Exchange of the Coronavirus Replicase Polyprotein Cleavage Sites Alters Protease Specificity and Processing

Coronavirus nonstructural proteins 1 to 3 are processed by one or two papain-like proteases (PLP1 and PLP2) at specific cleavage sites (CS1 to -3). Murine hepatitis virus (MHV) PLP2 and orthologs recognize and cleave at a position following a p4-Leu-X-Gly-Gly-p1 tetrapeptide, but it is unknown whether these residues are sufficient to result in processing by PLP2 at sites normally cleaved by PLP1. We demonstrate that exchange of CS1 and/or CS2 with the CS3 p4-p1 amino acids in engineered MHV mutants switches specificity from PLP1 to PLP2 at CS2, but not at CS1, and results in altered protein processing and virus replication. Thus, the p4-p1 residues are necessary for PLP2 processing but require a specific protein or cleavage site context for optimal PLP recognition and cleavage.Coronaviruses are positive-strand RNA viruses that translate their first open reading frames (ORF1a and ORF1b) into polyproteins that are processed by viral proteases into intermediate and mature nonstructural proteins (nsp1 to -16) (Fig. ​(Fig.11 A) (4, 7, 17, 20). nsp1, -2, and -3 are liberated at cleavage sites (CSs) between nsp1-2 (CS1), nsp2-3 (CS2), and nsp3-4 (CS3) by one or two papain-like protease (PLP) activities encoded within nsp3 (1, 2, 12, 13, 15) (Fig. ​(Fig.1B).1B). Murine hepatitis virus (MHV) and human coronavirus 229E (HCoV-229E) use two PLPs (PLP1 and PLP2) to process at CS1 to -3, while severe acute respiratory syndrome coronavirus (SARS-CoV) and avian infectious bronchitis virus (IBV) use a single PLP each (PLpro and PLP2, respectively) (10, 20, 25, 26). The factors determining the evolution and use of one versus two PLPs by different coronaviruses for processing of nsp1, -2, and -3 are unknown. Mutations at MHV CSs or within PLP1 alter replication and protein processing in surprising ways (8, 13). Loss of processing at MHV CS1 and CS2 by CS deletion or mutation results in changes in the timing and extent of virus replication. Inactivation of MHV PLP1 is more detrimental for virus replication than deletion of CS1 and CS2 or than inactivation of PLP1 combined with the CS deletions, even though not all of the mutant viruses process at CS1 or CS2 or display similar protein processing phenotypes. In contrast to MHV results, the HCoV-229E PLP1 and PLP2 have both been shown to process at CS1 and CS2, albeit at different efficiencies (Fig. ​(Fig.1B)1B) (24). Finally, the single SARS-CoV PLP2 homolog (PLpro) mediates efficient processing at CS1 to -3, each of which has an upstream position 4-Leu-X-Gly-Gly-position 1 (p4-LXGG-p1) amino acid motif implicated in PLpro processing (10, 16, 18). MHV possesses a p4-LXGG-p1 sequence only at CS3 and is cleaved by PLP2. These results suggest that p4-LXGG-p1 may be the critical determinant of recognition by PLP2/PLpro, but this hypothesis has not been tested in studies of replicating virus. Thus, it remains unknown whether the differences in PLP/CS recognition and processing are determined by the proximal p4-p1 residues (22).Open in a separate windowFIG. 1.MHV replicase organization, coronavirus PLP-mediated processing, and experimental design of cleavage site replacement viruses. (A) ORF1 of MHV genome RNA is shown, with overlapping ORF1a and ORF1b. The ORF1ab polyprotein is shown with nonstructural proteins (nsp1 to -16) indicated by vertical lines and numbers. Viral papain-like protease domains in nsp3 are shown as a white box containing black letters (PLP1) and a black box containing white letters (PLP2), and the nsp5 protease (3CLpro) is indicated as a gray box with a white number. Cleavage sites for PLP1 (CS1 and CS2 [shown as white arrowheads]), PLP2 (CS3 [shown as a black arrowhead]), and nsp5 (CS4 to -14 [shown as gray arrowheads]) are indicated. (B) The organization of nsp1 to nsp4 is shown for representative coronaviruses. PLPs are indicated, with the hatched box in IBV indicating a probable catalytically inactive remnant of PLP1. Processing events that were confirmed as occurring in vitro or during infection are shown by arrows with solid lines and large arrowheads, indicating single or dominant protease activity. The dashed lines and small arrowheads indicate minor or secondary cleavage activities. The CS amino acid sequences from position 4 (p4) to p1′ are shown for each CS, with a space and arrow representing the site of proteolytic processing. (C) The CS substitution viruses were engineered to replace the original CS amino acid sequences at CS1 and/or CS2 with that of the CS3 amino acid sequence p4-LKGG-p1. Both CS substitutions were also engineered into a catalytically inactive PLP1 (P1ko) background. PLPs are shown as numbers in boxes within nsp3. Engineered catalytically inactivated PLP1 is shown as a hatched box. Arrowheads indicate cleavage events of the WT virus and are linked to the enzyme predicted to mediate processing at the CS, as indicated by white boxes containing black characters (PLP1) or black boxes containing white characters (PLP2). The p4 through p1 amino acid residues for each CS are shown below each diagram. White and black vertical bars show the respective predicted PLP1 and PLP2 cleavage sites. Engineered substitutions are indicated in bold characters. Asterisks indicate engineered mutant genomes that could not be recovered as infectious virus.In this study, we used MHV as a model to test whether PLP/CS specificities could be switched by an exchange of CS amino acid sequences and to determine the impact of CS exchange on protein processing and virus replication. Replacement of the CS3 p4-LKGG-p1 at CS2, but not at CS1, was sufficient for a switch in protease specificity from PLP1 to PLP2. Some combinations of CS exchange could not be recovered with inactive PLP1, and recovered mutant viruses had altered protein processing and/or impaired growth compared to the wild type (WT). The results confirm that p4-LXGG-p1 amino acid sequences are necessary determinants of cleavage by PLP2 but also indicate that a larger cleavage site or a different protein context is required for efficient recognition and processing. Finally, the results support the conclusion that complex relationships with respect to the timing and extent of PLP/CS interactions are essential for successful replication and, likely, for virus fitness.     

