Crystal structure of SopA, a Salmonella effector proteinmimicking a eukaryotic ubiquitin ligaseJianbo Diao1, Ying Zhang1, Jon M Huibregtse2, Daoguo Zhou1 & Jue Chen1

Bacterial pathogens deliver virulence proteins into host cells to facilitate entry and survival. Salmonella SopA functions as anE3 ligase to manipulate the host proinflammatory response. Here we report the crystal structure of SopA in two conformations.Although it has little sequence similarity to eukaryotic HECT-domain E3s, the C-terminal half of SopA has a bilobal architecturethat is reminiscent of the N- and C-lobe arrangement of HECT domains. The SopA structure also contains a putative substrate-binding domain located near the E2-binding site. The two structures of SopA differ in the relative orientations of the C lobe,indicating that SopA possesses the conformational flexibility essential for HECT E3 function. These results suggest that SopA isa unique HECT E3 ligase evolved from the coevolutionary selective pressure at the bacterium-host interface.

Covalent attachment of ubiquitin to proteins regulates a broad range ofcritical cellular processes, including cell-cycle progression, immuneresponses, postreplication DNA repair and transcription. Ubiquitina-tion can have several types of effects on substrate proteins, the mostcommon being degradation by the 26S proteasome1. Ubiquitination isachieved through an enzyme cascade consisting of E1, E2 and E3enzymes1. E1 activates the C terminus of ubiquitin by forming athioester bond with the terminal carboxyl group of ubiquitin. Theactivated ubiquitin is transferred from E1 to the active site cysteine ofan E2 enzyme in a transthiolation reaction, preserving the thioesterlinkage. E3s interact with both the substrates and E2s, facilitatingtransfer of ubiquitin to lysine residues of substrate proteins. Most E3sbelong to either the RING-domain (really interesting new gene) orHECT-domain (homologous to the carboxyl terminus of E6AP)families of proteins, which are quite distinct in sequence, structureand catalytic properties. RING E3s share a characteristic globularstructure formed by B70 residues, with a set of cysteine and histidineresidues coordinating two zinc ions. HECT E3s share a B350-residueC-terminal domain having a strictly conserved catalytic cysteine residuepositioned B35 residues upstream of the C terminus2. Whereas RINGligases function as molecular scaffolds to bring E2 and the substratesinto close proximity, HECT E3s participate directly in the chemistry ofubiquitination by accepting ubiquitin from an E2 enzyme, again in theform of a ubiquitin-thioester intermediate, and directly catalyzingprotein ubiquitination3. Crystal structures determined for threeeukaryotic HECT domains4–6 show a common fold of two lobes linkedby a flexible hinge. The structure of the E6AP HECT domain incomplex with an E2 enzyme, UbcH7, demonstrates that E2 binds to ahydrophobic pocket on the N-terminal lobe (N lobe), at the oppositeend from the C-terminal lobe (C lobe)4. Because the catalytic cysteines

in E2 and E3 are 41 A apart, it is evident that conformational changeswould be required to bring the two cysteine residues in proximityfor the transthiolation reaction. Compared with E6AP, the two otherknown HECT domain structures show different relative orientationsof the C lobe and the N lobe5,6. The three different HECT structurescan be interconverted by rigid-body movements of the C lobe arounda four-residue hinge loop. Mutations that reduce the flexibilityof the hinge loop abolish or reduce E3 ligase activity6, indicatingthat movement of the C lobe may be essential for HECT E3 ligasesto carry out the ubiquitin transfer reaction. However, such structuralrearrangements have not been observed for any particular HECT E3ligase, and the orientation of the E6AP N and C lobes is not altered byE2 binding4.Salmonella enterica serovar typhimurium (S. typhimurium), a

bacterial pathogen responsible for over one billion human infectionseach year, has evolved to coexist with its eukaryotic hosts. Using acomplex machinery named the type III secretion and translocationsystems (T3SS), the bacterium delivers a variety of effector proteinsinto the host cell to promote bacterial entry and survival7. Animportant mechanism by which microbial pathogens manipulatehost cellular function is mimicry of the activities of host enzymes8.We have recently reported that Salmonella SopA, a T3SS effectorprotein, functions as an E3 ligase regulating host inflammatoryresponses9. To understand the molecular basis of how bacterialSopA mimics eukaryotic HECT E3s, we determined the crystalstructure of SopA in two conformations. Although it has many uniquecharacteristics, SopA shares an overall structural similarity to HECTE3s. Notably, this bacterial protein has obtained, through apparentconvergent evolution, several key elements of the HECT domain,including the sequence of the active site loop, the bilobal architecture,

