiquitin chains.

  • The structural features of thioester intermediates are conserved between K63-specific Nedd4 family ligases and Huwe1.

  • NMR data provide evidence for conformational changes of the C-terminal residues upon transthiolation.

  • The catalytic mechanisms of HECT domains seem to diverge after thioester formation.

Abstract

Ubiquitin (Ub) ligases (E3s) catalyze the attachment of Ub chains to target proteins and thereby regulate a wide array of signal transduction pathways in eukaryotes. In HECT-type E3s, Ub first forms a thioester intermediate with a strictly conserved Cys in the C-lobe of the HECT domain and is then ligated via an isopeptide bond to a Lys residue in the substrate or a preceding Ub in a poly-Ub chain. To date, many key aspects of HECT-mediated Ub transfer have remained elusive. Here, we provide structural and functional insights into the catalytic mechanism of the HECT-type ligase Huwe1 and compare it to the unrelated, K63-specific Smurf2 E3, a member of the Nedd4 family. We found that the Huwe1 HECT domain, in contrast to Nedd4-family E3s, prioritizes K6- and K48-poly-Ub chains and does not interact with Ub in a non-covalent manner. Despite these mechanistic differences, we demonstrate that the architecture of the C-lobe ~ Ub intermediate is conserved between Huwe1 and Smurf2 and involves a reorientation of the very C-terminal residues. Moreover, in Nedd4 E3s and Huwe1, the individual sequence composition of the Huwe1 C-terminal tail modulates ubiquitination activity, without affecting thioester formation. In sum, our data suggest that catalysis of HECT ligases hold common features, such as the β-sheet augmentation that primes the enzymes for ligation, and variable elements, such as the sequence of the HECT C-terminal tail, that fine-tune ubiquitination activity and may aid in determining Ub chain specificity by positioning the substrate or acceptor Ub.

Introduction

The post-translational modification of proteins with ubiquitin (Ub) plays a key role in cellular signaling [1]. The ubiquitination reaction is catalyzed by an enzymatic cascade consisting of an activating (E1), a conjugating (E2) and a ligating (E3) enzyme. Ub can be attached to substrates as a monomer at a single or multiple Lys residues (mono-/multi-ubiquitination) or as a poly-Ub chain (poly-ubiquitination) by the repeated action of the ubiquitination enzyme cassette. All seven lysines (K6, K11, K27, K29, K33, K48 and K63) and the N-terminal M1 residue can serve as linkage points in the distal (acceptor) Ub for isopeptide bond formation with the C-terminal carboxyl group of the donor Ub (UbD) molecule during chain elongation [2]. Notably, many E3 enzymes seem to prioritize a certain Lys residue in the acceptor Ub to form poly-Ub chains [3], [4]. The information encoded in the linkage types dictates the cellular fate of the target protein. K11- and K48-linked poly-Ub chains direct substrates to proteasomal degradation [5], [6], while mono-ubiquitination and K63-linked poly-Ub chains have non-proteolytic functions in the assembly and trafficking of cell surface receptors and signaling complexes [7]. It is thus important to understand how E3 ligases catalyze Ub chain assembly and what determines Ub chain length and linkage.

Homologous to the E6-AP carboxyl terminus (HECT)-type E3 ligases use a two-step mechanism to ubiquitinate substrates. First, the carboxyl group at the Ub C-terminus is transferred from an E2 thioester intermediate to the catalytic Cys of the HECT domain in a transthiolation reaction [8], [9]. Then the HECT ~ Ub thioester is attacked by the ε-amino group of a Lys in the substrate or the acceptor Ub to ultimately form an isopeptide bond [10]. For this last reaction step, HECT domains require a strictly conserved Phe or Tyr at the − 4 position relative to the HECT C-terminus [11]. Although HECT-type ligases contain a diverse array of domains upstream of the catalytic domain [12], the HECT domain alone can perform both catalytic steps in the absence of N-terminal domains. Moreover, due to the two-step mechanism, linkage specificity is largely determined by the HECT-type ligase itself, in particular by the C-terminal 60 aa of the HECT domain [13]. We have recently shown that exchanging even only the three C-terminal residues in neural precursor cell-expressed developmentally downregulated gene 4 (Nedd4) to those of the K48-specific E6AP E3 is sufficient to alter Ub chain specificity from K63 to a mixture of K48- and K63-linked chains [4]. However, the exact mechanisms underlying HECT-mediated poly-Ub chain formation are still unclear since the catalytically important C-terminal residues could not be resolved in crystal structures of HECT domain intermediates.

Among HECT-type ligases, the Nedd4 family forms the largest group with nine members in humans. This enzyme family contains an N-terminal C2 domain, two to three central WW domains and the C-terminal HECT domain [14]. Nedd4-family E3s predominantly synthesizes K63-linked poly-Ub chains [4], [15], and in contrast to other HECT ligases, the structures of their HECT domains have been characterized at multiple steps along their catalytic pathway [4], [16], [17]. HECT domains consist of two lobes that are tethered by a flexible hinge loop [18]. The larger N-terminal lobe (N-lobe) of the HECT domain engages the E2 [18] and contains a non-covalent Ub binding surface that is important for Ub chain elongation [19], [20], [21], [22], [23]. The smaller C-lobe bears the catalytic Cys that forms the thioester intermediate with the Ub C-terminus. Interestingly, both the orientation of the donor Ub toward the C-lobe and the relative arrangement of the N- and C-lobes are preserved upon Ub transfer from the E2 to the E3 [4], [17]. The energy for pushing the transthiolation reaction forward is provided by the formation of an additional β-strand between the C-lobe and the C-terminus of the UbD. For isopeptide formation, the Ub-loaded C-lobe rotates as a whole with respect to the N-lobe to present the thioester to a substrate Lys for nucleophilic attack [16]. Once the isopeptide bond has been formed, the C-lobe most likely discharges the substrate-conjugated Ub after ligation and subsequently switches back into a conformation that allows it to accept another Ub from the E2-thioester. Once a substrate is mono-ubiquitinated, a non-covalent Ub binding surface on the N-lobe facilitates chain elongation [19], [20], [21], [22], [23]. However, the mechanistic details underlying this phenomenon are unclear. Although these studies have remarkably furthered our understanding of the catalytic mechanism underlying HECT-mediated Ub transfer, they fall short of providing insights as to how the C-terminal region enables isopeptide formation and contributes to Ub chain selectivity. Moreover, so far all structural information on catalytic intermediates stems exclusively from K63-specific Nedd4 family members. Therefore, it is unclear whether these mechanisms apply to other if not all HECT domains.

