Brucella Immunogenic BP26 Forms a Channel-like Structure

Brucella Immunogenic BP26 Forms a Channel-like Structure

Daegeun Kim1, Jihye Park1, Soo Jin Kim2, Young-Min Soh1, Ho Min Kim2, Byung-Ha Oh1 and Ji-Joon Song1,3 1 - Department of Biological Sciences, KI for the BioCentury, KAIST, 335 Gwahangno, Daejeon 305–701, Korea 2 - Graduate School of Medical Science and Engineering, KAIST, 335 Gwahangno, Daejeon 305–701, Korea 3 - Graduate School of Nanoscience and Technology (WCU), KAIST, 335 Gwahangno, Daejeon 305–701, Korea

Correspondence to Ji-Joon Song: Department of Biological Sciences, KI for the BioCentury, KAIST, 335 Gwahangno, Daejeon 305–701, Korea. [email protected] http://dx.doi.org/10.1016/j.jmb.2013.01.015 Edited by J. Johnson

Abstract An outer membrane protein BP26/OMP28 of Brucella, BP26, is identified as a major immunodominant antigen and widely used as a diagnostic marker and for vaccination against Brucellosis. BP26 belongs to the family of proteins that contains a SIMPL (signaling molecule that associates with the mouse pelle-like kinase) domain, whose structure and function have been unknown. Here, we present the crystal structure of BP26 revealing that 16 BP26 molecules form a novel channel-like assembly as also shown by electron microscopy analysis. Eight BP26 molecules forming a ring structure contain a hole at the center of the octamer, and another octamer interacts with each other to form a channel having a large internal cavity. BP26 is found to be structurally similar to a bacteriophage protein involved in infection, implicating that BP26 might function during Brucella infection. In addition, the BP26 structure suggests that the protein functions as a multimeric channellike form and provides a canonical model for the SIMPL domains. © 2013 Elsevier Ltd. All rights reserved.

Introduction Brucella is a Gram-negative bacterium belonging to the α-proteobacteria division and is the cause of Brucellosis, one of the common zoonotic diseases. 1,2 Brucella infects host phagocytic and dendritic cells and escapes the host innate immune system to establish the chronic infection. 3–5 Despite that Brucella causes economic loss and occasionally infects human, early diagnosis of Brucellosis is difficult because of the poor specific symptom in early stage of infection and a lack of easy detection method. 6,7 The periplasmic space of Brucella between inner membrane and outer membrane contains peptidoglycan and soluble proteins. 8,9 Lipopolysaccharide (LPS) on the outer membrane is the major virulence factor of Brucella and has been used as an antigen for vaccination and as a diagnostic marker due to the strong immunoreactivity. 9 However, serological diagnosis of Brucellosis based on LPS detection has a problem of false positive reactivity with other Gram-negative bacteria. 10 Outer membrane pro-

teins (OMPs) in the periplasm also interplay with the host immune system so that OMPs are also considered as good candidates for vaccine development and diagnosis. 11 Brucella OMPs are divided into three groups according to their molecular masses. 12 Group 3 OMPs having molecular mass in the 25- to 31-kDa range have been extensively studied for diagnostics and vaccine development. 13–16 OMP28, one of the Group 3 OMP, is conserved throughout the Brucella genus. The transmembrane sequence of OMP28 is cleaved off and the resulting protein is called BP26. BP26 was initially identified as a major immunoreactive protein in animals infected by Brucella and is predicted to have a “SIMPL” (signaling molecule that associates with the mouse pelle-like kinase) domain with unknown function. Because BP26 is highly conserved in Brucella genus but not in other genera, it has been used as a diagnostic marker specific for Brucellosis. 17–19 Moreover, BP26 has been used as an alternative to LPS to develop vaccines for Brucellosis. 20,21 Indeed, BP26 was shown to have a

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J. Mol. Biol. (2013) 425, 1119–1126

1120

BP26 Forms a Channel-like Structure

protective antigenic function and adjuvant potency and to promote humoral and cellular responses. 21–23 So far, many studies on BP26 have been focused on diagnostics and vaccine development and little is known about molecular feature of BP26. We determined the crystal structure of BP26, revealing that the BP26 monomer is composed of two domains with a similar protein fold. Notably, 16 molecules of BP26 form a peculiar channel-like structure with a hole size of about 30 Å. This structure provides the first view of the SIMPL domains and reveals an unexpected structural similarity with the pilus-binding domain of a bacteriophage protein.

