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Crystal structure of the periplasmic domain of TssL, a key membrane component of Type VI secretion system
Accepted Manuscript Crystal structure of the periplasmic domain of TssL, a key membrane component of Type VI secretion system
Xiangbei Wang, Bo Sun, Mengxue Xu, Shenshen Qiu, Dongqing Xu, Tingting Ran, Jianhua He, Weiwu Wang PII: DOI: Reference:
S0141-8130(18)34277-6 doi:10.1016/j.ijbiomac.2018.09.166 BIOMAC 10601
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
16 August 2018 15 September 2018 25 September 2018
Please cite this article as: Xiangbei Wang, Bo Sun, Mengxue Xu, Shenshen Qiu, Dongqing Xu, Tingting Ran, Jianhua He, Weiwu Wang , Crystal structure of the periplasmic domain of TssL, a key membrane component of Type VI secretion system. Biomac (2018), doi:10.1016/j.ijbiomac.2018.09.166
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ACCEPTED MANUSCRIPT
Crystal structure of the periplasmic domain of TssL, a key membrane component of Type VI secretion system Xiangbei Wang1#, Bo Sun2#, Mengxue Xu1, Shenshen Qiu1, Dongqing Xu1, Tingting Ran1*, Jianhua He2*, Weiwu Wang1*
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1. Key Laboratory of Microbiological Engineering of Agricultural Environment of Ministry of Agriculture,
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Department of Microbiology, College of Life Sciences, Nanjing Agricultural University, Nanjing, China
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2. Shanghai Institute of Applied Physics, Chinese Academy of Sciences, 201204 Shanghai, PR China
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# These authors contribute equally.
Dr. Tingting Ran, E-mail: [email protected]
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*Correspondence authors:
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Prof. Dr. Jianhua He, E-mail: [email protected]
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Prof. Dr. Weiwu Wang, Tel,: +86-25-84395003; fax: +86-25-84396542; E-mail: [email protected]
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Abstract Type VI secretion system (T6SS), as a macromolecular system, is commonly found in Gram-negative bacteria and responsible for exporting effectors. T6SS consists of 13 core proteins. TssL is a component of the membrane complex and plays a pivotal role in T6SS. Here, we report the crystal structure of the C-terminal
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periplasmic domain of TssL (TssLCter) from Serratia marcescens FS14. The TssLCter (310-503) contain a five-
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stranded anti-parallel β-sheet flanked by five α-helices and a short N-terminal helix. Structural comparisons
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revealed that it belongs to the OmpA-like family with a remarked difference in the conformation of the loop35. In OmpA-like family, the corresponding loop is located close to loop2-3, forming a cavity with a small
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opening together with the longest α5, whereas in TssLCter, loop3-5 flipped away from this cavity region. In
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addition, significant differences in the peptidoglycan (PG) binding site suggest that big conformational change must take place to accomplish the PG binding for TssLCter. Furthermore, a long flexible loop between helices
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α1 and α2 is unique in TssL. TssL would have a big conformational change during the delivery of the Hcp
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needle and effectors. So we speculate that the long flexible endows TssL the adaptation of its evolutionary new function.
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Keywords: TssL; Type VI secretion system; Crystal structure; Peptidoglycan binding site
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1. Introduction T6SS is a new type of secretion system in Gram-negative bacterial, initially described in Vibrio cholerae in 2006[1]. T6SS is encoded by gene clusters of contiguous genes that often have a variable composition but typically contain at least 13 core genes, which encode the essential core components of the apparatus[2-4]. In
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addition to the core components, some of the gene clusters contain some regulatory components such as a
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Ser/Thr protein kinase (PpkA), phosphatase (PppA), TagF and some effector proteins[5-8].
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The core structure of T6SS consists of 13 core components (TssA-TssM), which assemble a macromolecular complex[9]. Based on their proposed functions, the core components of T6SS can be divided into two parts:
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phage-like subunits and trans-membrane complex [6, 9, 10]. The membrane complex consists of TssJ, TssL
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and TssM[2]. TssJ is an outer-membrane lipoprotein[11, 12]. TssM consists of an N-terminal region, three transmembrane helices and a C-terminal region that connects the inner and outer membranes[13, 14]. The
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cytoplasm and periplasmic domain of TssM interact with TssL and TssJ, respectively[2, 15, 16]. TssL is an
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integral membrane protein anchored to the inner-membrane through its single transmembrane fragment[17]. The crystal structure of TssL cytoplasmic domain (TssLCyto) has been determined in different species, such as
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Escherichia coli, Francisella tularensis, and Vibrio cholerae[18-20]. The crystal structure revealed that
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TssLCyto consists of two three-helix bundles and showed a hook-like structure in the cytoplasm. Two helices of TssLCyto associate to form a functional complex, which interacts with the cytoplasmic portion of TssM, TssE and TssK baseplate components[13, 18, 21, 22]. But the structural information of the C-terminal domain of TssL (TssLCter) is still lacking. In this study, we cloned and expressed the gene of the TssLCter (residues 310 to 503) from S. marcescens FS14. We report the crystal structure of the TssLCter at 2.3Å, the structure exhibits a mixed α/β-fold with five-stranded anti-parallel β-sheet flanked by five short α-helices and a short N-terminal helix. Structural comparisons
ACCEPTED MANUSCRIPT revealed that TssLCter belongs to the OmpA-like family but with remarked differences in the conformation of the loop between β3-α5 (loop 3-5) and the peptidoglycan (PG) binding residues, and a long flexible loop between helices α1 and α2 of TssLCter is unique which provides structural basis for the big conformational change during the ejection process of T6SS.
