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Urm1: a Non-Canonical UBL

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Urm1: a Non-Canonical UBL biomolecules Review Urm1: A Non-Canonical UBL Martin Termathe 1 and Sebastian A. Leidel 2,* 1 Institute of Biochemistry, Protein Biochemistry and Photobiocatalysis, University of Greifswald, Felix-Hausdorff-Strasse 4, 17489 Greifswald, Germany; [email protected] 2 Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland * Correspondence: [email protected] Abstract: Urm1 (ubiquitin related modifier 1) is a molecular fossil in the class of ubiquitin-like proteins (UBLs). It encompasses characteristics of classical UBLs, such as ubiquitin or SUMO (small ubiquitin-related modifier), but also of bacterial sulfur-carrier proteins (SCP). Since its main function is to modify tRNA, Urm1 acts in a non-canonical manner. Uba4, the activating enzyme of Urm1, contains two domains: a classical E1-like domain (AD), which activates Urm1, and a rhodanese homology domain (RHD). This sulfurtransferase domain catalyzes the formation of a C-terminal thiocarboxylate on Urm1. Thiocarboxylated Urm1 is the sulfur donor for 5-methoxycarbonylmethyl- 2-thiouridine (mcm5s2U), a chemical nucleotide modification at the wobble position in tRNA. This thio-modification is conserved in all domains of life and optimizes translation. The absence of Urm1 increases stress sensitivity in yeast triggered by defects in protein homeostasis, a hallmark of neurological defects in higher organisms. In contrast, elevated levels of tRNA modifying enzymes promote the appearance of certain types of cancer and the formation of metastasis. Here, we summarize recent findings on the unique features that place Urm1 at the intersection of UBL and SCP and make Urm1 an excellent model for studying the evolution of protein conjugation and sulfur-carrier systems. Keywords: 2-thiolation; tRNA modification; thiocarboxylate; rhodanese; sulfur-carrier protein; non-canonical UBL; ubiquitin-like protein Citation: Termathe, M.; Leidel, S.A. Urm1: A Non-Canonical UBL. Biomolecules 2021, 11, 139. https:// doi.org/10.3390/biom11020139 1. Introduction Urm1 (ubiquitin related modifier 1) is a non-canonical ubiquitin-like protein (UBL). Academic Editor: Elah Pick Classical UBLs, like ubiquitin, SUMO (small ubiquitin-related modifier) or Nedd8, are Received: 3 December 2020 conjugated to target proteins following an intricate enzymatic cascade. First, UBLs are Accepted: 19 January 2021 adenylated at their conserved C-terminal diglycine motif by a dedicated activating enzyme Published: 22 January 2021 (E1). Subsequently, a transthioesterification reaction with their conjugating enzyme (E2) occurs. Finally, an E3 ligase catalyzes the formation of an isopeptide bond between the C- Publisher’s Note: MDPI stays neutral terminus of the UBL and a lysine side chain of a target protein [1]. This protein conjugation with regard to jurisdictional claims in can trigger different downstream processes depending on the type of UBL or whether it published maps and institutional affil- forms chains with specific linkages. UBL attachment can mark proteins for proteasomal iations. degradation, but can also alter their localization or activity. Urm1 was first identified in a homology search in the Saccharomyces cerevisiae genome [2]. The goal of the study was to identify unknown UBLs distantly related to ubiquitin. There- fore, the query did not use known UBL. Bacterial sulfur-carrier protein (SCP) systems Copyright: © 2021 by the authors. share an identical activating mechanism with UBLs. The bacterial MoaD (molybdopterin Licensee MDPI, Basel, Switzerland. synthase sulfur carrier subunit) and ThiS (thiamin biosynthesis protein) were chosen in This article is an open access article the query to search for distantly related ubiquitin-like conjugation systems [1,2]. The distributed under the terms and MoaD-MoeB and ThiS-ThiF systems provide sulfur for the biosynthesis of the cofactors conditions of the Creative Commons molybdopterin and thiamin, respectively. In brief, MoaD and ThiS are adenylated by Attribution (CC BY) license (https:// their cognate E1-like activating enzyme MoeB (molybdopterin-synthase sulfurylase) and creativecommons.org/licenses/by/ ThiF and subsequently thiocarboxylated at their C-terminus. These bacterial SCPs share 4.0/). Biomolecules 2021, 11, 139. https://doi.org/10.3390/biom11020139 https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, 139 2 of 15 Biomolecules 2021, 11, x FOR PEER REVIEW 2 of 15 approximately 20% sequence similarity with Urm1 in contrast to ubiquitin, which exhibits onlyonly weak weak similarity similarity with with Urm1. Urm1. However, However, Urm1, Urm1, UBLs, UBLs, and and SCPs SCPs share share a characteristic a characteristic diglycinediglycine motif motif at attheir their