the transmembrane sequence was determined at 3.5 Å resolution using Se single-wavelength anomalous dispersion (SAD) method (Table 1). A clear electron density map was generated and 10 Semethionines in each BP26 molecule allowed unequivocal registration of amino acids into the electron density (Supplementary Fig. 1). The crystal belongs to the I4122 space group and there are four BP26 molecules in the asymmetric unit. The four BP26 molecules are nearly identical with each other with less than 1 Å pairwise r.m.s.d. The BP26 monomer is composed of two domains (domain I and domain II) (Fig. 1b and c). Interestingly, the two domains have a similar structural organization. Both domains are composed of one βsheet and two α-helices (Fig. 1b and c), and they can be indeed coarsely superimposed with an r.m.s.d. of 3.7 Å (Fig. 1d). In the domain I, the β-sheet is composed of three antiparallel β-strands. Two β-strands come from the N-terminal (β1, residues 39–50) and the C-terminal (β6, residues 235–250) portions of BP26. The third

Results Monomeric structure Homologues of Brucella BP26 are found in most of proteobacteria (Fig. 1a). The crystal structure of Brucella abortus BP26 (29–250 amino acids) lacking

(a)

Transmembrane domain

Brucella (1-100) Vibrio (1-100) Ochrobactrum (1-100) Agrobacterium (1-98) Rhizobium (1-98) Ahrensia (1-91) SIMPL (1-98)

MN T RAS N F L AAS F MNNRA T T L L A T S F MNNRA T T L L A T S F - - MK T KQAR L F AL - - MK T KS AR L F AL - - - - - - - MR T L S I - - M I TLKLKV I AL

Brucella (101-198) Vibrio (101-198) Ochrobactrum (101-198) Agrobacterium (99-197) Rhizobium (99-197) Ahrensia (92-191) SIMPL (99-192)

I I I F F F L

Brucella (199-250) Vibrio (199-251) Ochrobactrum (199-251) Agrobacterium (198-247) Rhizobium (198-247) Ahrensia (192-243) SIMPL (193-242)

VE VE VE I Q I Q TQ I S

•

P A F A Q E N QM T T Q P A R P A L A Q E N QM T K Q P A R P A L A Q E N QM T K Q P A R S GQV L AQD AN P R E A T S GQ L L AQE A T P R E A T PA I AEDT - - T PKPGV AAP AQAQE L P ADP P T

3

5-2

I I I I I I L

TGYSVS TS L TGY TVSNS L TGY TVSNS L TGYQV S NGL TGYQV S NGL VGY V V T NGL TGYRASNTV

5-3

VRVRE VRVRD VRVRD VRVRD VRVRD VRVRD V TVRD

4

•

L N L S V L RQAK L NL SV L REAK L NL SV L REAK L S F S V V KQAE L S F S V V KQAE L N LMVMRE AE L S LGVVVQAK

T A R E AM T A N N E AM T K V T A R E AM T A N N E AM A K V T A R E AM T A N N E AM A K V T AAAAL T ENSKAL ADV T AAAAL SENSKAL ADV T A R A A L D A N T Q AM A A V T A A E A K K A N A E RMA A V

L ANVGK I L AKVGNV L AKVGNV LKKLGT I L KKLGA I LGK LGT I L DKLGE L

L L L L L L L

L D AM K K AG L E AMK K AG L E AMK K AG LKALKEAA LKALKEAA L T DMK A E G I AA L KKAG

I I I I I I I

E DRD L Q T GG E DRD L Q T GG E DRD L Q T GG EDRDLQTSN EDRDLQTSN DERDLQTSN AKKD I QT SN

3

2 T T T T T T E

D E S V T L GV NQGGD L N D E S V T L G I NQGGD L N D E S V T L GV NQGGD L S D T S V K L G I NQGGD I S D T S V K L G I NQGGD I S DR S V T L GV N S GGN V R DE AV KAGANQ I NG I S