Received 23 June; accepted 18 October; published online 9 December 2007; doi:10.1038/nsmb1346

1Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA. 2Molecular Genetics and Microbiology, Institute for Cellular andMolecular Biology, The University of Texas at Austin, Austin, Texas 78712, USA. Correspondence should be addressed to J.C. (chenjue@purdue.edu) orD.Z. (zhoud@purdue.edu).

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and the conformational flexibility of the C lobe. In addition, thecrystal structure of SopA contains a putative substrate-bindingdomain that is located near the E2 binding site, which may suggesthow the substrate-binding and HECT domains are spatially organized.

RESULTSUbiquitination activity of SopAWhen incubated with ubiquitin, E1, E2 (UbcH7) and ATP, a glu-tathione S-transferase (GST)-SopA56–782 fusion protein forms a thio-ester intermediate9 and catalyzes autoubiquitination in vitro (Fig. 1a).The active site of SopA, Cys753, is 30 residues from the C terminus,and C753S mutation completely abolishes the autoubiquitinationactivity (Fig. 1). Although Salmonella SopA shares little sequencesimilarity with eukaryotic HECT E3s, an alignment of residuessurrounding the active site indicates that, besides the catalytic cysteine,a leucine six residues before Cys753 and a threonine that immediatelyprecedes Cys753 are conserved between SopA and all eukaryoticHECT E3s (Fig. 1b). Whereas mutation of Leu747 to alanine yieldedonly slightly lower activity, mutation of Thr752 to alanine reduced theautoubiquitination activity substantially (Fig. 1c). Consistent with thisresult, the corresponding T819A mutant of E6AP, a human HECT E3,also had reduced activity4. This conserved threonine residue is locatedat the interface of the N and C lobes and was suggested to be

important in stabilizing a catalytically competent conformation4–6.However, several other highly conserved and functionally importantresidues in the active site loop of E6AP, such as His818, Phe821 andAsp822, are not found in SopA.

Crystal structure of SopAWhereas the full length SopA was prone to proteolysis, a constructcontaining residues 163–782 was stable during purification and wasactive in ubiquitination (Fig. 1a). We obtained two structures of SopAfrom two different crystal systems. Native protein SopA163–782 crystal-lized in a tetragonal system diffracted to 2.1 A, and selenomethionine(SeMet)-substituted SopA165–782 crystallized in a monoclinic systemdiffracted to 2.8 A. SopA163–782 consists of three structural domains:an N-terminal b-helix, a central elongated region and a C-terminalglobular domain (Fig. 2a and Supplementary Fig. 1 online). Exceptfor the marked differences in the relative orientations of theC-terminal globular domain, the domain structures of SopA werevery similar in the two crystal forms.

56–7

82

56–7

82 (C

/S)

163–

782

56–7

82

56–7

82 (C

/S)

163–

782

Size(kDa)

250

150

100

Size(kDa)250

150

100

Anti-GST

SopAE6-APNedd-4UPL-2Oxi-1Smurf-1Rsp5p

StHsMmAtCeDmSc

Anti-Ub

WT L/A

Anti-GST Anti-Ub

GST-SopA

GST-SopA-(Ub)n

GST-SopA-(Ub)n

T/A C/S WT L/A T/A C/S

a

b

c

Figure 1 E3 ligase activity of SopA. (a) Western blots showing auto-

ubiquitination by GST-SopA56–782, SopA56–782(C/S) or SopA163–782.

(b) Local sequence alignment of S. typhimurium (St) SopA with eukaryotic

HECT E3 ligases human (Hs) E6AP, mouse (Mm) Nedd-4, Arabidopsis

thaliana (At) UPL-2, Caenorhabditis elegans (Ce) Oxi-1, Drosophila

melanogaster (Dm) Smurf1 and Saccharomyces cerevisiae (Sc) Rsp5p.

