The Crystal Structure of the YknZ Extracellular Domain of ABC Transporter YknWXYZ from Bacillus amyloliquefaciens

Bacillus possesses the peptide toxin Sporulation-Delaying Protein (SDP), which can kill cells within a biofilm to support continued growth, thereby delaying the onset of biofilm sporulation. The four-component transporter YknWXYZ acts as a major SDP efflux pump to protect cells against the endogenous SDP toxin, for which YknYZ is a non-canonical ATP-binding cassette (ABC)-type transporter. YknYZ consists of the following two components: (1) an individual protein (YknY) and (2) a respective permease (YknZ). To date, the crystal structure, molecular function, and mechanism of action of the integral membrane protein YknZ remain to be elucidated. In this study, to characterize the structural and biochemical roles of YknZ in the functional assembly of YknWXYZ, we predicted and overexpressed the YknZ extracellular domain. We determined the crystal structure of B. amyloliquefaciens YknZ at a resolution of 2.0 Å. The structure revealed that the YknZ extracellular region exhibits significant structural similarity with the MacB periplasmic domain, which is a non-canonical ABC-type transporter in the tripartite macrolide-specific efflux pump in Gram-negative bacteria. We also found that the YknZ extracellular domain can directly bind to an extracellular component of YknX. This structural and biochemical study provides insights into the assembly of YknWXYZ, which may be relevant to understanding cannibalistic peptide toxin resistance in Bacillus and controlling bacterial growth.


Introduction
The Gram-positive bacterium Bacillus subtilis cannibalistic toxin Sporulation-Delaying Protein (SDP) kills a subset of Bacillus cells within a biofilm to support continued growth, thereby delaying the onset of biofilm sporulation [1,2]. Mature SDP is a product of the sdpC gene, and recent analyses of the structure and activity of SDP have shown that this toxin is a 42-aminoacid peptide containing a single intramolecular disulfide bond [3]. The four-component transporter YknWXYZ in Bacillus has been implicated in the protection against SDP-dependent killing by expelling invading SDP toxin from the cytosol [4].
YknWXYZ contains the ATP-binding cassette (ABC) transporter YknYZ, in which an individual protein YknY dimer associates with a respective permease (YknZ) that utilizes ATP hydrolysis as a driving force [5]. The activity of YknYZ strictly depends on two accessory proteins, YknX and YknW. The membrane fusion protein (MFP) YknX is located in the extracellular region and anchors to the cytoplasmic membrane via an N-terminal anchor [6]. YknX is anchored to the cytoplasmic membrane via an uncleavable N-terminal signal peptide and is connected to YknZ [4]. YknW, a Yip1 family protein that is highly conserved in Bacillus species, can modulate the assembly of the YknXYZ complex by forming a composite interface with YknX and YknYZ [4].
Recently, YknWXYZ homologues were identified in various Gram-negative bacteria [4]. In Escherichia coli, macrolide-specific efflux transporters, such as MacAB-TolC, have also been implicated in enterotoxin secretion and macrolide antimicrobial compound discharge [7,8]. The MFP MacA connects an ABC-type transporter, MacB, and the outer membrane factor TolC to pump out macrolide antibiotics [9]. The non-canonical ABC-type transporter MacB was first identified as a macrolide antibiotic-specific transporter in E. coli [10]. Notably, fulllength MacB is believed to act as a homodimeric protein and has a novel architecture that consists of an N-terminal nucleotide binding domain, a four-transmembrane segment, and two putative extracellular regions [7,8,11]. Some research groups have revealed that the inner membrane component of MacB directly interacts with MFP MacA [12]. In Gram-negative bacteria, MacA exists as a funnel-like hexamer and forms an intermeshing cogwheel by interacting with TolC [9,13,14]. Several lines of evidence for a mechanism for the ABC-type transporter MacB have been suggested [12], but the reported studies have focused on Gram-negative bacteria instead of Gram-positive bacteria.