L VNDNP F VNDNP F VNDNP F TNDKP F TNDKP F ANDDP FGVDDP

SAV I NEARKRAVANA I AAT I NEARKRAV TDA I AAT I NEARKRAVADA I E A T V T E ARKQAV ADAL E A T V T E ARKQAV ADAL A E A I N Q A R K N AM E N A I E AAV QQARK AAV AD A I

100 100 100 98 98 91 98

5-1 AKAK T L AKAK T L AKAK T L AKAK T L SKAK T L AKATML AKAQA L

ADAAGVGL GRV ADAAGVG I GR L ADAAGVG I GRV T E AAGV K L GRV T E AAGV K L GRV T E T AG I GL AR I AS ALGVK LGRV

198 198 198 197 197 191 192

6

•

S E L S R P PMPMP I ARGQ F R TM L AAAP DN S V P N E QS R P PMPMP I AR AQ F K TMAD AAP QGS V P NEQS RP PMPMP I ARAQY K TMAAAAP E DAV P S E NMQ R - - P M P V P Q GMM R A AM A K E A D S - V P T E N S Q R - - P M P V P Q AMM R A S M A K E A D S - V P S E Q N R G - R P R P Q AM AM A R S KM A A D E A A P V P S E NS S NPQPMP I AR AAAAAAP AGAV A T - - P

(b)

A V T G E GMM T A S P DM A I A V T G E G TM T A S P DMA I A V T G E G TM T A S P DMA I S V T G E GQ A A I A P DMA I S V S G E GQ A A I A P DMA I S V S A T GN A D V A P DMA I T V S G E G E V T A V P DMA T

4

D I QP I YV YPDDKNN - - L KEP T N I QP RY V Y P DDKNG - - L KE P S N I QP RY V Y P DDKNG - - L KE P S S VQP L YKHY E P KDG - V Y V AP E S VQP L YKHY E P KDG - V Y V AP E N I NP RY F Y P P RKNDGTQKP P R S VQPQYDY P E - - NG - - - - E P E

I I I I I I I

I I I I I I I

1

2

1

S T I ML VGAF S L S A I ML AGA L T L S A I ML AGA L T L T A L A A A AM I P L T AM T A A AMM P L AA I I L AATAS I AA L AGAAA L A L

(c)

I AAGE NS Y NV S V NV V F E I K V E AGE NS Y NV S V NV V F E I KE V AAGE NS Y NV S V NV V F E I KE I ASGENS YSVVVNV T F ALGE I ASGENS YSVVVNV T F AL EQ I A AG E N S Y S V T V N V TWE L AQ I EPGENT VGASV T VV F E L T -

(d)

250 251 251 247 247 243 242

(e)

(f)

β5-1

β5-2

β6

β1

β4

β2

β5-3

Domain I

Domain I

α3

α4 α2

β3

Domain II

Domain II

α1

Domain I Domain II

Domain I G3P pilus binding domain

Domain II G3P pilus binding domain

Fig. 1. The crystal structure of BP26 monomer. Multiple sequence alignment of BP26 homologues from B. abortus, Vibrio cholerae, Ochrobactrum anthropi, Agrobacterium tumefaciens, Rhizobium leguminosarum and Ahrensia sp. R2A130 and the SIMPL domain (COG2968) shows high degree of sequence conservations. The secondary structures are shown above the alignment based on the crystal structure. Disordered region is indicated in a dashed line, and the transmembrane sequence, which is not included in the construct used, is indicated by a bracket (a). Ribbon diagram of the BP26 monomer structure with α-helices in red or pink and with β-strands in yellow, orange and dark orange. BP26 is composed of domain I and domain II (b). A topology diagram for the BP26 monomer structure with the same color scheme as in (b). The domain I and the domain II have a similar topology (c). Superimposition between the domain I and the domain II with an r.m.s.d. of 3.7 Å (d). Superimpositions between the domain I and Ike G3P pilus-binding domain and between the domain II and Ike G3P pilus-binding domain with an r.m.s.d. values of 3.4 and 2.1 Å, respectively (e and f).

