bioRxiv preprint doi: https://doi.org/10.1101/2020.03.10.984922; this version posted March 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Molecular basis of ubiquitination catalyzed by the bacterial transglutaminase MavC 2 1,2* 3* 1,2* 1,2* 3 1,2 3 Hongxin Guan , Jiaqi Fu , Ting Yu , Zhao-Xi Wang , Ninghai Gan , Yini Huang , 1 1,2 3† 1,2† 4 Vanja Perčulija , Yu Li , Zhao-Qing Luo and Songying Ouyang 5 1 6 The Key Laboratory of Innate Immune Biology of Fujian Province, Provincial University 7 Key Laboratory of Cellular Stress Response and Metabolic Regulation, Biomedical 8 Research Center of South China, Key Laboratory of OptoElectronic Science and 9 Technology for Medicine of the Ministry of Education, College of Life Sciences, Fujian 10 Normal University, Fuzhou, China 2 11 Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine 12 Science and Technology (Qingdao), Qingdao, China 3 13 Purdue Institute for Inflammation, Immunology and Infectious Disease and Department of 14 Biological Sciences, Purdue University, West Lafayette, IN, USA 15 16 *These authors contributed equally to this work. 17 †Corresponding authors: [email protected] (ZQL); [email protected] (SO) 18 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.10.984922; this version posted March 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 19 Summary 20 The Legionella pneumophila effector MavC is a transglutaminase that carries out atypical 21 ubiquitination of the ubiquitin (Ub) E2 conjugation enzyme UBE2N by catalyzing the 22 formation of an isopeptide bond between Gln40 of Ub and Lys92 (or to a less extent, Lys94) 23 of UBE2N, which results in inhibition of UBE2N signaling in the NF-κB pathway. In the 24 absence of UBE2N, MavC deamidates Ub at Gln40 or catalyzes self-ubiquitination. 25 However, the mechanisms underlying these enzymatic activities of MavC are not fully 26 understood at molecular level. In this study, we obtained the structure of the 27 MavC-UBE2N-Ub ternary complex that represents a snapshot of covalent cross-linking of 28 UBE2N and Ub catalyzed by MavC. The structure reveals the unique way by which the 29 cross-linked catalytic product UBE2N-Ub binds mainly to the Insertion and the Tail 30 domains of MavC prior to its release. Based on our structural, biochemical and mutational 31 analyses, we proposed the catalytic mechanism for both the deamidase and the 32 transglutaminase activities of MavC. Finally, by comparing the structures of MavC and 33 MvcA, the homologous protein that reverses MavC-induced UBE2N ubiquitination, we 34 identified several key regions of the two proteins responsible for their opposite enzymatic 35 activity. Our results provide insights into the mechanisms for substrate recognition and 36 ubiquitination mediated by MavC as well as explanations for the opposite activity of MavC 37 and MvcA. 38 39 Keywords 40 Legionella pneumophila; effectors; transglutaminase; ubiquitination; deamidase 41 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.10.984922; this version posted March 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 42 Introduction 43 Signal transduction in cells is often mediated by posttranslational modifications 44 (PTMs), which impact the activity of existing proteins to allow rapid responses to upstream 1,2 45 cues. Among more than 200 different types of PTMs identified so far , ubiquitination is 46 one of the most widely used. Canonical ubiquitination requires the activities of the E1, E2 47 and E3 enzymes that respectively activate, conjugate and ligate the 76-residue ubiquitin 3 48 (Ub) to modify proteins . Ubiquitination itself is further regulated by ubiquitination and other 49 types of PTMs such as phosphorylation, acetylation and ADP-ribosylation that target Ub, 4 50 components of the ubiquitination machinery, or both . This complex crosstalk among 51 various PTMs allows cells to achieve better fine-tuning of their response to various stimuli, 4,5 52 particularly under disease conditions . 53 Pathogens have evolved diverse mechanisms to co-opt host functions to promote their 54 fitness. One such mechanism is the acquisition of virulence factors capable of effective 6 55 modulation of cellular processes by various PTMs . Legionella pneumophila, the causative 56 agent of Legionnaires’ disease, is one such example. The intracellular life cycle of this 57 bacterium utilizes the Dot/Icm type IV secretion system that injects hundreds of virulence 7,8 58 factors known as effectors into host cells . These effectors extensively modulate cell 59 signaling hubs such as small GTPases and the Ub network to create a niche permissive for 9 60 intracellular replication of the L. pneumophila . 