BRIZ1 and BRIZ2 Proteins Form a Heteromeric E3 Ligase Complex Required for Seed Germination and Post-germination Growth in Arabidopsis thaliana

Ubiquitin pathway E3 ligases are an important component conferring specificity and regulation in ubiquitin attachment to substrate proteins. The Arabidopsis thaliana RING (Really Interesting New Gene) domain-containing proteins BRIZ1 and BRIZ2 are essential for normal seed germination and post-germination growth. Loss of either BRIZ1 (At2g42160) or BRIZ2 (At2g26000) results in a severe phenotype. Heterozygous parents produce progeny that segregate 3:1 for wild-type:growth-arrested seedlings. Homozygous T-DNA insertion lines are recovered for BRIZ1 and BRIZ2 after introduction of a transgene containing the respective coding sequence, demonstrating that disruption of BRIZ1 or BRIZ2 in the T-DNA insertion lines is responsible for the observed phenotype. Both proteins have multiple predicted domains in addition to the RING domain as follows: a BRAP2 (BRCA1-Associated Protein 2), a ZnF UBP (Zinc Finger Ubiquitin Binding protein), and a coiled-coil domain. In vitro, both BRIZ1 and BRIZ2 are active as E3 ligases but only BRIZ2 binds ubiquitin. In vitro synthesized and purified recombinant BRIZ1 and BRIZ2 preferentially form hetero-oligomers rather than homo-oligomers, and the coiled-coil domain is necessary and sufficient for this interaction. BRIZ1 and BRIZ2 co-purify after expression in tobacco leaves, which also requires the coiled-coil domain. BRIZ1 and BRIZ2 coding regions with substitutions in the RING domain are inactive in vitro and, after introduction, fail to complement their respective mutant lines. In our current model, BRIZ1 and BRIZ2 together are required for formation of a functional ubiquitin E3 ligase in vivo, and this complex is required for germination and early seedling growth.     

The Cullin-4 Complex DCDC Does Not Require E3 Ubiquitin Ligase Elements To Control Heterochromatin in Neurospora crassa

The cullin-4 (CUL4) complex DCDC (DIM-5/-7/-9/CUL4/DDB1 complex) is essential for DNA methylation and heterochromatin formation in Neurospora crassa. Cullins form the scaffold of cullin-RING E3 ubiquitin ligases (CRLs) and are modified by the covalent attachment of NEDD8, a ubiquitin-like protein that regulates the stability and activity of CRLs. We report that neddylation is not required for CUL4-dependent DNA methylation or heterochromatin formation but is required for the DNA repair functions. Moreover, the RING domain protein RBX1 and a segment of the CUL4 C terminus that normally interacts with RBX1, the E2 ligase, CAND1, and CSN are dispensable for DNA methylation and heterochromatin formation by DCDC. Our study provides evidence for the noncanonical functions of core CRL components.     

Detection of Nonstructural Protein 6 in Murine Coronavirus-Infected Cells and Analysis of the Transmembrane Topology by Using Bioinformatics and Molecular Approaches