Conserved residues are boxed. (c) The functional roles of conserved

residues in the active site loop. Western blots show autoubiquitination

of GST-SopA56–782 and its derivatives L747A (L/A), T752A (T/A) or

C753S (C/S) after incubation with ubiquitin, E1 and E2 at 35 1C for 60 min.

163

β-helix domain Central domain C-terminal domain

370 590 612 782

C lobe28 Å

25 Å

55 Å 60 Å

N lobe

SopA

Cys753

Cys820C (Ala782)

N (Glu370) N

C

E6AP

Gln369

Ala163

90°

a

c

b

Cys753 Cys753

Figure 2 SopA crystal structure. (a) Stereo view of SopA163–782, consisting

of three domains: an N-terminal b-helix domain (cyan), a central domain

(purple) and a C-terminal domain (gold), connected to the central domain

by a helical linker (green). The catalytic cysteine (Cys753) is indicated (red).Terminal residues (Ala163, Ala782) are also labeled. (b) Ribbon diagram

of SopA residues 163–348, the putative substrate-binding domain. Side

chains that form the hydrophobic ladders are shown in the right-hand view.

(c) Structural comparison of SopA with the human E6AP HECT domain.

Only residues 370–782 of SopA are shown. Secondary-structure elements

are distinguished by color: a-helix, blue; b-sheet, red; loops, yellow. The

catalytic cysteine residues are shown in ball-and-stick models (orange).

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Previous functional studies of E6AP and other HECT E3s indicatethat the region upstream of the HECT domain is required forsubstrate recognition10. The corresponding region in SopA showed acylindrical structure termed a parallel b-helix (Fig. 2b). The centralpart of the cylinder was formed by five coils stacking upon each other.The N-terminal end was capped by an a-helix of 22 A in length; theC-terminal end was covered by strands and two short helices. The coilsof the cylinder had four faces, and the central residue of each face wasinvariably hydrophobic, with side chains pointing into the center ofthe cylinder to form four ‘hydrophobic ladders’ (Fig. 2b). Residues onthe exterior of the cylinder were randomly oriented and variablein their chemical compositions. Besides the hydrophobic sidechain–stacking interactions, the b-helix was further stabilized byextensive hydrogen-bonding interactions between the peptide back-bone atoms of neighboring coils. The b-helix motif has been found inabout 30 proteins, including pectate lyase C from a plant pathogen,the phage P22 tailspike protein, the P69 pertactin toxin fromBordetella pertussis and several polysaccharide-degrading enzymes11.Most of the b-helix–containing proteins are involved in binding tocarbohydrates, and the sugar binding site, a cleft formed by protrudingloops, is found on the exterior of the cylinder11. Notably, a cleft is alsofound on the surface of the b-helix domain of SopA. In the differenceFourier map there is strong unidentified electron density locatedinside the cleft (Supplementary Fig. 2 online). Although it is possiblethat the first 162 residues removed for crystallization also participatein substrate binding, the presence of a b-helix domain upstream ofthe catalytic HECT domain suggests that SopA may recognize carbo-hydrate-modified substrate proteins.

The overall molecular architecture of the C-terminal half of theprotein, residues 370–782, resembled that of eukaryotic HECTdomains. This region had a bilobal structure, with an elongateddomain (N lobe) connected to a globular C-terminal lobe (C lobe).The N and C lobes were connected by a 33-residue a-helical linker(Fig. 2a,c), whereas the linker in eukaryotic HECT domains is short(three or four amino acids) and unstructured. The N lobe of SopA,containing about 220 residues, had a rod shape with dimensions ofB55 A � 25 A � 30 A. The structure of the C lobe (residues 612–782)was composed entirely of eight a-helices and connecting loops(a11 to a18). The N lobe of SopA was similar in size and shape tothose of the eukaryotic HECTs. As in the N lobe of E6AP, which can bedivided into two subdomains linked through two extendedstrands4, there were two different packing arrangements within thecentral domain of SopA. The smaller subdomain consisted of ahelical hairpin (a1 and a2,), and the larger subdomain was made ofsix a helices (a4 to a9) and three b strands (b1 to b3). The twosubdomains were connected by a helix (a3), which packed diagonally