1121

BP26 Forms a Channel-like Structure

Table 1. Data collection and refinement statistics

Data collection Space group Unit cell dimensions a, b, c (Å) α, β, γ (º) Wavelength (Å) Resolution (Å) Unique reflections Rsym I/σ(I) Completeness (%) Redundancy

Native

Se-methionine SAD

I4122

I4122

203.086, 203.086, 207.295 90, 90, 90 0.97941 50.00–3.50 (3.56–3.50)a 26,996 8.0 (53.1) 31.9 (3.8) 97.9 (99.3) 5.9 (6.1)

202.367, 202.367, 208.236 90, 90, 90 0.97941 50.00–4.15 (4.22–4.15) 16,612 16.3 (61.4) 46.4 (10.5) 100 (100) 28.2 (27.8)

Refinement Resolution used (Å) 30.0–3.5 Number of atoms Protein 6340 0.237 (24,457) Rwork (reflections) 0.283 (2448) Rfree (reflections) Ramachandran plot (%) Core 86.2 Allowed 13.8 r.m.s.d. Bond lengths (Å) 0.024 Angles (º) 2.0 Rwork = ∑||Fobs| − |Fcalc||/∑|Fobs|. Rfree = ∑||Fobs| − |Fcalc||/∑|Fobs|, where 10% of randomly selected data were used. Rsym = ∑|Ihkl − 〈Ihkl〉|/∑ Ihkl, where 〈Ihkl〉 is the mean intensity of all reflections equivalent to refection hkl. a The values in parentheses are the statistics from the highestresolution shell.

β-strand is disrupted by a loop containing multiple proline residues (Fig. 1a). The domain I has one long α-helix (α3) and one short α-helix (α4). The long αhelix sits on the β-sheet at a 40° angle relative to the parallel axes of the β-strands (Fig. 1b). The short αhelix is located the other side of the β-sheet. The domain II also has an antiparallel β-sheet composed of three β-strands and two α-helices (Fig. 1b). Both α-helices sit on the same side of the β-sheet with an orientation almost parallel with the β-sheet. The domain I and domain II are connected by two loops. The first loop links β1 and β2 and the second loop links α2 and α4 (Fig. 1c). While BP26 exhibits no apparent sequence homology to any functionally annotated protein, a search for structural homologies through the Dali server showed that the N-terminal pilus-binding domain of phage Ike gene 3 protein (G3P) from filamentous phage is highly similar to both domain I and domain II with r.m.s.d. values of 3.4 and 2.1 Å, respectively (Fig. 1e and f). 24,25 The pilus-binding domain is involved in the initial step of infection by interacting with the F pilus of Escherichia coli, suggesting that BP26 might be involved in infection by interacting with a host protein. 26

Channel-like structure of BP26 A prominent feature of the BP26 structure is a higher-order multimerization. Sixteen molecules of BP26 from four asymmetric units form a channel-like structure (referred to as BP26COM) (Fig. 2a and b). Eight molecules of BP26 form a ring structure having an 8-fold symmetry, and two ring structures interact with each other in a bottom-to-bottom mode (Fig. 2). Therefore, the two domains of BP26 generate a fourlayered structure (I-II-II-I) for BP26COM (Fig. 2c). The overall dimensions of BP26COM are 120 Å high and 115 Å wide. The first layer is composed of 24 antiparallel βstrands composed of the β-sheets in the domain I from eight molecules of BP26 that are aligned side by side to form a β-barrel structure. The long αhelices in the domain I stack diagonally to form an outer frame and reinforce the interactions between the β-sheets (Fig. 3). The short α-helices in the domain I, which are located at the lower part of βbarrel between the β-sheets of BP26 molecules, serve as wedges between BP26s to make the lower part (77 Å) much wider than the upper part (38 Å) of the β-barrel structure (Fig. 3, I). The second layer is composed of the β-strands of the domain II that are aligned to form another β-barrel structure. In addition, the connecting loop between α2 and α3 also aligns with these β-strands. A total of 16 α-helices, two α-helices from each BP26 domain II, also stack diagonally to clamp the second β-barrel structure (Fig. 3, II). The third and the fourth layers organized by another eight BP26 molecules as are identical with the first and the second layers, respectively. The interaction between the second layer and the third layer is through exchanging the loops between β3 and β4 in the domain IIs from the two octameric rings (Fig. 3, III). Like locked fingers, 16 loops coming from different BP26 molecules cross alternatively and hold the two octameric ring structures. The interactions among BP26 monomers are mostly through conserved hydrophobic residues (Supplementary Fig. 2), suggesting that the oligomeric state of BP26COM is well maintained in solution. With this structural arrangement, BP26COM generate a large internal cavity. In order to examine if the structure of BP26COM is maintained in solution and to rule of a possibility that BP26COM structure is due to a crystal packing, we obtained two-dimensional (2D) class-average electron microscopic image with 5520 negatively stained particles and compared the image with the projection of the crystal structure (Fig. 2d and Supplementary Fig. 3). It clearly shows that the 2D class-average structure is similar to the projection of the crystal structure indicating that hexadecameric BP26 is an obligate oligomer, which reflects the physiological structure of BP26.