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2. Materials and Methods
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2.1 Cloning, protein expression, and protein purification
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The C-terminal domain of TssL (TssLCter) gene encoding amino-acid residues 310-503 was amplified through PCR method with the chromosomal DNA of serratia sp. FS14 as template. PCR was carried out with pfu DNA
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polymerase with the forward primer 5’-atacatatgGATCAGTCCCGCGCGCT-3’ and the reverse primer 5’-
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tctaagcttGTTATCCTCGGGGACCTCG-3’. The restriction sites, Nde I and Hind III, in the primers are underlined, respectively. The PCR product was digested with Nde I and Hind III, and then cloned into the
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expression vector pET-28a with a maltose-binding protein (MBP) gene, which is digested with the same
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restriction enzymes to create the expression construct. This plasmid contains the TssLCter gene with 6×Histagged at the C-terminus. The construct was verified by restriction-enzyme digestion and DNA sequencing.
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The verified plasmid was then transformed into E. coli C43 (DE3) for protein expression.
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The fresh colony of E. coli C43 (DE3) with the pET28a-MBP-TssLCter was inoculated overnight in LB medium supplemented with 30 µg/ml kanamycin with shaking (180 rev/min) at 37 ℃. The overnight cultures of the transformant were diluted 1:50 and grown to an OD600 of ~1.0 at 37 ℃, then the cells were induced with 0.5 mM IPTG (isopropyl β-D-thiogalactopyranoside) and grown for another 3 h at 28 ℃. The cells were harvested by centrifugation at 5000 rev/min (Hitachi Rotor R20A2) for 5 min and the cell pellet was then resuspended in binding buffer (50 mM K2HPO4/KH2PO4 pH 7.6, 300 mM NaCl and 5 mM imidazole), supplemented with 1% PMSF, After sonication, and the cell debris was clarified by centrifugation at 12000 rev/min for 30 min
ACCEPTED MANUSCRIPT (Hitachi Rotor R20A2), the recombinant protein was purified by immobilized metal-affinity chromatography (IMAC) on Ni-NTA Superflow (Qiagen). The supernatant was loaded onto a 2 ml bed volume Ni-NTA column pre-equilibrated with 6 bed volumes of binding buffer. Then the column was washed with binding buffer containing 5 mM, 20 mM, 50 mM, 100 mM
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and 250 mM imidazole (6 bed volumes each) at 4 ℃, respectively, the target protein was mainly eluted in the
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buffer with 50 mM imidazole. Peak fractions were pooled and concentrated to approximately 1 ml by
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centrifugation with an Amicon Ultra-15 filter (Millipore). The concentrated protein sample was then loaded onto a Superdex G-200 (Pharmacia Biotech) column pre-equilibrated with 20 mM Tris-HCl pH 8.8, 300 mM
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NaCl. The column was then eluted with 1.2 bed volumes of the same buffer. The elution pattern shows a single
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monodisperse peak. The sample was analyzed by SDS-PAGE to check the protein purity by gel staining with Coomassie Brilliant Blue R250.