to the helical hairpin (Fig. 2a,c). There was no apparent sequencesimilarity between the N lobes of E6AP and SopA, and thefirst residues of the N lobes of E6AP and SopA were located atopposite ends of the molecule. Whereas the smaller subdomainof SopA was formed by N-terminal residues of the N lobe, thecorresponding domain of E6AP, comprising the E2 binding site, isinserted into a segment that makes up the larger subdomain(Fig. 2c). The C lobe of SopA was larger than a canonical HECTC lobe, consisting of 170 residues instead of 100. Instead of thea/b structure found in eukaryotic HECT domains, the C lobe ofSopA consisted entirely of a-helices. The helices were folded roughlyinto a spherical shape, with a central four-helix bundle (a14 to a17)flanked by four peripheral helices (a11, a12, a13 and a18). The activesite loop (residues 747–755) was located on the surface of the moleculefacing the b-helix domain. The C-terminal residue (Ala782) waspartially buried at the cleft between the central domain and theC-terminal domain.

Conformational flexibilityThe two SopA structures determined in two different crystal systemsdiffered in the orientation of the N and C lobes (Fig. 3). The nativeSopA163–782 adopted a more open form, with the C lobe looselypacked to the N lobe. Except a few interactions with the linker helix,the C lobe had no contacts with the N lobe or the b-helix domain.Alignment of the structures showed that, compared to the nativeprotein, the C lobe of the SeMet-substituted SopA165–782 was rotatedapproximately 251 toward the N lobe, moving the active site Cys753 by14 A. The lobe rotated by bending the linker helix around residueGln598. Consequently, the N and C lobes formed new contacts,burying about 300 A2 of solvent-accessible surface at the interface.The active site cysteine remained exposed to solvent in both con-formations. The C lobe movement was much smaller than that ineukaryotic HECT domain structures, where a 31-A displacement ofthe active site cysteine was observed between WWP1 and E6AP HECTdomains4,6. In contrast to changes at the N and C lobe interface, theinteraction between the N lobe and the b-helix domain was essentiallyunchanged between the native and SeMet forms (Fig. 3).

Interactions with E2Because the molecular surface of SopA was completely different fromthat of eukaryotic HECT E3s, it was not possible to model theE2 binding site based on the E6AP-UbcH7 structure4. The b-helixdomain of SopA was positioned very near the site that binds E2 inE6AP, raising the possibility that the b-helix might preclude inter-actions with an E2 protein because of steric hindrance or, alternatively,might form part of the E2 binding site. To map the SopA E2interaction site, we tested several truncations for their ability tointeract with UbcH7. Two constructs, one consisting of residues163–782 and one of residues 370–782, each bound stably to UbcH7in GST pull-down experiments (Fig. 4a). To quantify the interactionsof SopA with UbcH7, we labeled UbcH7 with fluorescein-5-maleimideand measured the change in fluorescence polarization upon titrationof SopA (Fig. 4b). The dissociation constants (Kd) determined forSopA163–782 and SopA370–782 were identical (32.4 mM), indicating thatthe b-helix domain neither participates in nor interferes withE2 binding. Compared with E6AP, which has a Kd around 6 mM(refs. 12,13), the affinity of SopA for UbcH7 is approximately five-foldweaker. A shorter construct containing residues 470–782 did notbind UbcH7 in a GST pull-down experiment (Fig. 4a), suggestingthat the distal end of the N lobe (a1 to a5), which would correspondmost closely with the E2 binding site of E6AP, is key for E2

25° 25°

Figure 3 Conformational flexibility. Superimposed SopA structures from two

crystal forms in stereo view. Native SopA structure, red; SeMet-substituted

structure, blue. The active site cysteines are shown as balls.