1122

BP26 Forms a Channel-like Structure

(a)

(b)

115 Å

120 Å 1

(c)

30 Å

(d)

Fig. 2. Structure of hexadecameric BP26 complex. A side view of hexadecameric BP26 complex structure. Each monomer BP26 is shown in a different color. The overall dimensions are 120 Å in its height and 115 Å in its width (a). A top view of hexadecameric BP26 complex structure. Two octamers generate holes at the top and at the bottom with 30 Å in its diameter (b). Two BP26 monomers (shown in magenta and orange) are shown in the hexadecameric BP26 complex structure (c). 2D class-average EM structure of BP26 (top) and the matched projection of the crystal structure (bottom) (d).

I

β5-2

I

II β1

β6

α3

β4

α3 α2

β3

α1 β2

II α4

III

IV

III α1 α2 β4 β3 β3 β4

α1

IV Fig. 3. Detailed interactions between BP26 monomers. Detailed interaction between the domain I showing that the βsheets in the domain I form a β-barrel structure (I). Detailed interaction between the domain II showing that the β-strands of the domain II align to form another β-barrel structure (II). Detailed interaction between two BP26 octamers showing that there are crossovers among the loops between β3 and β4 in the domain IIs (III). Detailed view of the hole is showing the well-ordered eight Pro37 residues (shown in red as a ball-and-stick model, IV).

1123

BP26 Forms a Channel-like Structure

Another prominent feature of BP26COM is the hole generated by the BP26 octamer. The diameter of the hole is about 30 Å, and the two holes in the two octameric rings are aligned to render BP26COM to have a channel (Fig. 2). The rim of the hole consists of eight Pro37 residues that are well ordered (Fig. 3, IV). Electrostatic surface representation of BP26COM shows that the outer surface of BP26 is highly charged without a notable local basic or acidic patch (Fig. 4a). However, the rim of hole is highly basic due to the presence of Lys39, Arg197 and Lys250, suggesting that the hole might interact with negatively charged molecules (Fig. 4b). The inner surface of the complex is also highly charged. Interestingly, there are six positively charged layers: the rim of the hole makes the first layer, eight Arg206 residues on the inner wall make the second layer, eight Arg215 residues make the third layer and the second octameric ring generates the other three layers (Fig. 4c). Collectively, 16 molecules of BP26 form a channel-like structure with highly charged surfaces and highly positively charged holes.

Discussion The crystal structure of BP26 presented in this study reveals for the first time that BP26 forms a novel channel-like structure. The crystal structure and electron microscopy (EM) analysis of BP26 indicates that BP26COM is an obligate complex and that BP26 functions in this multimeric form. Considering that the transmembrane sequence is located at the end of the N-terminus of BP26, the transmembrane sequences would be located at both ends of BP26COM. Therefore, it is likely that BP26COM forms after the N-terminal transmembrane sequence is cleaved off. Alternatively, there is a possibility for another form of BP26 multimer such as a “cup”shaped structure with a BP26 octamer with the Nterminal transmembrane sequences.

