Stabilization of an unusual salt bridge in ubiquitin by the extra C-terminal domain of the proteasome-associated deubiquitinase UCH37 as a mechanism of its exo specificity

Abstract

Ubiquitination is countered by a group of enzymes collectively called deubiquitinases (DUBs); ∼100 of them can be found in the human genome. One of the most interesting aspects of these enzymes is the ability of some members to selectively recognize specific linkage types between ubiquitin in polyubiquitin chains and their endo and exo specificity. The structural basis of exo-specific deubiquitination catalyzed by a DUB is poorly understood. UCH37, a cysteine DUB conserved from fungi to humans, is a proteasome-associated factor that regulates the proteasome by sequentially cleaving polyubiquitin chains from their distal ends, i.e., by exo-specific deubiquitination. In addition to the catalytic domain, the DUB features a functionally uncharacterized UCH37-like domain (ULD), presumed to keep the enzyme in an inhibited state in its proteasome-free form. Herein we report the crystal structure of two constructs of UCH37 from Trichinella spiralis in complex with a ubiquitin-based suicide inhibitor, ubiquitin vinyl methyl ester (UbVME). These structures show that the ULD makes direct contact with ubiquitin stabilizing a highly unusual intramolecular salt bridge between Lys48 and Glu51 of ubiquitin, an interaction that would be favored only with the distal ubiquitin but not with the internal ones in a Lys48-linked polyubiquitin chain. An inspection of 39 DUB-ubiquitin structures in the Protein Data Bank reveals the uniqueness of the salt bridge in ubiquitin bound to UCH37, an interaction that disappears when the ULD is deleted, as revealed in the structure of the catalytic domain alone bound to UbVME. The structural data are consistent with previously reported mutational data on the mammalian enzyme, which, together with the fact that the ULD residues that bind to ubiquitin are conserved, points to a similar mechanism behind the exo specificity of the human enzyme. To the best of our knowledge, these data provide the only structural example so far of how the exo specificity of a DUB can be determined by its noncatalytic domain. Thus, our data show that, contrary to its proposed inhibitory role, the ULD actually contributes to substrate recognition and could be a major determinant of the proteasome-associated function of UCH37. Moreover, our structures show that the unproductively oriented catalytic cysteine in the free enzyme is aligned correctly when ubiquitin binds, suggesting a mechanism for ubiquitin selectivity.

Figure 1

(a) Schematic structures representing inhibition of UCH37 by UbVME (I and II); Definition of proximal and distal ubiquitin in a diubiquitin substrate (III); Schematic structure of the acyl-enzyme intermediate formed during deubiquitination catalyzed by a cysteine DUB (IV). The UbVME adduct (II) mimics the acyl-enzyme intermediate (IV), as shown in yellow shading. (b) Domain diagrams of TsUCH37 constructs compared to other UCH family members with UCH domains boxed in grey and additional domains boxed and labeled as shown. (c) Kinetic assay of UbAMC hydrolysis by TsUCH37^cat. (d) Analytical ultracentrifugation profiles of TsUCH37^cat (left) and TsUCH37^cat-UbVME complex (right), indicating that both are monomeric in solution.

Figure 2. Crystal structures of TsUCH37 constructs bound to UbVME

(a) Dimeric structure of TsUCH37^cat bound to UbVME (orange) in crystals. Monomers are shown in teal (chain A) and grey (chain C). Inset shows the disulfide bridge that links the two subunits via Cys71. The electron density is rendered from the 2Fo-Fc map contoured at 1σ. (b) Monomer of TsUCH37^cat -UbVME structure. (c) Structure of TsUCH37^ΔC46-UbVME, with TsUCH37^ΔC46 shown in olive and UbVME in orange.

Figure 3. Secondary structures of TsUCH37 constructs

TsUCH37^ΔC46-UbVME and TsUCH37^cat-UbVME are superposed with α-helices and 3₁₀-helices shown in pale yellow, β-sheets in blue, loops in green, and UbVME in orange. Arrows indicate where the TsUCH37^ΔC46-UbVME structure lacks density, compared to TsUCH37^cat-UbVME, from residues 57-71.