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interactions. Accordingly, the autoubiquitination activity of SopA470–782

was substantially lower than that of SopA370–782, and the controlconstruct SopA570–782 showed no activity (Supplementary Fig. 3online). The structure of the E6AP–Ubc7 complex showed that ahighly conserved phenylalanine residue of UbcH7, Phe63, is key inmediating interactions with eukaryotic HECT domains4. Alteration ofthis residue abolishes or severely weakens the ability of E2s to functionwith HECT E3s12,14, with no defects in the interactions with E1 nor inthe formation of a ubiquitin thioester13,14. Consistently, the UbcH7F63A mutant bound to SopA with less affinity (Kd 4 400 mM,Fig. 4a,b). Moreover, compared with the wild-type UbcH7, higherconcentrations of the F63A mutant were required for the autoubiqui-tination activity of SopA (Fig. 4c). SopA was able to use both wild-type UbcH7 and the F63A mutant at concentrations greater than0.2 mM; however, SopA had a clear preference for wild-type UbcH7over the F63A mutant at lower concentrations of E2 (Fig. 4c). Thus,Phe63 in UbcH7 may be critical in recognition of SopA also.

DISCUSSIONPreviously we showed that Salmonella SopA forms an ubiquitin-thioester intermediate9, indicating that it carries out ubiquitina-tion by a mechanism similar to that of HECT E3 ligases. Thecrystal structures showed that SopA contained three domains, withthe C-terminal two resembling the bilobal architecture of HECT E3s.Comparison of different HECT domain structures has led to a

model in which the conformational flexibility of the C loberelative to the N lobe is essential for catalyzing substrate ubiquitina-tion6, and the two alternative structures of SopA are consistentwith this model. The sequence similarity between SopA andHECT E3s is limited to the activity loop, and the folding of theN and C lobes is different from that in eukaryotic HECT E3 ligases.Therefore, one cannot completely rule out the possibility thatSopA belongs to a previously unidentified class of E3 ligases.

An intriguing feature of SopA is the proximity of the putativesubstrate-binding domain to the E2-binding site (Fig. 5). Thisarrangement is in marked contrast to the simplest predicted modelfor eukaryotic HECT E3 ligases, in which the substrate-bindingdomain would be at the end of the molecule opposite the E2-bindingsite15. No structural information is available to indicate how thesubstrate-binding and HECT domains of eukaryotic HECT E3sare spatially organized, and it will therefore be particularly interestingto see whether the SopA model is applicable to eukaryotic HECTE3s. Confirmation of the SopA model awaits identification of abona fide substrate and establishment of the functional role of theb-helix domain.

Recent studies show that a common strategy used by microbialpathogens is to exploit host cellular pathways through functionalmimicry8. In many cases, the bacterial proteins show little sequencenor structural similarity to the host cell protein, except for a few keyfeatures preserved to perform the same function8. Here we show thatSalmonella SopA represents another case of such mimicry. A BLASTsearch identified a SopA homolog in enterohemorrhagic Escherichiacoli O157:H7 (GI:13361024), with 26% amino acid sequence identity.In addition to SopA, two other bacterial T3SS effectors, AvrPtoB fromPseudomonas syringae16 and IpaH from Shigella17, function as ubiqui-tin ligases. Neither AvrPtoB nor IpaH shares sequence similaritywith SopA. Structural studies have shown that AvrPtoB is a RINGE3 ligase16, and biochemical evidence suggests that IpaH belongs toa new class of E3 ligases17. Studies of how these proteins functionwill shed light on how pathogens exploit the host ubiquitinationpathways and provide a unique angle on the molecular mechanismsunderlying ubiquitination.

METHODSCloning, expression and purification. We cloned DNA encoding

S. typhimurium SopA163–782 into a pET15b plasmid with an N-terminal His6

tag and transformed the plasmid into E. coli strain BL21 (DE3) (Novagen).

Bacteria were grown to log phase in LB medium at 37 1C and protein

expression was induced with 0.1 mM IPTG at 16 1C for 20 h. We purified

protein with a cobalt column (Clontech, TALON metal affinity resin) followed

by gel filtration (Amersham Biosciences, Superdex 200) and ion exchange

C lobe C lobe

Smurf2

E6AP

C lobe

WWP1

N lobe N lobe

Eukaryotic HECT-E3Bacterial HECT-E3

Substrate-bindingdomain

E2 E2

Figure 5 Comparison of bacterial and eukaryotic HECT E3 domains. Left,

bacterial HECT E3 ligase. The two locations of the C lobe are shown based

on the two conformations of SopA determined in this study. Modeled E2 is

shown in dotted lines. Right, eukaryotic HECT E3 ligase. The conformationalflexibility of the C lobe is modeled based on crystal structures of E6AP

(ref. 4), Smurf2 (ref. 5) and WWP1 (ref. 6).