C-terminus, C-terminus, which which en ensuressures their their recognition recognition for for activation activation (see (see FigureFigure 1A).1A). Despite Despite the the low low similarity similarity of of th thee primary primary sequence, sequence, these these proteins proteins all all share share a a β-graspβ-grasp fold, fold, consisting consisting of of five five ββ-sheets-sheets arranged arranged around around a a ce centralntral helixhelix (see(see FigureFigure1 B)1B) [ 3]. [3].In In evolutionary evolutionary terms, terms, it it is is thought thought that that UBL UBL and and SCP SCP may may havehave emergedemerged fromfrom aa single,sin- gle,multifunctional multifunctional ancestral ancestral systemsystem capable of of both both adenylating adenylating and and thiolating thiolating activities. activities. Moreover,Moreover, UBLs UBLs may may have have subs subsequentlyequently acquired acquired the the ability ability for for protein protein conjugation activ-ac- tivity;ity; whereaswhereas SCP, SCP, may may have have further further specialized specialized with with sulfur-transfer sulfur-transfer reactions reactions [3,4 ].[3,4]. Urm1 Urm1possesses possesses both both sequence sequence and and structural structural features features of of the the last last common common ancestor; ancestor; therefore, there- fore,Urm1 Urm1 is proposedis proposed to beto be a molecular a molecular fossil fossil [3 ].[3]. Consequently, Consequently, this this has has allowed allowed us us to to use useUrm1 Urm1 as as a modela model to to study study the the specialization specialization of of a a UBL UBL on on tRNA tRNA thio thio modification modification at at the theexpense expense of of the the lack lack of of protein protein conjugation conjugation [3 [3,4].,4]. A rm1_P_2qjlAScUrm1 1 MVNVKVEFLGGLDAI FGKQRVHKIKMDKEDPVTVGDL IDHI VSTMINNPNDVSI F IEDDS 60 biquitin_P_3cmmBScUbiquitin 1 ---MQIFV-----KTLTG-KTITLEVES--SDTIDNVKSKIQDKEGIPP----------- 38 oaD_P_1jw9DEcMoaD 1 --MIKVLFFAQVRELVGT-DATEVAAD---FPTVEALRQHMAAQSDRWALA--LED---- 48 AMP1_P_3po0AHvSAMP1 1 ----MWKADA----VAGS-RTVRVDVDG--DATVGDA-DA-VGAH---ASR---VGDDG- 40 rm1_P_2qjlAScUrm1 61 IRPGI ITL INDTDWELEGEK- - - - -DYILEDGDI ISFTSTLHGG 99 biquitin_P_3cmmBScUbiquitin 39 - - DQQRL I FAGKQLE - - - - DGRTLSDYNIQKESTLHLVLRLRGG 76 oaD_P_1jw9DEcMoaD 49 --GKLLAAVNQTLVSF---------DHPLTDGDEVAFFPPVTGG 81 AMP1_P_3po0AHvSAMP1 41 -YDH--NVRNGAAAG------------ATAAGD------AVSGG 63 B N N C C ScUrm1 overlay N N N C C C ScUbiquitin EcMoaD HvSAMP1 FigureFigure 1. Comparison 1. Comparison of Urm1, of Urm1, ubiquitin, ubiquitin, SAMP1, SAMP1, and and MoaD. MoaD. (A) ( AStructure-based) Structure-based sequence sequence align- align- mentment of ofUrm1 Urm1 (UniProt (UniProt entry entry P40554), P40554), ub ubiquitiniquitin (UniProt entryentry P0CH08)P0CH08) from fromSaccharomyces Saccharomyces cerevisiae cere- , visiae,SAMP1 SAMP1 (UniProt (UniProt entry entry D4GUF6) D4GUF6) from fromHaloferax Haloferax volcanii volcaniiand and MoaD MoaD (UniProt (UniProt entry entry P30748) P30748) from fromEscherichia Escherichia coli. coli.The The degree degree of conservation of conservation is reflected is reflected by color by color intensity. intensity. Sequences Sequences were alignedwere alignedwith PROMALS3Dwith PROMALS3D [5] and [5] visualized and visualized using Jalviewusing Jalview [6]. (B )[6]. Richardson (B) Richardson ribbon ribbon diagrams diagrams of the three- of thedimensional three-dimensional schematic schematic representation representation of protein of structuresprotein structures of ScUrm1 of (PDBScUrm1 entry (PDB 2QJL), entryScUbiquitin 2QJL), ScUbiquitin (PDB entry 3CMM), EcMoaD (PDB entry 1JW), and HvSAMP1 (PDB entry 3PO0). Struc- (PDB entry 3CMM), EcMoaD (PDB entry 1JW), and HvSAMP1 (PDB entry 3PO0). Structures were tures were superimposed and visualized using PyMol [7] and their N- and C-terminus are indicated. superimposed and visualized using PyMol [7] and their N- and C-terminus are indicated. TheThe Urm1-Uba4-system Urm1-Uba4-system is not is notonly only restricted restricted to eukaryotes, to eukaryotes, but also but is also found is found in ar- in chaealarchaeal systems. systems. Ubiquitin-like Ubiquitin-like SAMPs SAMPs (small archaeal (small archaeal modifier modifier proteins) proteins) were first were identi- first fiedidentified in Haloferax in Haloferax volcanii [8–11]. volcanii The[8– SAMP1,11]. The 2, SAMP1, and 3 are 2, andconj 3ugated are conjugated to their protein to their targets protein bytargets an isopeptide by an isopeptide bond. The bond.SAMP-activating The SAMP-activating enzyme UbaA enzyme is critical UbaA for is protein critical forconjuga- protein tionconjugation as well as sulfur as well mobili as sulfurzation mobilizationduring tRNA duringthiolation tRNA and thiolationmolybdopterin
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