Coronaviruses encode large replicase polyproteins which are proteolytically processed by viral proteases to generate mature nonstructural proteins (nsps) that form the viral replication complex. Mouse hepatitis virus (MHV) replicase products nsp3, nsp4, and nsp6 are predicted to act as membrane anchors during assembly of the viral replication complexes. We report the first antibody-mediated Western blot detection of nsp6 from MHV-infected cells. The nsp6-specific peptide antiserum detected the replicase intermediate p150 (nsp4 to nsp11) and two nsp6 products of approximately 23 and 25 kDa. Analysis of nsp6 transmembrane topology revealed six membrane-spanning segments and a conserved hydrophobic domain in the C-terminal cytosolic tail.Coronaviruses are enveloped, positive-stranded RNA viruses that sequester host cell membranes to assemble viral replication complexes in the cytoplasm of infected cells (2, 21). In the case of murine coronavirus mouse hepatitis virus (MHV), three viral proteases process the replicase polyproteins (3, 4, 5, 9, 12, 13, 14, 16, 18, 19, 24, 26) into intermediates and 16 mature nonstructural protein (nsp) products (Fig. ​(Fig.1A).1A). It is unclear whether the intermediate forms or the mature nsps are responsible for assembly of the viral replication complex. The replicase proteins nsp3, nsp4, and nsp6 contain transmembrane (TM)-spanning sequences that are proposed to be important for sequestering endoplasmic reticulum (ER) membranes to form the double-membrane vesicles which are the site of viral RNA synthesis (11, 17). However, the mechanism used by the nsps to generate double-membrane vesicles is not yet understood. Recent reports (8, 15, 22, 23, 28) and the study presented here have unraveled the membrane topology of these nsps. nsp4 is a glycoprotein with four TM domains (8, 22, 23, 28). nsp3 anchors its 213-kDa multidomain protein to ER membranes, likely using two TM domains (15, 22). Recently, nsp6 was shown to contain six TM domains (22); however, the authors were unable to resolve which of two C-terminal hydrophobic domains can act as the final membrane-spanning region.Open in a separate windowFIG. 1.Schematic diagram of MHV RNA genome, indicating the proteolytic processing scheme of the replicase polyprotein and Western blot detection of MHV nsp6. (A) MHV-A59 linear RNA genome with the canonical representation of replicase, structural, and accessory genes. The replicase polyprotein intermediates and mature nsps generated during processing are depicted. The mature nsp6 replicase protein (hatched box) and the antibodies used to detect nsp6 and nsp8 (solid black boxes) are indicated. aa''s, amino acids. (B) Western blot analysis of nsp6. Whole-cell lysates were prepared from mock-infected (M) and MHV-infected (I) HeLa-MHVR cells, and the lysates were separated by 12.5% SDS-PAGE. Products were detected by probing with nsp6- or nsp8-specific antibodies.In this report, we show the first antibody-mediated detection of MHV-A59 nsp6 in virally infected cells. We also report the TM topology of nsp6, as determined by glycosylation tagging and N-linked glycosylation sequence insertion mutagenesis approaches, providing evidence that nsp6 contains six membrane-spanning segments with a large C-terminal tail exposed to the cytosol. Multiple alignment of the nsp6 amino acid sequences from each coronavirus group revealed a high level of conservation at the C-terminal end, suggesting an evolutionarily conserved function.To detect nsp6 replicase protein in MHV-A59-infected cells, we used a polyclonal rabbit antiserum directed against a peptide (PLGVYNYKISVQEL) from the C-terminal region of nsp6. We detected the replicase intermediate p150 (nsp4 to nsp11) and two nsp6-specific products of 23 and 25 kDa (Fig. ​(Fig.1B,1B, lane 2) in MHV-infected HeLa-MHVR (25) cells by Western blot analysis. We found similar mature products of nsp6 in MHV-infected murine cell lines 17Cl-1 and DBT (data not shown). The same MHV-infected cell lysate was used to detect nsp8 replicase protein with a specific antibody that also recognizes p150 (Fig. ​(Fig.1B,1B, lane 4). The reason for the existence of multiple forms of nsp6 is currently unknown, although posttranslational modification or alternative processing of nsp6 cannot be ruled out at this point. Future experiments will be directed at purification and analysis of the two forms of nsp6 detected here.To develop a framework for understanding the membrane topology of nsp6, we first performed nsp6 bioinformatics analysis. Five out of the seven bioinformatics tools predicted that nsp6 would encode seven TM domains, whereas two programs predicted that it would encode eight TM domains (Fig. ​(Fig.2).2). However, because both the N and C termini of nsp6 must be processed in the cytosol by the viral 3C-like protease (3CLpro), we expected nsp6 to encode an even number of TM domains and established a consensus TM domain prediction for nsp6 (Fig. ​(Fig.2,2, bottom row). The consensus provided a working model for generating plasmid DNA constructs for evaluating the membrane topology of MHV nsp6. First, we employed enhanced green fluorescent protein (EGFP) glycosylation tagging (EGFPglyc) experiments as previously used for determining the membrane topology of other viral replicase TM proteins (20, 22). This approach allowed us to determine the localization of the tagged protein based on the differences in the mobility of the endoglycosidase H (endo H)-treated protein versus that of the untreated protein by the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Based on the consensus topology model (Fig. ​(Fig.3A)3A) suggesting a maximum of eight TM domains, we generated a series of plasmid DNA constructs starting with the N-terminal putative TM1 domain, and successively larger constructs were fused at their carboxyl terminus in frame with EGFPglycV5. The plasmid DNAs were individually transfected into BsrT7 cells (6), and the newly synthesized fusion proteins were radiolabeled with 100 μCi of [³⁵S]methionine-cysteine per ml from 20 to 22 h posttransfection. Chimeric proteins expressed from the cell lysates were immunoprecipitated with V5 antibody, either endo H treated or mock treated, separated using 12.5% SDS-PAGE, and analyzed by autoradiography as described previously (15).Open in a separate windowFIG. 2.Summary of TM predictions for MHV nsp6 obtained from membrane topology bioinformatics tools. The nsp6 amino acid sequence (amino acids 3637 to 3923 in the MHV A59 genome are numbered 1 to 287 for simplicity) was analyzed for TM-spanning domains by the use of various bioinformatics tools, and the residue numbers with predicted TM domains are displayed. The consensus TM topology of MHV nsp6 used as a basis for the topology experiments is depicted at the bottom row (shaded in gray).Open in a separate windowFIG. 3.Determining the topology of nsp6 by the use of EGFPglyc and insertion of glycosylation consensus sites. (A) Schematics of a working topology model of MHV nsp6 (obtained from our consensus experiments) and nsp6-EGFPglycV5 fusion constructs generated for endo H assay. (B) Metabolic labeling and endo H treatment of nsp6-EGFPglycV5 fusion proteins. The nsp6-EGFPglycV5 fusion proteins expressed in transfected BsrT7 cells were radiolabeled from 20 to 22 h posttransfection, and then cell lysates were subjected to immunoprecipitation with V5 antibody, treated with endo H or left untreated, separated by 12.5% SDS-PAGE, and analyzed by autoradiography. (C) Map of plasmid DNA construct showing the sites of inserted glycosylation acceptor consensus sequences (NXS). The locations of glycosylation insertion in the nsp6-V5 construct are represented, with the amino acid number at the site of insertion. (D) Metabolic labeling and endo H analysis of glycosylation sequence insertion expression constructs of nsp6-V5. The plasmid DNAs (iNsp6-V5 constructs) were transfected and analyzed as described for panel B. (E) MHV-A59 nsp6 topology model, summarizing the results of EGFPglycV5 and glycosylation sequence insertion experiments. Amino acid positions indicated by the symbol “Y” were glycosylated and were positive by endo H assay, whereas those positions tested but found not glycosylated and negative by endo H assay are depicted by solid black horizontal lines. The inserted glycosylation acceptor sequence positions precede the letter i. Selected charged residues are shown in white characters on a black background. K, lysine residues; R, arginine residues; E, glutamic acid residues.We found that fusion protein products expressed from the reporter constructs (nsp6-35glycV5, nsp6-86glycV5, and nsp-165glycV5) were glycosylated, as shown by sensitivity to endo H treatment, indicating that the C-terminal end of these chimeric proteins must extend into the ER lumen (Fig. ​(Fig.3B,3B, lanes 4, 8, and 10). In contrast, the remaining reporter constructs were not sensitive to endo H treatment; therefore, the C-terminal end of the chimeric constructs must extend into the cytoplasm (Fig. ​(Fig.3B,3B, lanes 6, 12, 14, 16, and 18). Thus, these results indicate the presence of three luminal loops in nsp6. Identical results were obtained when we used PNGaseF (data not shown), which indicates that the lack of endo H sensitivity was not attributable to the protein transiting through the Golgi body, thereby rendering the protein insensitive to endo H treatment.To further investigate nsp6 topology in detail, we exploited a glycosylation sequence insertion mutagenesis approach (7) to create acceptor sequences in the region between amino acids 86 and 200 of nsp6 by the use of site-directed mutagenesis as described in reference 32 in order to independently investigate the topology, since bioinformatics predictions of the TM domains within this region differ (Fig. ​(Fig.2).2). Consensus glycosylation acceptor sites (NXS) were generated at four sites in the nsp6-V5 plasmid backbone by introducing single-codon insertions as depicted in Fig. ​Fig.3C.3C. All the glycosylation insertion constructs were expressed and analyzed by use of the endo H assay as described above. As expected, the parental nsp6-V5 protein is not glycosylated and did not show a mobility shift after endo H treatment (Fig. ​(Fig.3D,3D, lanes 1 and 2). In contrast, expression of 99iNsp6-V5 revealed evidence of endo H sensitivity (Fig. ​(Fig.3D,3D, lanes 3 and 4), indicating the ER luminal localization of the N99 introduced into MHV nsp6. This result is in agreement with those obtained with the nsp6-86glycV5 construct that is also endo H sensitive (Fig. ​(Fig.3B,3B, lanes 7 and 8). The insertion of glycosylation acceptor sequences at other sites yielded endo H-negative results (lanes 6, 8, and 10), indicating the possibility that the introduced NX(S/T) motifs (i) are localized in the cytosol, (ii) are localized within the membrane, or (iii) are not used, as the glycosylation site is not at least 12 amino acids away from the end of the preceding TM and 14 amino acids away from the beginning of the following TM (12 + 14 rule), thus rendering it inaccessible for glycosylation (1, 7, 30). Our results confirm and extend the results of a recent study (22) in which authors were unable to resolve whether TM6 or TM7 acted as the final TM domain. Our results indicate that TM6 is the final TM domain for MHV nsp6. We propose a topology model of MHV-A59 nsp6 in Fig. ​Fig.3E3E which is in accordance with the distribution of positively charged residues (positive inside rule; reviewed in reference 31), depicting the higher number of lysine and arginine residues facing the cytosolic side of the membrane and the majority of charged residues excluded from the TM domain. Taken together, the results presented above are consistent with a six-TM domain model of MHV nsp6. This report provides new information on the membrane topology of nsp6 and provides potential clues with respect to the assembly of the coronavirus replication complex.To determine whether the experimentally determined six-TM-spanning domain topology of MHV-A59 is conserved among coronaviruses, we performed MUSCLE (10) and ClustalW (29) multiple sequence alignment of nsp6 amino acid sequences representing group 1, group 2, and group 3 coronaviruses obtained from PATRIC (http://patric.vbi.vt.edu/) (27). The most striking observations were the amino acid sequence conservation in the C terminus of all nsp6 proteins and the conservation in the hydrophobicities within the putative TM domains (Fig. ​(Fig.4).4). This analysis revealed several conserved sites that may be important for the function of nsp6. We designated the conserved region between TM2 and TM3 the “KH loop” because of the invariant lysine and histidine residues that are present in the cytosolic loop (Fig. ​(Fig.3E),3E), although the function of these amino acids is not yet known. We also designated the hydrophobic region in the C-terminal tail the “conserved hydrophobic domain” (Fig. ​(Fig.4).4). We speculate that cysteine residue(s) within the region we designated the “conserved G(X)C(X)G motif” may be modified by palmitoylation, indicating that this region of nsp6 may have important functions in establishing protein-protein or protein-membrane interactions during the assembly of the viral replication complex. Additionally, for the MHV-A59 nsp6 protein, the NetPhosK 1.0 server (http://www.cbs.dtu.dk/services/NetPhosK/) predicted serine and tyrosine residues (serine 244 and tyrosine 250; see Fig. ​Fig.4)4) at the C-terminal region as sites of possible phosphorylation by epidermal growth factor receptor kinase and protein kinase C, respectively. Both predicted sites are highly conserved in all coronavirus nsp6 proteins (Fig. ​(Fig.4).4). Overall, our analysis revealed conserved features in the nsp6 C-terminal region whose importance in viral replication can be investigated using a coronavirus reverse genetics system.Open in a separate windowFIG. 4.Multiple sequence alignment (MSA) and percent sequence identity of coronavirus nsp6. The nsp6 amino acid sequences of 18 different coronaviruses were obtained from PATRIC (http://patric.vbi.vt.edu/) and aligned using MUSCLE and ClustalW software. The experimentally determined TM domains of MHV-A59 nsp6 were used as a reference for alignment. Unshaded boxes indicate the conserved TM domains that aligned with other coronavirus nsp6 sequences; the conserved hydrophobic domain (CHD) predicted by all the topology programs is indicated by gray shading. The residues of the peptide against which the nsp6 antibody was raised are boxed, with residue designations shown in boldface. Putative sites for palmitoylation (cysteine residue[s]) within the GXCXG motif) and phosphorylation (serine 244 and tyrosine 250 in MHV-A59 nsp6) are indicated. Percent identity (% ID) values are indicated. In MSA, the following notations were used: asterisk indicate invariant amino acids, colons indicate highly similar amino acids, and dots indicate similar amino acids. HCoV, human coronavirus; PHEV, porcine hemagglutinating encephalomyelitis virus; BCoV, bovine coronavirus; BatSARS, bat severe acute respiratory syndrome coronavirus; BatCoV, bat coronavirus; SARSCoV, severe acute respiratory syndrome coronavirus; FIPV, feline infectious peritonitis virus; PRCoV, porcine respiratory coronavirus; TGEV, transmissible gastroenteritis virus; PEDV, porcine epidemic diarrhea virus; IBV, infectious bronchitis virus.     