In contrast to the Nedd4 family, structural and mechanistic information on reaction intermediates of other HECT-type ligases is sparse. Moreover, while Nedd4 ligases primarily synthesize K63-poly-Ub chains [4], [23], HECT domains outside this family seem to prioritize other linkages, for example, K48-chains in the case of E6AP [13], K48- and K29-chains for UBE3C (a.k.a. KIAA10 or RAUL) [15], [24], [25], K33- and K11-chains for AREL1 [15] and K6-chains for the HECT-like bacterial ligase NleL [26]. HECT, UBA and WWE domain containing 1 (Huwe1; a.k.a. MULE, ARF-BP1, Lasu1 or HECTH9) was shown to generate K6-, K48- and/or K11-linked chains [15], [27], [28], [29]. However, reports exist that Huwe1 also synthesizes K63-chains [30]. N-terminal to its HECT domain, Huwe1 contains two Armadillo-like repeat domains, a Ub-associated (UBA) domain, a UIM (Ub Interacting Motif), a WWE protein interaction domain and a BH3 motif for binding its bona fide substrate Mcl-1 [31], [52]. Huwe1 regulates important cellular processes including apoptosis and DNA damage repair and is closely linked to tumor development by targeting various key oncoproteins and tumor suppressors such as ARF, p53, Myc and Miz1, for proteasomal degradation [32], [33], [34], [52].

To further our understanding of HECT domains, we characterized the catalytic mechanism of the Huwe1 HECT domain and compared it in detail to the Nedd4-family member SMAD-specific E3 ubiquitin protein ligase 2 (Smurf2). We show that in contrast to Nedd4-family E3s, the Huwe1 HECT domain preferentially synthesizes K6- and K48-linked Ub chains and does not interact with monomeric Ub despite being capable of generating poly-Ub chains. Regardless of these mechanistic differences, we find that the crystal structures of the Smurf2 and Huwe1 C-lobe ~ UbD complexes are highly similar. By NMR spectroscopy, we found that in both cases, the C-terminal residues of the C-lobe are affected by thioester formation. As for Nedd4 [3], [4], we show that the presence and the protein sequence of the C-terminal residues are not crucial for transthiolation, but strongly affect isopeptide formation. Given that the orientation of the UbD with respect to the C-lobe is preserved among Nedd4 family members and Huwe1, this suggests that the variable sequence of the C-terminal residues may create HECT ~ UbD binding interfaces that are important for catalytic efficiency and potentially specific for certain Lys residues in the substrate or acceptor Ub.

Section snippets

The Huwe1 HECT domain forms preferentially K6- and K48-linked Ub chains

To gain insight into Huwe1 Ub chain specificity, we performed auto-ubiquitination assays with the isolated Huwe1 HECT domain using fluorescently labeled WT and single Lys Ub mutants (Fig. 1a). We found that the Huwe1 HECT domain was most active with WT Ub and readily produced poly-Ub chains with both K48- and K6-only Ub. For a more detailed analysis, we examined the Ub chain type specificity of the Huwe1 HECT domain with absolute-quantification (AQUA) assays [4], [35]. To this end, three

Discussion

HECT-type ligases are key regulators of various signal transduction pathways that play a role in cellular homeostasis and embryonic development but adversely also in carcinogenesis [32]. Although significant progress has been made over the past decade in elucidating the mechanisms underlying HECT-mediated ubiquitination in Nedd4-family E3s, many general questions remain unresolved such as whether the mechanisms underlying Ub transfer are conserved within the entire HECT family, which residues

Constructs and reagents

The nucleotide sequence of the human Huwe1 ligase (NP_113584.3) corresponding to aa 3993–4375 (hereafter referred to as aa 3–385 for clarity) was amplified by PCR from a cDNA vector purchased from Imagenes and ligated into a pETM-11 vector (EMBL Heidelberg) to express a Huwe1 HECT domain with an N-terminal His6-tag followed by a TEV protease cleavage site. To generate a Huwe1 HECT domain where all Cys were replaced except for the catalytic Cys (hereafter referred to as “C1”), we performed the

Acknowledgments

We are grateful to Marcus Hartmann, Ancilla Neu and Iris Holdermann (MPI for Developmental Biology, Tübingen, Germany) for help with data collection and determination of the crystal structures and to Paolo Soffientini and the Proteomics/MS Technological Development Unit at Cogentech/IFOM for the AQUA analysis. We also thank Samira Anders and Mira Schütz-Stoffregen (MPI for Developmental Biology, Tübingen, Germany) for cloning and purification of proteins used in this work. S.W. acknowledges

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