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. 2016 Jul 7;63(1):146-55.
doi: 10.1016/j.molcel.2016.05.009. Epub 2016 Jun 9.

MINDY-1 Is a Member of an Evolutionarily Conserved and Structurally Distinct New Family of Deubiquitinating Enzymes

Syed Arif Abdul Rehman  1 , Yosua Adi Kristariyanto  1 , Soo-Youn Choi  1 , Pedro Junior Nkosi  1 , Simone Weidlich  1 , Karim Labib  1 , Kay Hofmann  2 , Yogesh Kulathu  3
Affiliations

MINDY-1 Is a Member of an Evolutionarily Conserved and Structurally Distinct New Family of Deubiquitinating Enzymes

Syed Arif Abdul Rehman et al. Mol Cell. .

Abstract

Deubiquitinating enzymes (DUBs) remove ubiquitin (Ub) from Ub-conjugated substrates to regulate the functional outcome of ubiquitylation. Here we report the discovery of a new family of DUBs, which we have named MINDY (motif interacting with Ub-containing novel DUB family). Found in all eukaryotes, MINDY-family DUBs are highly selective at cleaving K48-linked polyUb, a signal that targets proteins for degradation. We identify the catalytic activity to be encoded within a previously unannotated domain, the crystal structure of which reveals a distinct protein fold with no homology to any of the known DUBs. The crystal structure of MINDY-1 (also known as FAM63A) in complex with propargylated Ub reveals conformational changes that realign the active site for catalysis. MINDY-1 prefers cleaving long polyUb chains and works by trimming chains from the distal end. Collectively, our results reveal a new family of DUBs that may have specialized roles in regulating proteostasis.