61 Co-option of the host Ub network by L. pneumophila appears to be of particular 62 importance for modulating host cellular immune process to facilitate its intracellular 63 replication. More than 10 effectors with E3 Ub ligase activity have been identified. Although 64 their target proteins remain elusive in most cases, theseeffectors cooperate with E1 and E2 10 65 enzymes in host cells to form active ubiquitination machineries (Fig. S1A). A paradigm 66 shift discovery was made by the study of the SidE effector family (SidEs) that includes + 67 effectors such as SdeA, which catalyze a NAD -dependent ubiquitination. This mechanism 68 involves Ub activation via ADP-ribosylation and phosphodiesterase (PDE)-mediated 69 ligation of phosphoribosylated ubiquitin (PR-Ub) onto serine residues of substrate proteins 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.10.984922; this version posted March 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 11-13 70 (Fig. S1B). Interestingly, two research groups recently reported that DupA and DupB, 71 the two highly homologous PDE domain-containing deubiquitinases from L. pneumophila, 14 72 similarly reverse phosphoribosyl serine ubiquitination on their substrates (Fig. S1B). 73 Moreover, the activity of SidEs is regulated by SidJ, another effector which inhibits the 15-17 74 mono-ADP-ribosyltransferase activity by calmodulin-dependent glutamylation (Fig. 75 S1B). 76 The modification of the E2 enzyme UBE2N by MavC represents another atypical 77 ubiquitination mechanism. In this reaction, UBE2N, which exists in cell primarily as a 18,19 78 UBE2N~Ub conjugate linked by a thioester bond , is ligated to Ub via an isopeptide 79 bond formed between Gln40 of Ub and Lys92 (i.e. γ-glutamyl-ε-Lys bond between UbGln40 20 80 and UBE2NLys92) or, to a lesser extent, Lys94 of UBE2N . This ligation is mediated by 21 81 transglutamination, a reaction that does not require exogenous energy (Fig. S1C-D). 82 Analogously to other transglutaminases that function as deamidases in the absence of 22 83 their target substrates , MavC uses catalytic Cys74 that is crucial for both enzymatic 23 84 activities (Fig. S1E). Ubiquitination at Lys92 abolishes the activity of UBE2N, which in 85 turn curbs the formation of K63-type polyubiquitin chains through canonical ubiquitination 20 86 otherwise mediated by UBE2N, E1 and UVE1, thereby inhibiting NF-κB activation (Fig. 87 S1A). 23 88 MavC and its homolog MvcA are structurally similar to the canonical ubiquitin 89 deamidase cycle inhibiting factor (Cif) effectors from enteropathogenic Escherichia coli and 24,25 90 its homolog in Burkholderia pseudomallei (CHBP) (Fig. 1). Both MavC and MvcA have 91 ubiquitin deamidase activity but only MavC is able to induce monoubiquitination of UBE2N. 92 Furthermore, we recently found that MvcA counteracts the trangslutamination activity of 21 93 MavC by removing ubiquitin from UBE2N-Ub . However, although the structures of MavC 23 94 and its homolog MvcA have been solved (Fig. 1C), the mechanism underlying 95 transglutaminase-induced UBE2N ubiquitination by MavC and the molecular basis for their 96 opposite catalytic activities both remain elusive. Here, by solving the structure of the 97 MavC-UBE2N-Ub ternary complex and comparing it to other available structures of MavC 98 and MvcA, we illustrate the structural basis for substrate recognition by MavC and the 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.10.984922; this version posted March 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 99 mechanism that mediates the formation of the isopeptide bond between Lys92 in UBE2N 100 and Gln40 in Ub. In addition, structural comparison of the MavC and MvcA in their apo form 101 and in ternary complex has allowed us to gain insights into the basis of the opposite 102 biochemical activity exhibited by these two highly similar proteins in terms of regulation of 103 UBE2N ubiquitination. 104 105 Results 106 The Insertion domain of MavC is essential for UBE2N ubiquitination but not for 107 self-ubiquitination and ubiquitin deamidation activities of MavC 108 Purified MavC from E. coli exists primarily as a mixture of monomers and dimers in 109 solution, both of which interact with UBE2N in size-exclusion chromatography (SEC) (Fig.