Figure 4. ULD-ubiquitin interactions

(a) Sequence alignment of the ULD of UCH37 highlighting conserved residues in UCH37 homologs. Glu265 and Asn272 (according to Ts numbering) are absolutely conserved, highlighted in red. (b) Superposition of TsUCH37^ΔC46 -UbVME (the ULD in olive, UbVME in orange), human UCH37 (the ULD in purple, PDB ID 3IHR), and TsUCH37 with the entire ULD modeled (cyan) based on the structure of the ULD in human UCH37. The model was generated using SwissModel and MD simulation (please see Materials and Methods). This model is taken from a snapshot collected at 1.3 ns during an MD simulation run of 2 ns. (c) The structure of TsUCH37-ubiquitin complex with the entire ULD modeled as shown in (b), showing that the conserved residues of the ULD could make additional contacts with ubiquitin. The regions marked i and ii are expanded in the panels below. The UCH domain is surface-rendered in grey.

Figure 5. Ubiquitin recognition by TsUCH37

(a) Surface rendering of TsUCH37^cat shown in cyan with ubiquitin binding regions highlighted. The distal site is shown in yellow, the active-site cysteine is shown in red, and resolved portions of the crossover loop are shown in pink. (b) Surface rendering of TsUCH37^ΔC46 shown in green with ubiquitin binding regions highlighted as in (a), except with additional C-terminal tail ubiquitin binding residues highlighted in blue. (c) Interactions near the active site cleft with the C-terminal hexapeptide tail of ubiquitin. UbVME residues are shown in orange, TsUCH37 residues are shown in teal, and human UCH37 residues in purple. (d) Interactions of Arg72 of ubiquitin with surrounding residues of TsUCH37^cat. Density from the 2Fo-Fc map is contoured at 1σ, shown in blue mesh. (e) UCH37 distal site binding residues, with TsUCH37 in teal and human UCH37 in purple. (f) Ile44 patch interacting residues, with UbVME in orange, TsUCH37 in teal, human UCH37 in purple. Waters involved in binding are also shown, enveloped with density from 2Fo-Fc map contoured at 1σ. Sequence alignment of this region in TsUCH37 compared to human UCH37 is shown as an inset. (g) Active site of TsUCH37 (in teal), showing the catalytic residues, compared to human UCH37 (in purple), with UbVME in orange.

Figure 6. ULD of TsUCH37 binding to ubiquitin

(a) TsUCH37^ΔC46 (in olive) ULD residues interacting with UbVME (in orange). Inset shows Arg261 and Tyr262 interactions with UbVME, as well as the intra-molecular salt bridge formed between Lys48 and Glu51 of ubiquitin. Density rendered from 2Fo-Fc map contoured to 0.7σ. (b-e) Comparison of the Lys48-Glu51 distance in ubiquitin observed in other DUB-ubiquitin structures. (b) Lys48 and Glu51 form a 3.7Å salt bridge in TsUCH37^ΔC46 – UbVME structure (olive), but not in TsUCH37^cat-UbVME structure (9.9 Å). (c) The same distance in all other UCH-ubiquitin structures are ≥9Å: UCHL3-UbVME in yellow (PDB ID 1XD3), UCHL1-UbVME in red (PDB ID 3KW5), Yuh1-Ubal in pink (PDB ID 1CMX), PfUCHL3-UbVME in purple (PDB ID 2WDT), TsUCH37^cat-UbVME in teal. (d) The same distance in OTU-ubiquitin structures: Otu1-ubiquitin in dark red (PDB ID 3BY4) is 8.7 Å, and in DUBA-Ubal in pale yellow (PDB ID 3TMP) is 6.0 Å. (e) The same distance in HAUSP/USP7-Ubal (PDB ID 1NBF in blue) is 10Å and in USP14-Ubal (PDB ID 2AYO in brown) is 10.9Å.

Figure 7

Sequence alignment of TsUCH37 and other homologs: human UCH37, S. pombe Uch2, and P. falciparum UCH54. Secondary structures for the two TsUCH37 structures are annotated above (e.g. α1 = alpha helix 1, β1 = beta sheet 1, η1 = 3₁₀ helix 1). α2’ and η2 are not resolved in the TsUCH37^ΔC46 –UbVME structure, and helices α7 and α8 are not present in the TsUCH37^cat-UbVME construct.