Ubiquitin Regulates GGA3-mediated Degradation of BACE1

BACE1 (β-site amyloid precursor protein-cleaving enzyme 1) is a membrane-tethered member of the aspartyl proteases, essential for the production of β-amyloid, a toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. The BACE1 C-terminal fragment contains a DXXLL motif that has been shown to bind the VHS (VPS27, Hrs, and STAM) domain of GGA1–3 (Golgi-localized γ-ear-containing ARF-binding proteins). GGAs are trafficking molecules involved in the transport of proteins containing the DXXLL signal from the Golgi complex to endosomes. Moreover, GGAs bind ubiquitin and traffic synthetic and endosomal ubiquitinated cargoes to lysosomes. We have previously shown that depletion of GGA3 results in increased BACE1 levels and activity because of impaired lysosomal degradation. Here, we report that the accumulation of BACE1 is rescued by the ectopic expression of GGA3 in H4 neuroglioma cells depleted of GGA3. Accordingly, the overexpression of GGA3 reduces the levels of BACE1 and β-amyloid. We then established that mutations in the GGA3 VPS27, Hrs, and STAM domain (N91A) or in BACE1 di-leucine motif (L499A/L500A), able to abrogate their binding, did not affect the ability of ectopically expressed GGA3 to rescue BACE1 accumulation in cells depleted of GGA3. Instead, we found that BACE1 is ubiquitinated at lysine 501 and is mainly monoubiquitinated and Lys-63-linked polyubiquitinated. Finally, a GGA3 mutant with reduced ability to bind ubiquitin (GGA3L276A) was unable to regulate BACE1 levels both in rescue and overexpression experiments. These findings indicate that levels of GGA3 tightly and inversely regulate BACE1 levels via interaction with ubiquitin sorting machinery.     