To gain insight into the functional and structural properties of the YknZ respective permease, we aimed to determine the three-dimensional structure of YknZ. Here, we present the first crystal structure of the extracellular domain of YknZ from Bacillus amyloliquefaciens. Biochemical experiments demonstrated that the extracellular domain of YknZ has a strong affinity for the MFP YknX.

Expression and purification of the recombinant YknZ ED
Expression and purification of the recombinant proteins of YknZ ED and YknX were performed using similar protocols. The constructs were transformed into BL21 cells (DE3), and the proteins were expressed in the cytosol as hexa-histidine-tag fusion proteins. A single colony was used to inoculate 25 ml of LB broth supplemented with 50 μg ml -1 ampicillin, which was incubated overnight at 310 K with shaking. Cultured YknZ ED expression cells were then transferred to 1.5 liters of LB broth with 50 μg ml -1 ampicillin at 310 K until reaching an OD 600 between 0.6 and 0.8. In particular, the recombinant Ba YknZ ED protein was expressed using M9 medium supplemented with an amino acid mixture containing L-(+)-selenomethionine at 303 K until reaching an OD 600 between 0.6 and 0.8. After induction with 0.5 mM isopropyl-Dthiogalactoside, the cultures were grown for 6 h at 303 K. The BL21 cells (DE3) were harvested by centrifugation and then stored at 193 K until use. Cell pellets were resuspended in a lysis buffer containing 20 mM Tris (pH 8.0), 150 mM NaCl, and 2 mM 2-mercaptoethanol, and the cells were lysed by sonication on ice. The cell debris was removed by centrifugation at 45,000 g for 30 min at 277 K. The resulting supernatant was then stirred for 30 min at 4°C and loaded onto a Ni-NTA affinity resin (Qiagen, Netherlands). After the resin was washed with lysis buffer supplemented with 20 mM imidazole, the recombinant hexa-histidine-tagged Ba YknZ ED was eluted with 30 ml of lysis buffer supplemented with 250 mM imidazole. After heating, the eluted fractions were determined by SDS-PAGE analysis and pooled, and 2-mercaptoethanol was added to a final concentration of 10 mM. The hexa-histidine tag was removed by incubating the recombinant protein overnight at 298 K with recombinant TEV protease. The solution was diluted 3-fold with 20 mM Tris (pH 8.0) buffer and then loaded onto a Q anionexchange column (HiTrap-Q; GE Healthcare, USA). The protein was eluted from the column using a linear gradient of 0 to 1 M NaCl in 20 mM Tris buffer (pH 8.0). The collected fractions containing the target recombinant proteins were further purified and separated on a HiLoad Superdex 200 gel-filtration column (GE Healthcare, USA) pre-equilibrated with a buffer consisting of 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 2 mM 2-mercaptoethanol. The fractions corresponding to the peaks were subjected to SDS-PAGE on a 15% acrylamide gel and stained with Coomassie blue. The purified proteins were concentrated to 12 mg ml -1 and stored at 193 K until use.

Circular dichroism (CD) spectroscopy
To evaluate the structural integrity of the Ba YknZ ED recombinant protein, CD spectroscopy was performed using a JASCO-J1500 spectropolarimeter (JASCO, Japan). Concentrated Ba YknZ ED protein and BSA were diluted to 0.1 mg/ml in 20 mM HEPES (pH 7.5) buffer containing 150 mM NaCl. The absorption spectra were measured in the spectral range between 200 and 280 nm at 298 K. Three consecutive scans were performed and averaged, and the solvent signal was subtracted from all of the spectra.
Crystallization, data collection, and refinement structure The pooled, purified Ba YknZ ED-SeMet samples were concentrated to 12 mg ml -1 using a Vivaspin centrifugal concentrator fitted with a 10 kDa molecular-weight cutoff filter (Millipore, USA) prior to crystallization. The search for crystallization conditions was performed with a Crystal Screen HT high-throughput sparse-matrix screening kit (Hampton Research, USA) using the sitting-drop vapor-diffusion method at various temperatures (277, 279, 288 and 295 K). The final crystallization conditions consisted of 22.5% (w/v) polyethylene glycol 4000, 0.125 M ammonium acetate, and 0.1 M sodium acetate trihydrate (pH 4.6) at 288 K. Immediately after the crystal was removed from the drop, it was soaked in 10 μl of cryoprotectant solution consisting of well solution supplemented with 25% (v/v) glycerol for 3 to 5 s and was then flashcooled in liquid nitrogen.