(a)

(b)

The unexpected structural similarity between the BP26 monomer and the N-terminal pilus-binding domain of phage Ike G3P raises a possibility that BP26 may play a role in infection. Johnson et al. recently observed that BP26 binds extracellular matrix proteins: fibronectin, vitronectin and collagen, but not laminin (Poster presentation, Brucellosis 2011 International Research Conference), supporting an idea that BP26 might be involved in infection step. Another functional clue of BP26 is that it belongs to the SIMPL superfamily according to the Conserved Domain Database (Supplementary Fig. 4). There are only a few eukaryotic proteins containing a SIMPL domain. Among them, IRAK1 (interleukin-1 receptor-associated kinase 1) binding protein 1 (IRAK1BP1) possesses a SIMPL domain and is involved in regulating IRAK1 leading to NF-κB activation. Although there is a marginal sequence conservation between IRAK1BP1 and BP26 (Supplementary Fig. 4) and there is no experimental evidence that BP26 is involved in NF-κB pathway or that IRAK1BP1 forms a multimeric complex, BP26 might function in an innate immune response pathway, where IRAK1 and IRAK1BP1 are involved. However, the biological function of BP26 and whether BP26 is involved in immune response and during infection need to be further investigated. A BLAST search with the BP26 sequence shows that BP26 homologues are found in most of αproteobacteria and some γ-proteobacteria and that they are well conserved (Fig. 1a). The oligomeric structure of BP26 seems to be conserved in other species as we observed that BP26 of Mycobacterium tuberculosis shows a similar size by a sizeexclusion chromatography indicating that multimerization is a general feature of BP26 (data not shown). Mapping of highly conserved residues across different species on the presented structure indicates that these residues seem to be important for forming the channel-like structure (Supplementary Fig. 5). This observation suggests that a

(c) layer 1 layer 2 layer 3

layer 4 layer 5 layer 6

Fig. 4. Electrostatic surface representation of hexadecameric BP26 complex. Side view of hexadecameric BP26 complex shows highly charged surface throughout the molecule (a). Top view of hexadecameric BP26 complex reveals highly basic charged hole (b). Slice cut at the middle of the BP26 complex shows highly charged surface at the inside of the molecule (c).

1124 channel-like structure itself, rather specific residues, might be important for the function of BP26. Consistent with this, the basic residues located at the rim of the hole and the inside of the BP26COM are diversified. Therefore, each species seems to have a BP26 protein that is quite distinctive in the primary sequence but similar in the overall structure. The fact that BP26 has a species-specific sequence with high immunogenic property and that most pathogenic bacteria have BP26 prompt us to suggest exploiting structural information of BP26 for development of a pathogen-specific diagnostic marker for pathogen infection. Specifically, BP26 or antibodies against BP26 may serve as a diagnostic marker for detection of infection by a specific pathogen. In summary, BP26 is composed of two similar domains and that it forms a novel channel-shape hexadecameric complex, which appears as the functional unit. In addition, our structure provides the first glimpse into the structure of the SIMPL domains.

Materials and Methods Recombinant BP26 protein expression and purification BP26 (29 ~ 250 amino acids), periplasmic portion without a signal peptide of B. abortus OMP28, was cloned into a modified pET28a vector, containing N-terminal Histag and tobacco etch virus protease cleavage site after the His-tag (Novagen). The plasmid containing BP26 was transformed into BL21 (DE3) RIPL E. coli competent cells. BP26 was expressed with 1 mM isopropyl-1-thio-β-Dgalactopyranoside for 16 h at 18 °C. The cells were harvested and resuspended using buffer A [50 mM Tris– HCl (pH 8.0) and 5% glycerol] containing 500 mM NaCl and phenylmethylsulfonyl fluoride. The resuspended cells were sonicated and centrifuged at 18,000 rpm for 1 h. The supernatant was incubated with 5 ml of Ni-NTA resin (Qiagen). The protein was eluted with the buffer A containing 300 mM NaCl and 100 ~ 200 mM imidazole after washing with the buffer A containing 1 M NaCl. Nterminal His-tag was cleaved with tobacco etch virus protease during dialysis in a buffer containing 50 mM Tris– HCl (pH 8.0), 100 mM NaCl, 0.5 mM ethylenediaminetetraacetic acid and 1 mM DTT. The cleaved His-tag and uncleaved BP26 were removed with Ni-NTA resin. BP26 was applied to a tandem HiTrap Q-SP column. BP26 in the flow-through fraction was further purified with Superdex 200 26/60 size-exclusion column (GE Healthcare) equilibrated with 50 mM Tris–HCl (pH 8.0), 100 mM NaCl and 20 mM β-mercaptoethanol. BP26 was finally concentrated to 15.2 mg/ml. Se-substituted protein was expressed and purified as the wild-type protein. Crystallization, data collection and structure determination Initial crystals of BP26 were grown in a buffer containing 2.5 M ammonium sulfate (AMS) and 0.1 M Tris–HCl