Crystal Structure of the Human Ubiquitin-activating Enzyme 5 (UBA5) Bound to ATP: MECHANISTIC INSIGHTS INTO A MINIMALISTIC E1 ENZYME*

E1 ubiquitin-activating enzymes (UBAs) are large multidomain proteins that catalyze formation of a thioester bond between the terminal carboxylate of a ubiquitin or ubiquitin-like modifier (UBL) and a conserved cysteine in an E2 protein, producing reactive ubiquityl units for subsequent ligation to substrate lysines. Two important E1 reaction intermediates have been identified: a ubiquityl-adenylate phosphoester and a ubiquityl-enzyme thioester. However, the mechanism of thioester bond formation and its subsequent transfer to an E2 enzyme remains poorly understood. We have determined the crystal structure of the human UFM1 (ubiquitin-fold modifier 1) E1-activating enzyme UBA5, bound to ATP, revealing a structure that shares similarities with both large canonical E1 enzymes and smaller ancestral E1-like enzymes. In contrast to other E1 active site cysteines, which are in a variably sized domain that is separate and flexible relative to the adenylation domain, the catalytic cysteine of UBA5 (Cys²⁵⁰) is part of the adenylation domain in an α-helical motif. The novel position of the UBA5 catalytic cysteine and conformational changes associated with ATP binding provides insight into the possible mechanisms through which the ubiquityl-enzyme thioester is formed. These studies reveal structural features that further our understanding of the UBA5 enzyme reaction mechanism and provide insight into the evolution of ubiquitin activation.     

Evolution of the B3 DNA Binding Superfamily: New Insights into REM Family Gene Diversification

Background

The B3 DNA binding domain includes five families: auxin response factor (ARF), abscisic acid-insensitive3 (ABI3), high level expression of sugar inducible (HSI), related to ABI3/VP1 (RAV) and reproductive meristem (REM). The release of the complete genomes of the angiosperm eudicots Arabidopsis thaliana and Populus trichocarpa, the monocot Orysa sativa, the bryophyte Physcomitrella patens,the green algae Chlamydomonas reinhardtii and Volvox carteri and the red algae Cyanidioschyzon melorae provided an exceptional opportunity to study the evolution of this superfamily.

Methodology

In order to better understand the origin and the diversification of B3 domains in plants, we combined comparative phylogenetic analysis with exon/intron structure and duplication events. In addition, we investigated the conservation and divergence of the B3 domain during the origin and evolution of each family.

Conclusions

Our data indicate that showed that the B3 containing genes have undergone extensive duplication events, and that the REM family B3 domain has a highly diverged DNA binding. Our results also indicate that the founding member of the B3 gene family is likely to be similar to the ABI3/HSI genes found in C. reinhardtii and V. carteri. Among the B3 families, ABI3, HSI, RAV and ARF are most structurally conserved, whereas the REM family has experienced a rapid divergence. These results are discussed in light of their functional and evolutionary roles in plant development.     

The MLLE Domain of the Ubiquitin Ligase UBR5 Binds to Its Catalytic Domain to Regulate Substrate Binding

E3 ubiquitin ligases catalyze the transfer of ubiquitin from an E2-conjugating enzyme to a substrate. UBR5, homologous to the E6AP C terminus (HECT)-type E3 ligase, mediates the ubiquitination of proteins involved in translation regulation, DNA damage response, and gluconeogenesis. In addition, UBR5 functions in a ligase-independent manner by prompting protein/protein interactions without ubiquitination of the binding partner. Despite recent functional studies, the mechanisms involved in substrate recognition and selective ubiquitination of its binding partners remain elusive. The C terminus of UBR5 harbors the HECT catalytic domain and an adjacent MLLE domain. MLLE domains mediate protein/protein interactions through the binding of a conserved peptide motif, termed PAM2. Here, we characterize the binding properties of the UBR5 MLLE domain to PAM2 peptides from Paip1 and GW182. The crystal structure with a Paip1 PAM2 peptide reveals the network of hydrophobic and ionic interactions that drive binding. In addition, we identify a novel interaction of the MLLE domain with the adjacent HECT domain mediated by a PAM2-like sequence. Our results confirm the role of the MLLE domain of UBR5 in substrate recruitment and suggest a potential role in regulating UBR5 ligase activity.     

SARS-CoV-2 Nsp3 unique domain SUD interacts with guanine quadruplexes and G4-ligands inhibit this interaction

The multidomain non-structural protein 3 (Nsp3) is the largest protein encoded by coronavirus (CoV) genomes and several regions of this protein are essential for viral replication. Of note, SARS-CoV Nsp3 contains a SARS-Unique Domain (SUD), which can bind Guanine-rich non-canonical nucleic acid structures called G-quadruplexes (G4) and is essential for SARS-CoV replication. We show herein that the SARS-CoV-2 Nsp3 protein also contains a SUD domain that interacts with G4s. Indeed, interactions between SUD proteins and both DNA and RNA G4s were evidenced by G4 pull-down, Surface Plasmon Resonance and Homogenous Time Resolved Fluorescence. These interactions can be disrupted by mutations that prevent oligonucleotides from folding into G4 structures and, interestingly, by molecules known as specific ligands of these G4s. Structural models for these interactions are proposed and reveal significant differences with the crystallographic and modeled 3D structures of the SARS-CoV SUD-NM/G4 interaction. Altogether, our results pave the way for further studies on the role of SUD/G4 interactions during SARS-CoV-2 replication and the use of inhibitors of these interactions as potential antiviral compounds.     

Structure and Function of the PLAA/Ufd3-p97/Cdc48 Complex

PLAA (ortholog of yeast Doa1/Ufd3, also know as human PLAP or phospholipase A2-activating protein) has been implicated in a variety of disparate biological processes that involve the ubiquitin system. It is linked to the maintenance of ubiquitin levels, but the mechanism by which it accomplishes this is unclear. The C-terminal PUL (PLAP, Ufd3p, and Lub1p) domain of PLAA binds p97, an AAA ATPase, which among other functions helps transfer ubiquitinated proteins to the proteasome for degradation. In yeast, loss of Doa1 is suppressed by altering p97/Cdc48 function indicating that physical interaction between PLAA and p97 is functionally important. Although the overall regions of interaction between these proteins are known, the structural basis has been unavailable. We solved the high resolution crystal structure of the p97-PLAA complex showing that the PUL domain forms a 6-mer Armadillo-containing domain. Its N-terminal extension folds back onto the inner curvature forming a deep ridge that is positively charged with residues that are phylogenetically conserved. The C terminus of p97 binds in this ridge, where the side chain of p97-Tyr⁸⁰⁵, implicated in phosphorylation-dependent regulation, is buried. Expressed in doa1Δ null cells, point mutants of the yeast ortholog Doa1 that disrupt this interaction display slightly reduced ubiquitin levels, but unlike doa1Δ null cells, showed only some of the growth phenotypes. These data suggest that the p97-PLAA interaction is important for a subset of PLAA-dependent biological processes and provides a framework to better understand the role of these complex molecules in the ubiquitin system.     