We used a single-wavelength anomalous dispersion (SAD) phasing method to resolve the structure. Diffraction data were measured to 2.0 Å, with the wavelength tuned to 0.9795 Å, using an ADSC Q310 CCD detector on the BL17U beamline at the Shanghai Synchrotron Radiation Facility (SSRF) (Shanghai, People's Republic of China). All of the data were collected at 100 K using a nitrogen stream. An oscillation range of 1°per frame over a 360°rotation was implemented, the exposure time was 5 sec per frame, and the detector distance was 340 mm. The intensity data were processed, merged and scaled using the HKL-2000 program [15].
The structure was solved via SAD-phasing using AUTOSOL from the PHENIX package, and the nearly complete model was improved after refinement with REFINE from the PHENIX package [16]. The Ba YknZ ED model was refined to convergence, resulting in an R factor of 18.1% and an R free of 24.9% to a resolution of 2.0 Å. Ramachandran plots and root-meansquare deviations from ideality for bound angles were determined using PHENIX [16]. One molecule of Ba YknZ ED-SeMet constituted the asymmetric unit, and each contained six SeMet residues. The model building and fit to electron-density maps were performed with the graphics software COOT [17]. All of the figures containing molecular structures were prepared using PyMOL [18]. The data collection and refinement statistics are given in Table 1.  Size exclusion chromatography for the analysis of the Ba YknZ ED oligomeric state Size-exclusion chromatography was performed to determine whether Ba YknZ ED forms dimeric structures in solution. Purified Ba YknZ ED protein (0.3 mg) was injected at a flow rate of 0.5 ml/min into a Superdex 200 10/300 GL column (GE Healthcare) that was pre-equilibrated with column buffer (20 mM Tris buffer pH 8.0, 150 mM NaCl, 2 mM 2-mercaptoethanol) at room temperature (295 K). The same column buffer was also used for the elution. The molecular weight of each protein was estimated according to the elution volume.
In vitro binding assay for Ba YknZ ED and Ba YknX Ba YknZ ED-coupled resin was prepared using CNbr-activated resin (50 μg; GE Healthcare) and Ba YknZ ED protein (0.5 mg) according to the manufacturer's instructions; the Ba YknZ ED-uncoupled CNbr-activated region was similarly used as a control. Purified YknX protein (0.6 mg) and the coupled resin (20 μl) or uncoupled resin were incubated in 200 μl of buffer (20 mM HEPES pH 7.5, 150 mM NaCl, and 2 mM 2-mercaptoethanol) for 2 h at 277 K, and the mixture was then centrifuged at 2000 rpm for 2 min. The resin was collected and thoroughly washed five times with 500 μl of 20 mM HEPES (pH 7.5) buffer containing 150 mM NaCl and 2 mM 2-mercaptoethanol. To analyze the YknX bound to Ba YknZ ED protein, the beads were boiled with SDS sample buffer, applied to SDS-PAGE on a 15% gel and stained with Coomassie blue.