SopA163–782 versus UbcH7 WTSopA370–782 versus UbcH7 WTSopA163–782 versus UbcH7 F63A

Nor

mal

ized

fluo

resc

ence

pola

rizat

ion

1.2

1.0

0.8

0.6

0.4

0.2

0.0

–0.20 50 100

[SopA] (µM)150 200 250

GST pull-down

F63A

163–

782

163–

782

370–

782

470–

782

GST –

UbcH7:

GST-SopA:

WT WT WT WT WT

Ubc

H7

+

Concentration:

WT UbcH7:

GST-SopA-(Ub)n

UbcH7(F63A):Size (kDa)

250

150

100

– + – + – + –

– + – + – + – +

b

a

c

Figure 4 SopA-E2 interactions. (a) GST pull-down. Immunoblot with anti-

UbcH7 of eluate from glutathione beads coated with immobilized GST-SopA,

incubated with wild-type UbcH7 or the F63A mutant. (b) Determination

of the dissociation constants of SopA-UbcH7 interactions. The change in

fluorescence polarization of fluorescein-labeled UbcH7 is plotted as a

function of SopA concentration. (c) Immunoblot with anti-ubiquitin showing

SopA autoubiquitination with wild-type UbcH7 and the F63A mutant.

Concentrations of UbcH7 or the F63A mutant (1 mM, 200 nM, 40 nMand 8 nM) decrease from left to right.

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(Amersham Biosciences, Source 15Q) chromatography. We expressed SeMet-

substituted protein in the methionine auxotroph strain B834 (DE3) and

purified it similarly to the native protein except that we added 10 mM

b-mercaptoethanol to purification buffers. The N-terminal His6 tag was

removed by protease before crystallization.

Crystallization and structural determination. Proteins were concentrated to

18 mg ml–1 and buffer exchanged to 20 mM Tris, pH 8.0. We grew crystals by

mixing protein solution with a reservoir solution containing 0.2 M sodium

potassium phosphate, 0.1 M Bis-Tris propane, pH 6.5, 20% (w/v) PEG3350 at

1:1 ratio in sitting drops at 20 1C. Crystals were stabilized in the reservoir

solution by adding ethylene glycol to 20% (v/v) and flash-frozen in liquid

nitrogen. Data were collected at 100 K on beam line 23-ID at the Advanced

Photon Source (Structural Biology Center, Argonne National Laboratory) and

processed with HKL2000 (ref. 18). We obtained experimental phasing using

SHARP19 and improved it by density modification20 (Supplementary Fig. 4

online). The initial model was built using program Coot21 and refined in CNS22

and REFMAC5 in CCP4 (ref. 20). We determined the native protein structure

by molecular replacement using a shorter fragment of the SeMet model

(residues 165–580). The structures are refined to good geometry, with no

residues in the disallowed region in the Ramachandran plots (Table 1).

GST pull-down assay. We cloned DNA encoding SopA163–782, SopA370–782,

SopA470–782 and UbcH7into plasmid pGEX-KG and expressed them as

GST-fusion proteins. We generated the UbcH7 F63A mutant using the

Quick-Change Site-Directed Mutagenesis-II kit (Stratagene). GST was

removed from UbcH7 and the F63A mutant by thrombin. GST-SopA

fragments were incubated with tag-free UbcH7 in 1:1 molar ratio for 1 h at

4 1C. We then added glutathione beads and incubated for 20 min. After

extensive washing, we loaded bound proteins onto

SDS-PAGE and visualized them by western blot

using mouse antibody to UbcH7 (BD Biosciences).