Lysine 63-linked Polyubiquitination of the Dopamine Transporter Requires WW3 and WW4 Domains of Nedd4-2 and UBE2D Ubiquitin-conjugating Enzymes

RNA interference screen previously revealed that a HECT-domain E3 ubiquitin ligase, neuronal precursor cell expressed, developmentally down-regulated 4-2 (Nedd4-2), is necessary for ubiquitination and endocytosis of the dopamine transporter (DAT) induced by the activation of protein kinase C (PKC). To further confirm the role of Nedd4-2 in DAT ubiquitination and endocytosis, we demonstrated that the depletion of Nedd4-2 by two different small interfering RNA (siRNA) duplexes suppressed PKC-dependent ubiquitination and endocytosis of DAT in human and porcine cells, whereas knock-down of a highly homologous E3 ligase, Nedd4-1, had no effect on DAT. The abolished DAT ubiquitination in Nedd4-2-depleted cells was rescued by expression of recombinant Nedd4-2. Moreover, overexpression of Nedd4-2 resulted in increased PKC-dependent ubiquitination of DAT. Mutational inactivation of the HECT domain of Nedd4-2 inhibited DAT ubiquitination and endocytosis. Structure-function analysis of Nedd4-2-mediated DAT ubiquitination revealed that the intact WW4 domain and to a lesser extent WW3 domain are necessary for PKC-dependent DAT ubiquitination. Moreover, a fragment of the Nedd4-2 molecule containing WW3, WW4, and HECT domains was sufficient for fully potentiating PKC-dependent ubiquitination of DAT. Analysis of DAT ubiquitination using polyubiquitin chain-specific antibodies showed that DAT is mainly conjugated with Lys⁶³-linked ubiquitin chains. siRNA analysis demonstrated that this polyubiquitination is mediated by Nedd4-2 cooperation with UBE2D and UBE2L3 E2 ubiquitin-conjugating enzymes. The model is proposed whereby each ubiquitinated DAT molecule is modified by a single four-ubiquitin Lys⁶³-linked chain that can be conjugated to various lysine residues of DAT.     

Identification of a Novel Zn2+-binding Domain in the Autosomal Recessive Juvenile Parkinson-related E3 Ligase Parkin

Missense mutations in park2, encoding the parkin protein, account for ∼50% of autosomal recessive juvenile Parkinson disease (ARJP) cases. Parkin belongs to the family of RBR (RING-between-RING) E3 ligases involved in the ubiquitin-mediated degradation and trafficking of proteins such as Pael-R and synphillin-1. The proposed architecture of parkin, based largely on sequence similarity studies, consists of N-terminal ubiquitin-like and C-terminal RBR domains. These domains are separated by a ∼160-residue unique parkin sequence having no recognizable domain structure. We used limited proteolysis experiments on bacterially expressed and purified parkin to identify a new domain (RING0) within the unique parkin domain sequence. RING0 comprises two distinct, conserved cysteine-rich clusters between Cys¹⁵⁰–Cys¹⁶⁹ and Cys¹⁹⁶–His²¹⁵ consisting of CX₂-₃CX₁₁CX₂C and CX_4–6CX_10–16-CX₂(H/C) motifs. The positions of the cysteine/histidine residues in this region bear similarity to parkin RING1 and RING2 domains, as well as other E3 ligase RING domains. However, in parkin a 26-residue linker region separates the motifs, which is not typical of other RING domain structures. Further, the RING0 domain includes all but one of the known ARJP mutation sites between the ubiquitin-like and RBR regions of parkin. Using electrospray ionization mass spectrometry and inductively coupled plasma-atomic emission spectrometry analysis, we determined that the RING0, RING1, IBR, and RING2 domains each bind two Zn²⁺ ions, the first observation of an E3 ligase with the ability to bind eight metal ions. Removal of the zinc from parkin causes near complete unfolding of the protein, an observation that rationalizes cysteine-based ARJP mutations found throughout parkin, including RING0 (C212Y) that form cellular inclusions and/or are defective for ubiquitination likely because of poor zinc binding and misfolding. The identification of the RING0 domain in parkin provides a new overall domain structure for the protein that will be important in assessing the roles of ARJP mutations and designing experiments aimed at understanding the disease.Autosomal recessive juvenile Parkinson disease (ARJP)² is a neurodegenerative disorder arising from the loss of dopaminergic neurons in the substantia nigra of the midbrain. ARJP is characterized by the onset of Parkinsonian symptoms such as tremors, rigidity, and bradykinesia. It is distinguished from the idiopathic form of Parkinson disease by the onset of symptoms, prior to the age of forty. The hereditary nature of ARJP implicates a number of mutations in the genes encoding the proteins parkin, PINK1, LRRK2, and DJ-1 as the cause of dopaminergic neurodegeneration (1–4). A variety of deletion, truncation, and point mutations distributed throughout the park2 gene, which encodes the protein parkin, have been reported in ARJP patients (1, 5–18).Parkin functions as a ubiquitin ligase (E3) and belongs to a family of RBR (RING-between-RING) ubiquitin ligase enzymes involved in proteosome-mediated protein degradation (19–21). The currently accepted domain architecture of parkin, deduced from multiple sequence alignment, shows that the C terminus of the protein is characterized by two ∼50-residue RING (really interesting new gene) domains separated by a 51-residue IBR (In-Between-RING) domain (22, 23). The RING domains of parkin are proposed to interact with the ubiquitin-conjugating enzymes UbcH7, UbcH8, Ubc7, and Ubc13 and control parkin-mediated ubiquitination of a variety of substrates such as Pael-R, synphilin-1, Sept5, and PICK1 among others (24–31). Other members of the RBR family include the human homolog of Drosophila Ariadne (HHARI), DORFIN, and HOIL-1, which share close domain architecture (32–35). Traditionally, RING domains coordinate two Zn²⁺ ions through a C₃HC₄ metal-binding consensus sequence. However, the RING2 domain of HHARI binds a single Zn²⁺ (36), and because this is the only RING2 structure available for an RBR protein, it suggests that there may be variability in the number of Zn²⁺ ions coordinated by different RING domains. The recent three-dimensional structure of the parkin IBR domain (23) revealed a two-site zinc-binding motif with a novel fold compared with other zinc-binding motifs (37). However, despite the potential importance of zinc binding to the RING domains (or other portions) of parkin, the ability and capacity for zinc coordination or its impact on structure has not been identified for parkin.The N terminus of parkin comprises a ubiquitin-like domain (UblD) proposed to facilitate the delivery and degradation of ubiquitinated substrates by the 26 S proteosome via interactions with the S5a subunit (38, 39). The central ∼150 residues of parkin separating the UblD from the RBR region are referred to as the unique parkin domain (UPD). This segment of parkin is essential for function, and ARJP associated mutations within this region have been shown to lead to dysfunction of parkin E3 ligase activity (40, 41). However, the absence of any sequence similarity to other proteins or the identification of a distinct domain within the UPD has made these experiments difficult to interpret. Other than the isolated UblD and IBR domains of parkin, there has been limited success with the purification and characterization of parkin, especially when lacking affinity tags in the final purified form. Bacterially expressed parkin typically shows a heterogeneous mixture of full-length and degraded protein species, making characterization of the protein difficult (42). In this work we have used purified parkin to identify a novel zinc-binding C₄C₃(C/H) domain upstream of the RBR region and within the UPD. We have used limited proteolysis and electrospray ionization mass spectrometry (ESI-MS) to show that this domain coordinates two Zn²⁺ ions in addition to six other Zn²⁺ ions in the RBR C terminus. The presence of a new parkin-specific zinc-binding domain provides insight into the structure of parkin and opens the door to establish the importance of this domain in ARJP for this new subclass of RBR E3 ligases.     