Results
Plasmid construction, expression, and purification of the recombinant B. amyloliquefaciens YknZ ED The respective permease YknZ is a 397-amino-acid membrane protein. Functional and structural studies of the YknZ native state usually require the overexpression of full-length YknZ; however, conducting these studies is challenging because the expression yield of the full-length YknZ construct is usually too low [4]. In this study, we found that YknZ has a novel architecture that consists of a four-transmembrane segment and two putative extracellular regions ( Fig  1A, residues 44-273 and residues 345-358) as predicted by analysis using TMHMM Server V. 2.0 (www.cbs.dtu.dk) [19]. The second YknZ extracellular region (residues 345-358) appeared to be too short to form a structured motif because it consists of only 14 residues. Thus, because only the first YknZ extracellular region was expected to form a structured domain, the first YknZ extracellular region was named the extracellular domain (ED) in this study. To determine whether Ba YknZ ED forms dimeric structures in solution, we measured the molecular size of Ba YknZ ED using size-exclusion chromatography. We obtained data suggesting that the Ba YknZ ED protein is eluted as a monomer on a size-exclusion column (Fig 1B). The YknZ ED was constructed and successfully overexpressed, with a yield of 12 mg from one liter of Luria-Bertani (LB) medium culture. The purity of the protein sample was determined to be > 93% according to Coomassie staining following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (S1 Fig). To ascertain whether Ba YknZ ED behaved as a well-folded protein, the secondary structure of Ba YknZ ED was characterized by circular dichroism (CD) spectroscopy. The CD spectrum was very similar to the bovine serum albumin (BSA) CD spectrum (Fig 1C). The results indicated that YknZ ED has structural integrity because its spectrum matches the expected structure of YknZ in the absence of the membrane-embedded domain.

Structural determination of the recombinant Ba YknZ ED
The purified recombinant YknZ ED protein sample was successfully crystallized under several conditions via the sitting-drop vapor-diffusion method in initial crystallization screening trials. Ultimately, the optimal conditions were found to be 22.5% (w/v) polyethylene glycol 4000, 0.125 M ammonium acetate, and 0.1 M sodium acetate trihydrate (pH 4.6) at 288 K. Initial attempts to determine the structure of Ba YknZ ED using a molecular replacement method with the program MOLREP [21] and structure of the YknZ homologue MacB (PDB code 3FTJ) [12] as a search model were unsuccessful. Fortunately, the structure of Ba YknZ ED was determined using SAD phasing methods with SeMet-substituted protein crystals. The best crystals readily diffracted to better than 2.0 Å resolution. From the diffraction dataset, the crystals were found to belong to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters of a = 44.2, b = 46.2, and c = 87.5 Å. Based on the estimated Matthews coefficient (1.8 Å 3 Da -1 ) and the solvent-content prediction (31.66%), a monomer was expected in the asymmetric unit. The diffraction data set had a resolution range of 41.0-2.0 Å with 99.8% completeness, an R merge of 28.8%, an R factor of 19.44%, and an R free of 24.73%. In the Ramachandran plot, 96.69% of the model residues were found in favored regions, 3.31% in allowed regions, and 0.00% in disallowed regions. The data collection and the refinement statistics for the structure are presented in Table 1.

Overall structure of Ba YknZ ED
The refined model of Ba YknZ ED contains 188 residues from Asn 58 to Asn 256 , and the N-and C-terminal regions of Ba YknZ ED were disordered in the crystal. The disordered N-and Cterminal regions of Ba YknZ ED may play a role in the connection between Ba YknZ ED and the transmembrane segments in full-length YknZ. The crystal asymmetric unit of the crystal contains one molecule of Ba YknZ ED. We also performed size-exclusion chromatography to gain insight into the Ba YknZ ED oligomeric state and found Ba YknZ ED was eluted as a monomer similar to Actinobacillus actinomycetemcomitans MacB periplasmic core domain (Aa MacB PCD), which is a non-canonical ABC-type transporter within Gram-negative bacteria [12], To understand whether Ba YknZ ED have an affinity toward each other at high concentrations, we tested the potential of Ba YknZ ED to dimerize in solution by native page electrophoresis, and analyses of native gels indicate that Ba YknZ ED can dimerize (S2 Fig). This result suggests that Ba YknZ ED molecules have an affinity toward each other, like other homologous proteins.