Fluorescence polarization assay. We prepared E2s

labeled with fluorescein-5-maleimide (Molecular

Probes) according to manufacturer’s recommended

protocol. Briefly, we incubated untagged wild-type

UbcH7 and the F63A mutant with fluorescein-5-

maleimide at 1:25 molar ratio at 4 1C for 12 h. Free

fluorescent dye was removed by gel filtration chro-

matography followed by dialysis. GST-SopA was

treated with thrombin to remove the GST tag and

then dialyzed against 20 mM HEPES buffer,

pH 7.5, and 0.1 M NaCl. We measured fluorescence

anisotropy at 25 1C using a Beacon 2000 fluores-

cence polarization instrument (VWR) with the

excitation wavelength set at 488 nm and emission

wavelength set at 535 nm. Increasing amounts of

SopA were added to aliquots of 15-nM wild-type

UbcH7 or 12-nM F63A mutant. We averaged data

from three measurements and fitted them to a

single-site binding model using nonlinear regression

with SigmaPlot.

In vitro ubiquitination assay. We performed in vitro

ubiquitination experiments as previously des-

cribed23. Briefly, a reaction mixture containing

ubiquitin, E1, E2 (UbcH7) and GST-SopA was

incubated at 35 1C for 60 min and immunoblotted

with rabbit antibody to GST or monoclonal anti-

ubiquitin. To examine the SopA-E2 interaction,

reaction mixtures containing decreasing concentra-

tions (1 mM, 200 nM, 40 nM and 8 nM) of wild-type

UbcH7 or the F63A mutant were incubated with

ubiquitin, E1 and GST-SopA163–782 at 35 1C for

90 min. Immunoblotting was performed using

monoclonal anti-ubiquitin.

Table 1 Data collection, phasing and refinement statistics

Native SeMet

Data collection

Space group P4122 P21

Cell dimensions

a, b, c (A) 79.7, 79.7, 212.7 102.7, 68.5, 106.6

a, b, g (1) 90, 90, 90 90, 90.9, 90

Peak Inflection Remote1 Remote2

Wavelength 1.07223 0.97945 0.97964 1.06369 0.95373

Resolution (A) 50–2.1 50–3.0 50–3.0 50–2.8 50–3.0

(2.2–2.1) (3.1–3.0) (3.1–3.0) (2.9–2.8) (3.1–3.0)

Rsym 7.2 (33.8) 9.4 (55.2) 8.6 (54.1) 6.2 (44.0) 7.8 (49.8)

I / sI 14.8 (2.8) 14.9 (3.2) 16.8 (3.0) 27.6 (3.0) 19.8 (3.6)

Completeness (%) 99.7 (95.8) 99.8 (98.9) 99.7 (97.0) 94.6 (84.0) 99.9 (99.3)

Redundancy 12.7 (9.2) 6.6 (6.1) 6.6 (5.9) 6.8 (5.0) 6.8 (6.2)

Refinement

Resolution (A) 50–2.1 50–2.8

No. reflections 35,698 33,990

Rwork / Rfreea 20.4/24.9 27.0/29.1

No. atoms

Protein 4,990 9,346

Ligand/ion 24 –

Water 134 95

B-factors

Protein 49.0 57.2

Ligand/ion 55.6 –

Water 43.9 91.4

R.m.s. deviations

Bond lengths (A) 0.008 0.008

Bond angles (1) 1.19 0.99

Values in parentheses are for highest-resolution shell.aRfree was calculated for a randomly chosen 5% of reflections.

Accession codes. Coordinates and structure factors of SopA have been

deposited in the Protein Data Bank under accession numbers 2QYU (native

protein) and 2QZA (SeMet-substituted protein).

Note: Supplementary information is available on the Nature Structural & MolecularBiology website.

ACKNOWLEDGMENTS

We thank the staff at the Advanced Photon Source beam line 23-ID for assistancewith data collection. This work was supported by US National Institutes ofHealth grants (AI049978 to D.Z. and CA072943 to J.M.H.) and by a Pewscholarship (to J.C.).

AUTHOR CONTRIBUTIONSJ.D. determined the structures of SopA and contributed the data for Figure 4a,b.Y.Z. subcloned most of the constructs and contributed the data for Figures 1 and4c and Supplementary Figure 3. J.D., Y.Z., J.M.H., D.Z. and J.C. designedexperiments, analyzed data and prepared the manuscript.

Published online at http://www.nature.com/nsmb/

Reprints and permissions information is available online at http://npg.nature.com/

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