Nuclear Magnetic Resonance Structure of the Nucleic Acid-Binding Domain of Severe Acute Respiratory Syndrome Coronavirus Nonstructural Protein 3

The nuclear magnetic resonance (NMR) structure of a globular domain of residues 1071 to 1178 within the previously annotated nucleic acid-binding region (NAB) of severe acute respiratory syndrome coronavirus nonstructural protein 3 (nsp3) has been determined, and N- and C-terminally adjoining polypeptide segments of 37 and 25 residues, respectively, have been shown to form flexibly extended linkers to the preceding globular domain and to the following, as yet uncharacterized domain. This extension of the structural coverage of nsp3 was obtained from NMR studies with an nsp3 construct comprising residues 1066 to 1181 [nsp3(1066-1181)] and the constructs nsp3(1066-1203) and nsp3(1035-1181). A search of the protein structure database indicates that the globular domain of the NAB represents a new fold, with a parallel four-strand β-sheet holding two α-helices of three and four turns that are oriented antiparallel to the β-strands. Two antiparallel two-strand β-sheets and two 3₁₀-helices are anchored against the surface of this barrel-like molecular core. Chemical shift changes upon the addition of single-stranded RNAs (ssRNAs) identified a group of residues that form a positively charged patch on the protein surface as the binding site responsible for the previously reported affinity for nucleic acids. This binding site is similar to the ssRNA-binding site of the sterile alpha motif domain of the Saccharomyces cerevisiae Vts1p protein, although the two proteins do not share a common globular fold.The coronavirus replication cycle begins with the translation of the 29-kb positive-strand genomic RNA to produce two large polyprotein species (pp1a and pp1ab), which are subsequently cleaved to produce 15 or possibly 16 nonstructural proteins (nsp''s) (11). Among these, nsp3 is the largest nsp and also the largest coronavirus protein. nsp3 is a glycosylated (16, 22), multidomain (36, 51), integral membrane protein (38). All known coronaviruses encode a homologue of severe acute respiratory syndrome coronavirus (SARS-CoV) nsp3, and sequence analysis suggests that at least some functions of nsp3 may be found in all members of the order Nidovirales (11). Hallmarks of the coronavirus nsp3 proteins include one or two papain-like proteinase domains (3, 12, 16, 31, 56, 62), one to three histone H2A-like macrodomains which may bind RNA or RNA-like substrates (5, 9, 48, 54, 55), and a carboxyl-terminal Y domain of unknown function (13). An extensive bioinformatics analysis of the coronavirus replicase proteins by Snijder et al. (51) provided detailed annotations of the then-recently sequenced SARS-CoV genome (35, 47), including the identification of a domain unique to SARS-CoV and the prediction of the ADP-ribose-1″-phosphatase (ADRP) activity of the X domain (since shown to be one of the macrodomains).Only limited information is so far available regarding the ways in which the functions of nsp3 are involved in the coronavirus replication cycle. Some functions of nsp3 appear to be directed toward protein; e.g., the nsp3 proteinase domain cleaves the amino-terminal two or three nsp''s from the polyprotein and has deubiquitinating activity (4, 6, 14, 30, 53, 60). Most homologues of the most conserved macrodomain of nsp3 appear to possess ADRP activity (9, 34, 41-43, 48, 59) and may act on protein-conjugated poly(ADP-ribose); however, this function appears to be dispensable for replication (10, 42) and may not be conserved in all coronaviruses (41). The potential involvement of nsp3 in RNA replication is suggested by the presence of several RNA-binding domains (5, 36, 49, 54, 55). nsp3 has been identified in convoluted membrane structures that are also associated with other replicase proteins and that have been shown to be involved in viral RNA synthesis (16, 24, 52), and nsp3 papain-like proteinase activity is essential for replication (14, 62). Other conserved structural features of nsp3 include two ubiquitin-like domains (UB1 and UB2) (45, 49). We have also recently reported that nsp3 is a structural protein, since it was identified as a minor component of purified SARS-CoV preparations, although it is not known whether nsp3 is directly involved in virogenesis or is incidentally incorporated due to protein-protein or protein-RNA interactions (36).A nucleic acid-binding region (NAB) is located within the polypeptide segment of residues 1035 to 1203 of nsp3. The NAB is expected to be located in the cytoplasm, along with the papain-like protease, ADRP, a region unique to SARS-CoV (the SARS-CoV unique domain [SUD]), and nsp3a, since both the N and C termini of nsp3 were shown previously to be cytoplasmic (38). Two hydrophobic segments are membrane spanning (38), and the NAB is located roughly 200 residues in the N-terminal direction from the first membrane-spanning segment. This paper presents the next step in the structural coverage of nsp3, with the determination of the NAB structure. The structural studies included nuclear magnetic resonance (NMR) characterization of two constructs, an nsp3 construct comprising residues 1035 to 1181 [nsp3(1035-1181)] and nsp3(1066-1203), and complete NMR structure determination for the construct nsp3(1066-1181) (see Fig. ​Fig.8).8). The structural data were then used as a platform from which to investigate the nature of the previously reported single-stranded RNA (ssRNA)-binding activity of the NAB (36). Since no three-dimensional (3D) structures for the corresponding domains in other group II coronaviruses are known and since the SARS-CoV NAB has only very-low-level sequence identity to other proteins, such data could not readily be derived from comparisons with structurally and functionally characterized homologues.Open in a separate windowFIG. 8.Sequence alignment of the polypeptide segment nsp3(1066-1181) that forms the globular domain of the SARS-CoV NAB with homologues from other group II coronaviruses. Protein multiple-sequence alignment was performed using ClustalW2 and included sequences from SARS-CoV Tor2 (accession no. {"type":"entrez-protein","attrs":{"text":"AAP41036","term_id":"30795144","term_text":"AAP41036"}}AAP41036) and representatives of three protein clusters corresponding to three group II coronavirus lineages identified by a BLAST search: bat coronavirus HKU5-5 (BtCoV-HKU5-5; accession no. {"type":"entrez-protein","attrs":{"text":"ABN10901","term_id":"124389468","term_text":"ABN10901"}}ABN10901), BtCoV-HKU9-1 (accession no. {"type":"entrez-protein","attrs":{"text":"P0C6T6","term_id":"190360129","term_text":"P0C6T6"}}P0C6T6), and human coronavirus HKU1-N16 (HCoV-HKU1-N16; accession no. {"type":"entrez-protein","attrs":{"text":"ABD75496","term_id":"89515407","term_text":"ABD75496"}}ABD75496). Above the sequences, the positions in full-length SARS-CoV nsp3, the locations of the regular secondary structures in the presently solved NMR structure of the SARS-CoV NAB globular domain, and the residue numbering in this domain are indicated. Amino acids are colored according to conservation and biochemical properties, following ClustalW conventions. Residues implicated in interactions with ssRNA are marked with inverted black triangles. In the present context, the key features are that there is only one position with conservation of K or R (red) and that there are extended sequences with conservation of hydrophobic residues (blue) (see the text).     