The YknZ ED structure included a mixed α/β domain that was dispersed into upper and lower subdomains (Fig 2A). The upper subdomain was formed by residues 103-219 and was composed of a seven-stranded antiparallel β-sheet with four α-helices. A loop, in which significant electron density was not observed, connected β 2 to β 9 in this domain. The lower subdomain was formed by residues 58-102 and 219-256 and consisted of three α-helices and a three-stranded antiparallel β-sheet. A loop connected α 1 to α 2 in this subdomain and was disordered in the crystal structure of Ba YknZ ED. Additionally, no interactions were observed between the two distinct components, which were connected via a linker (β 2 and β 9 ) (Fig 2B).

Ba YknZ ED molecular architecture is analogous to homologous protein structures
The DaliLite server [22] was used to identify unique proteins with structural similarity to Ba YknZ ED. The DaliLite server revealed that the structure of Aa MacB PCD was highly similar to Ba YknZ ED, which was found to be the top match and showed a Z-score of 20.1 and sequence identity of up to 22%. As evident from the results, the structural overlays of the two proteins revealed highly similar overall topologies with a root mean square deviation (rmsd) of 1.6 Å between the corresponding 121 residues. Upon superposition of two proteins as references, we found the overall structures and relative domain orientations of Ba YknZ ED and Aa MacB PCD to be very similar but not identical; some minor structural differences were present (Fig 3, Left). In particular, the structure of Aa MacB PCD revealed that each monomer had a sickle shape that was dispersed into the following two distinct components: (1) the N-and Cterminal regions and (2) a mixed α/β domain [12]. In contrast, the structure of Ba YknZ ED only contained a mixed α/β domain with disordered N-and C-terminal regions. Additionally, the Aa MacB PCD β 6 was divided into two β-strands (β 6 and β 7 ) in the Ba YknZ ED structure. We previously found a shallow crevice in the MacB PCD structure; the mixed α/β domain is almost completely divided into two parts by this crevice. We also found this crevice in the middle of the mixed α/β domain in the Ba YknZ ED structure, where it virtually divided the domain into two parts (Fig 3, Upper-right). The overall structural architecture of the lower subdomain of Aa MacB PCD was similar to that of subdomains in the AcrB porter domain [12]. To understand whether the overall structural architecture of the Ba YknZ ED lower subdomain had high similarity to other homologous proteins, we compared the structure of the Ba YknZ ED lower subdomain with the Aa MacB PCD lower subdomain and the subdomains of the inner membrane transporter CusA pore domain from E. coli [23]. We found that the domain structures were generally similar, although there were slight differences. In Gramnegative bacteria, CusA is the inner membrane transporter of the heavy metal efflux pump CusCBA [23]. The most significant structural difference is the number of β-strands; the lower subdomain of Ba YknZ ED contained three β-strands, while the other structures each contain four β-strands. Whereas α 1 and α 2 were connected in the Aa MacB PCD structure, the corresponding region in the Ba YknZ ED structure was largely disordered, and the conformations of these helices were slightly different in Ba YknZ ED, as α 1 was tilted outwards compared with the Aa MacB PCD structure. We also observed that β 1 in the Ba YknZ ED N-terminal region was shorter than that of the other homologous proteins (Fig 3, lower-right).

Ba YknZ ED can directly bind to YknX
Yamada et al. used chemical cross-linking and co-purification experiments to demonstrate that full-length YknYZ can complex with YknX [4]. To test whether Ba YknZ ED directly binds YknX, a binding assay was performed using purified YknZ ED and YknX proteins, as described in the Materials and Methods section. As shown in Fig 4, the purified YknX protein was not bound to the CNbr-activated region, which is in sharp contrast to the strong binding of YknX to YknZ ED. This result indicated that Ba YknZ ED directly associated with YknX, which was consistent with a previous report in which full-length YknYZ was used [4].