BP26 Forms a Channel-like Structure

(pH 8.5) by the sitting-drop method using Mosquito (TTP LabTech) at 20 °C. Optimal crystals were obtained in 2.4 M AMS, 75 mM Tris–HCl (pH 8.5) and 3% (v/v) methanol by the hanging-drop method. Se-methioninesubstituted crystal condition was optimized as 2.4 M AMS, 175 mM Tris–HCl (pH 8.5) and 100 mM NaCl. For cryoprotection, crystals were soaked in gradually increasing the glycerol contents to 20% under crystallization condition. X-ray diffraction data sets were collected using a CCD Quantum 315r (ADSC) at beamline 5C (SB-II) in Pohang Accelerator Laboratory, Korea, and using a PAD Pilatus 2M-F at beamline BL1A in Photon Factory, Tsukuba, Japan. Data were processed using HKL2000 (HKL Research). Initial phases were obtained by SAD method using Autosol with PHENIX. Model building was conducted using the program O and COOT. The structure refinement was done with the program PHENIX and CNS using the higher-resolution data from native crystal. 27,28 EM analysis BP26 protein was negatively stained with 0.75% uranyl formate. Images were collected on a 4 K × 4 K Eagle HS CCD camera (FEI) on a Tecnai T120 microscope (FEI) operating at 120 kV. The defocus and nominal magnification for all images was − 1 μm and 67,000×, respectively. Particles were automatically selected with EMAN2 boxer 29 and bad particles were manually excluded. Image processing was performed using IMAGIC. 30 In brief, windowed particles were normalized, band-pass filtered between 15 and 100 Å. After reference-free alignment, multivariate statistical analysis (MSA) followed by MSA classification and averaging, classified 5520 particles into 30 classes. Five selected classes among 30 classes were used for further rounds of multi-reference alignment and MSA. After repeated multi-reference alignment/MSA, all data sets were classified into final 10 different classes. Each 10 classes were compared to forward projection images of the crystal structure, which are low-pass filtered at 10 Å. Database linking Database: Protein Data Bank† ID: 4HVZ.

Acknowledgements We thank the members of Song and Oh laboratories for discussions. The X-ray data for BP26 were collected at Pohang Accelerator Laboratory, Pohang, Korea, and at Photon Factory, Tsukuba, Japan. We thank Dr. MyungHee Kim for the help for the data collection. This work is partially supported by the Intelligent Synthetic Biology Center of Global Frontier Project funded by the Ministry of Education, Science and Technology (2011–0031955), by the World Class University program grant (R31-2008-001100710-0 to J.S.), a Gastric Cancer Basic Research Laboratory Program grant (2011–0020334 to J.S.), a

1125

BP26 Forms a Channel-like Structure

grant (2012R1A1A1010456 to H.M.K.) through the National Research Foundation of Korea and a grant by the Ministry of Knowledge Economy, Korea Institute for Advancement of Technology through the Inter-ER Cooperation Projects.

8. 9.

Supplementary Data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jmb.2013.01.015

10.

Received 7 November 2012; Received in revised form 31 December 2012; Accepted 14 January 2013 Available online 23 January 2013 Keywords: Brucella abortus; multimerization; SIMPL domain; infection; pilus-binding domain

11.

12.

† www.rcsb.org 13. Abbreviations used: OMP, outer membrane protein; SAD, single-wavelength anomalous dispersion; G3P, gene 3 protein; 2D, two-dimensional; EM, electron microscopy; IRAK1BP1, IRAK1 binding protein 1; MSA, multivariate statistical analysis.

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