Identification of a Peptide Inhibitor of the RPM-1·FSN-1 Ubiquitin Ligase Complex

The Pam/Highwire/RPM-1 (PHR) proteins include: Caenorhabditis elegans RPM-1 (Regulator of Presynaptic Morphology 1), Drosophila Highwire, and murine Phr1. These important regulators of neuronal development function in synapse formation, axon guidance, and axon termination. In mature neurons the PHR proteins also regulate axon degeneration and regeneration. PHR proteins function, in part, through an ubiquitin ligase complex that includes the F-box protein FSN-1 in C. elegans and Fbxo45 in mammals. At present, the structure-function relationships that govern formation of this complex are poorly understood. We cloned 9 individual domains that compose the entire RPM-1 protein sequence and found a single domain centrally located in RPM-1 that is sufficient for binding to FSN-1. Deletion analysis further refined FSN-1 binding to a conserved 97-amino acid region of RPM-1. Mutagenesis identified several conserved motifs and individual amino acids that mediate this interaction. Transgenic overexpression of this recombinant peptide, which we refer to as the RPM-1·FSN-1 complex inhibitory peptide (RIP), yields similar phenotypes and enhancer effects to loss of function in fsn-1. Defects caused by transgenic RIP were suppressed by loss of function in the dlk-1 MAP3K and were alleviated by point mutations that reduce binding to FSN-1. These findings suggest that RIP specifically inhibits the interaction between RPM-1 and FSN-1 in vivo, thereby blocking formation of a functional ubiquitin ligase complex. Our results are consistent with the FSN-1 binding domain of RPM-1 recruiting FSN-1 and a target protein, such as DLK-1, whereas the RING-H2 domain of RPM-1 ubiquitinates the target.     

Ankyrin Repeat Domain Protein 2 and Inhibitor of DNA Binding 3 Cooperatively Inhibit Myoblast Differentiation by Physical Interaction

Ankyrin repeat domain protein 2 (ANKRD2) translocates from the nucleus to the cytoplasm upon myogenic induction. Overexpression of ANKRD2 inhibits C2C12 myoblast differentiation. However, the mechanism by which ANKRD2 inhibits myoblast differentiation is unknown. We demonstrate that the primary myoblasts of mdm (muscular dystrophy with myositis) mice (pMB^mdm) overexpress ANKRD2 and ID3 (inhibitor of DNA binding 3) proteins and are unable to differentiate into myotubes upon myogenic induction. Although suppression of either ANKRD2 or ID3 induces myoblast differentiation in mdm mice, overexpression of ANKRD2 and inhibition of ID3 or vice versa is insufficient to inhibit myoblast differentiation in WT mice. We identified that ANKRD2 and ID3 cooperatively inhibit myoblast differentiation by physical interaction. Interestingly, although MyoD activates the Ankrd2 promoter in the skeletal muscles of wild-type mice, SREBP-1 (sterol regulatory element binding protein-1) activates the same promoter in the skeletal muscles of mdm mice, suggesting the differential regulation of Ankrd2. Overall, we uncovered a novel pathway in which SREBP-1/ANKRD2/ID3 activation inhibits myoblast differentiation, and we propose that this pathway acts as a critical determinant of the skeletal muscle developmental program.     

Structure of the Legionella Virulence Factor,SidC Reveals a Unique PI(4)P-Specific Binding Domain Essential for Its Targeting to the Bacterial Phagosome

The opportunistic intracellular pathogen Legionella pneumophila is the causative agent of Legionnaires’ disease. L. pneumophila delivers nearly 300 effector proteins into host cells for the establishment of a replication-permissive compartment known as the Legionella-containing vacuole (LCV). SidC and its paralog SdcA are two effectors that have been shown to anchor on the LCV via binding to phosphatidylinositol-4-phosphate [PI(4)P] to facilitate the recruitment of ER proteins to the LCV. We recently reported that the N-terminal SNL (SidC N-terminal E3 Ligase) domain of SidC is a ubiquitin E3 ligase, and its activity is required for the recruitment of ER proteins to the LCV. Here we report the crystal structure of SidC (1-871). The structure reveals that SidC contains four domains that are packed into an arch-like shape. The P4C domain (PI(4)P binding of SidC) comprises a four α-helix bundle and covers the ubiquitin ligase catalytic site of the SNL domain. Strikingly, a pocket with characteristic positive electrostatic potentials is formed at one end of this bundle. Liposome binding assays of the P4C domain further identified the determinants of phosphoinositide recognition and membrane interaction. Interestingly, we also found that binding with PI(4)P stimulates the E3 ligase activity, presumably due to a conformational switch induced by PI(4)P from a closed form to an open active form. Mutations of key residues involved in PI(4)P binding significantly reduced the association of SidC with the LCV and abolished its activity in the recruitment of ER proteins and ubiquitin signals, highlighting that PI(4)P-mediated targeting of SidC is critical to its function in the remodeling of the bacterial phagosome membrane. Finally, a GFP-fusion with the P4C domain was demonstrated to be specifically localized to PI(4)P-enriched compartments in mammalian cells. This domain shows the potential to be developed into a sensitive and accurate PI(4)P probe in living cells.