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Figures

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Graphical abstract
Figure 1
Figure 1
Identification of the MINDY Family of DUBs (A) Schematic representation of the domain structure of human FAM63A. (B) Halo-tagged FAM63A (388–426) coupled to HaloLink resin was incubated with tetraUb of the indicated linkage types. The captured materials were separated on 4%–12% SDS-PAGE gel and silver stained. (C–H) DUB assays testing activity and specificity of polyUb cleavage by FAM63A (C), putative catalytic domain of FAM63A (110–384) (D) (asterisk indicates FAM63A), full-length FAM63B (E), YPL191C/MIY1 (F), YGL082W (G), and full-length human FAM188A (H); 1.6 μM of DUBs were incubated with 2.2 μM of tetraUb chains for the indicated time. (I) Phylogenetic tree of MINDY family DUBs based on alignment of catalytic domains (Figure S2B). See also Figure S1.
Figure 2
Figure 2
Crystal Structure of MINDY-1cat (A) Structure of catalytic domain of FAM63A/MINDY-1cat (110–370). The Cys loop (cyan) and the catalytic residues are indicated. β sheets are colored red and 3_10 helices blue. (B) A close-up image of the MINDY-1cat catalytic site. Q131, C137, and H319 are shown. (C and D) Hydrolysis of 1.9 μM K48-linked triUb by 1.6 μM MINDY-1 wild-type (WT) and the indicated mutants of the active site residues (C) or Q131 that forms the oxyanion hole residue (D). (E) Sequence alignment of human FAM63A, FAM63B, FAM188A, and FAM188B. Secondary structure elements are shown for MINDY-1cat. The catalytic residues are highlighted with red asterisks. Residues 300–371 of FAM188A that form the EF hand domain have been omitted from the alignment. Fully conserved residues are shaded in red. See also Figures S2 and S3.
Figure 3
Figure 3
Structure of MINDY-1cat∼Ub (A) Overall structure of the catalytic core domain of MINDY-1 (pink) covalently bound to Ub. Ub exists in two alternate conformers in the structure that are rotated by ∼45° (cyan and orange). The vinylthioether linkage connecting UbPrg with MINDY-1 is shown in sticks. The Cys loop (β2-α1) is shown in blue. (B) Conserved residues on the surface of MINDY-1 based on the sequence alignment in Figure S1C generated with the Consurf server (http://consurf.tau.ac.il) are shown. While the backside of MINDY-1cat is not conserved, surfaces interacting with and around the distal Ub are conserved. (C) Close-up view of the catalytic groove where the C terminus of Ub sits, with coloring scheme as in (B). (D) An aromatic cage formed by V212, W240, Y258, and F315 interacts with L73 of Ub. Close-up view of the conserved hydrophobic pocket accommodating L73 colored as in (B). (E) DUB assays monitoring cleavage of 1.9 μM K48-triUb with 1.6 μM MINDY-1cat performed as in Figure 1C comparing activity of MINDY-1 and point mutants lining the L73 pocket: V210A, W240A, Y258A, and F315A. (F) Close-up view of ionic interactions between Ub and MINDY-1. (G) DUB assays comparing activity of MINDY-1 mutants that disrupt ionic interactions with Ub as performed in (E). (H) Close-up image of the MINDY-1 catalytic triad showing two alternate conformations for H319 and Q131. Distances to C137 are indicated by dotted lines. (I) Superposition of apo and complex states of MINDY-1cat shows movement of the Cys loop (apo in orange and MINDY-1cat∼Ub complex in pink). See also Figure S4.
Figure 4
Figure 4
MINDY-1 Cleaves PolyUb Chains in a Stepwise Manner (A) Time course of cleavage of 3.5 μM K48-pentaUb by 1.6 μM of full-length MINDY-1 and MINDY-1cat and 160 nM MIY1. Asterisks indicate MINDY-1. (B) Kinetics of cleavage of fluorescently labeled K48-linked diUb by MINDY-1cat, MIY1, and OTUB1. DUBs (1 μM) were incubated with 500 nM of K48-linked diUb that has been labeled with an infrared fluorescent dye at its distal Ub (green circle) for the indicated times. Fluorescent Ub was visualized using Odyssey LI-COR system at 800 nm channel. D, distal Ub, P, proximal Ub. (C) Quantification of K48-Ub2 hydrolysis by MINDY-1cat, MIY1, and OTUB1 in (B). Percentage of the formed Ub1 intensity is shown on the y axis (n = 3; mean ± SD). (D) DUB assays monitoring time-dependent cleavage of fluorescently labeled pentaUb by MINDY-1cat, MIY1, and OTUB1 as in (B). The proximal Ub of the chain (indicated by green circle) was labeled with an infrared fluorescent dye. (E) Quantification of cleavage of K48-linked pentaUb by MINDY-1cat, MIY1, and OTUB1 in (D). The percentage of the total intensities of Ub4, Ub3, Ub2, and Ub1 formed is shown on the y axis (n = 3; mean ± SD). See also Figure S5. (F) Steady-state kinetics of K48-linked pentaUb cleavage by MINDY-1cat. MINDY-1cat (15 nM) was incubated with 0.075–2.4 μM fluorescently labeled pentaUb (IR-K48-Ub5). The K48-Ub4 formed at the early time point (less than 10% of the substrate) was quantified to obtain initial velocities (V0). V0 was plotted against IR-K48-Ub5 concentration, and the data were fitted to the Michaelis-Menten equation to derive kcat and Km (n = 3; mean ± SD). (G) Flag pull-downs from extracts of HEK293 cells inducibly expressing the indicated full-length MINDY-1 constructs. ev, empty vector; MIU∗, MIU mutant, L415A/A416G. Immunoblotting with a K48-linkage specific antibody was performed to monitor captured polyUb material. (H) Time course comparing hydrolysis of K48-polyUb chains containing at least 5 Ub by full-length MINDY-1 and MINDY-1cat, which lacks the MIU. (I) Model depicting the synergy between different domains of MINDY-1, where the UBD mediates substrate targeting to result in trimming of the Ub chain from the distal end by the catalytic domain. See also Figure S5.
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