Discussion
In both Gram-positive and Gram-negative bacteria, ABC-type transporters are functional subunits of multi-component transporters that perform diverse physiological functions utilizing ATP hydrolysis as a driving force. Recently, an ABC transporter YknYZ and its homologues were identified in various bacteria [4,10]. Unlike most dimeric ABC-type transporters, MacB and YknYZ are characterized as non-canonical ABC-type transporters, which consist of a fourtransmembrane segment, an N-terminal nucleotide binding domain (NBD), and a large periplasmic domain [7,24]. In this study, we presented the first three-dimensional structure of the Ba YknZ extracellular domain, which is associated with YknY. The overall structure of the Ba YknZ ED monomer showed a structure similar to that of Aa MacB PCD. The sickle shape YknZ ED structure included a mixed α/β domain that was dispersed into upper and lower subdomains by a shallow crevice, and the structure of the Ba YknZ ED lower subdomain was generally similar to those in other homolog proteins such as the Aa MacB PCD lower subdomain and the subdomains of the inner membrane transporter CusA pore domain from E. coli. The upper subdomain of MacB or CusA may play a role in forming similar structures in the cognate binding partners of MacB or CusA [12]; thus, we speculate that the upper subdomain of YknZ fits into YknX and the lower subdomain of YknZ provides the substrate entrance site.
Recently, assembly models for the MacAB-TolC and CusCBA pumps have been proposed [13,22]. In these models, the inner membrane component (MacB and CusA, respectively) directly interacts with MFP (MacA and CusB, respectively), which acts as a hexameric protein.
However, these previous studies mentioned above did not reveal direct interactions between the YknZ dimer and the YknX oligomer or indicate which part of YknZ is important for YknX binding. In this study, we observed an interaction between YknZ ED and YknX using an in vitro binding assay, which was consistent with a previous report in which full-length YknYZ was used [4]. The MFP YknX is crucial for the functional assembly of the YknWXYZ transporter in the extracellular. Moreover, the structure and overall size of the YknX protein are similar to those of MacA (sequence identity 32%) (S3 Fig). In our previous work, we suggested that MacA is a funnel-like hexameric protein [9]. In this study, we observed that the Ba YknX residues Gle 227 and Tyr 280 were strictly conserved in the sequence alignment ( Fig 5);  these two residues are located within the β-barrel domains and play an important role in hexamerization in the crystal structure of MacA [9]. We speculate that YknX forms a similar oligomeric assembly in the functional complex despite previous biochemical data indicating that YknX likely forms a dimer or trimer [4]. In this study, we also found that YknZ ED can directly interact with MFP YknX, but we did not determine which region of YknZ is important for this interaction. Further structural and functional studies of the binary and ternary complexes (YknXYZ) are required to determine whether the upper subdomains of YknZ are essential for the interactions between YknZ and YknX and whether these interactions are similar or different in the assembly of the four-component transporter YknWXYZ. In addition, a previous study found that the individual homodimeric protein YknY can associate with YknZ, and the assembly of the complex is modulated by YknW in the YknWXYZ complex [4]. Based on these findings, we propose an assembly model in which the YknZ ED dimer directly fits into the hexameric YknX, which displays an upside-down, funnel-shaped orientation (Fig 6). Given the results presented in this study, we cannot draw conclusions regarding the relationship between the crystal structure of Ba YknZ ED and SDP. However, we speculate that the upper subdomain of YknZ fits into the funnel-mouth region of the YknX hexamer and that the lower subdomain of YknZ provides the SDP entrance site.
In conclusion, we purified YknZ ED and successfully determined the crystal structure of Ba YknZ ED at a resolution of 2.0 Å. Our structure of YknZ showed that each monomer includes a mixed α/β domain, which is dispersed into upper and lower subdomains in a manner similar to that observed with Aa MacB PCD. We also found that the Ba YknZ ED crystal structure was very similar to that of Aa MacB PCD. In vitro binding assays demonstrated that YknZ ED had an affinity toward MFP YknX. The YknX hexamer displayed a upside-down, funnel-shaped orientation similar to Aa MacA. Based on the above results, we proposed an assembly model for YknWXYZ that can offer a structural basis for understanding cannibalistic peptide toxin resistance in B. amyloliquefaciens. However, crystal structures of the complex are needed to reveal the assembly of this type of transporter at the atomic level.