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2.2 Crystallization, Date collection, and Structure determination
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The protein was concentrated to 60 mg/ml in 20 mM Tris-HCl pH 8.8, 300 mM NaCl using an Amicon Ultra15 filter (Millipore). Initial crystallization screens were performed using screening kits from Hampton
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Research and Microlytic company employing the sitting-drop vapour-diffusion method. All trials were stored
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at 4 ℃ and 22 ℃, respectively. The first crystal was obtained at 4 ℃ by mixing the protein solution in a 1:1 ratio with reservoir solution consisting of 0.1 M KSCN, 30% (w/v) PEG MME 2000. The initial obtained crystals were not suitable for data collection. So the condition was optimized by grid screening of the precipitate concentration, the pH of the crystallization condition, different concentrations of the protein sample combined with commercial additive and detergent screens (Hampton Research). After around three weeks, single crystals suitable for diffraction were obtained by adding 2.0 M NDSB-221 [3-(1-Methylpiperidinio)-1-propanesulfonate] as additive into the crystallization condition. Before data
ACCEPTED MANUSCRIPT collection, the crystals were looped out from the crystallization drop and quickly transferred into fresh mother liquor with 10% glycerol for a few seconds, and then the crystal was flash-cooled directly in liquid nitrogen. The X-ray diffraction data-sets were collected on beam line BL19U1 of Shanghai Synchrotron Radiation Facility (Shanghai, People’s Republic of China), using a Pilatus 6 M detector. The data were processed using
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the XDS to a resolution of 2.3 Å[23]. The structure of MBP-TssLCter fusion protein was solved by molecular
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replacement method with program Phaser using MBP as the search model[24] The final model of TssLCter was
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then manually built and adjusted with the program coot[25]. The structure was refined using REFMAC5 and Phenix[26, 27]. The data-collection and processing statistics are summarized in Table 1.
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3. Results and Discussion
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3.1 Overall structure of TssLCter
The TssLCter was crystallized in the space groups P212121 with unit-cell dimension parameters a=45.35 Å,
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b=108.89 Å, c=236.88 Å. The crystal structure of the MBP-TssLCter fusion protein was solved using the
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molecular replacement method with the fusing tag MBP as template[28]. The structure was refined to 2.3 Å resolution. Each asymmetric unit contains two molecules of the fusion protein MBP-TssLCter. The final model
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was refined to Rwork values of 18.30% (Rfree of 22.87%). The Ramachandran plot shows that 97.58% of the
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residues were in the favored region, 2.42% were in the allowed region. The superposition of TssLCter two monomers (monomer A and monomer B) shows a backbone rmsd (root mean square deviation) of 0.30 Å indicating that the two TssLCter monomers are almost identical. The data collection and structure refinement statistics are presented in Table 1. The final model has been deposited in the worldwide Protein Data Bank under the PDB ID code 6AEO. The overall structure of the MBP-TssLCter fusion protein is shown in Figure 1. In monomer A, no electron density is visible for the fragment from residues Pro327 to Lys329 in loop between helices α1-α2, in contrast, that loop is clearly resolved in monomer B. The loop between the β3-α5 is clearly
ACCEPTED MANUSCRIPT seen in monomer A, but no electron density is visible for residues from Arg411 to Ser414 in monomer B. These results suggest that these two loops are flexible in the TssLCter. The structure of the TssLCter (310-503 aa) consists of a five-strand β-sheet (β1-2-5-3-4) and six α-helices (α16). TssLCter is composed of a core domain and an N-terminal helix (α1) which is the extensional portion of the
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transmembrane helix. Helix α1 (311-317 aa) locates at the N-terminal and connects with the core domain by a
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long loop (318-339 aa). In the core domain, helices α2-α5 packs against the β–sheet on one side, helix α6
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locates just before the N-terminal of strand β5 and in the same surface level of the β-sheet. Helix α2 (340-346 aa) runs approximately parallel to α4 (384-393 aa); the shortest helix α3 (377-382 aa), which is separated only
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by an arginine residue with α4, runs parallel to the longest helix α5 (416-438 aa). These two parallel groups
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form an angle about 135º. In the β-sheet, β1 and β5 are parallel with each other and be approximately antiparallel to β2, β3 and β4. In the core domain, β2-α3, β3-α5 and β4-α6 are connected by long loops containing
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overall, a question mark shape (Fig. 2).
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13 residues (loop2-3), 11 residues (loop3-5) and 12 residues (loop4-6), respectively (Fig.2). TssLCter presents,
3.2 Structural comparisons indicate TssLCter belong to OmpA family
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The structure similarity search with the Dali server[29] revealed that TssLCter belongs to the OmpA-like family,
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which is conserved in many other Gram-negative organisms and contains the PGB domain[30]. The search retrieved the following structures with backbone rmsd from 2.0 to 3.3 Å: PomB (PDB code 3wpw)[31], YiaD (PDB code 2k1s)[32], YfiB (PDB code 5eb0)[33], and MotB (PDB code 3khn)[34] et al, with relative high Z scores (9.6-19.0) (Table 2). Despite their low sequence identity (≤31%), the core backbone fold of the TssLCter is similar to these proteins. TssLCter shares the highest sequence identity (31%) with PomB, TssLCter superposed very well with PomB (Fig. 3A), but remarked differences are found in the N-terminal helices α1, α3-4 and the loop between β3-α5 (loop3-
ACCEPTED MANUSCRIPT 5) (Fig.3A, 3B). α1 of TssLCter is an extra helix which is not presented in other similar structures and is the extensional portion of the transmembrane helix. A long loop connected the α1 helix with the core domain; this long loop and the α1 helix are unique in TssLCter. The orientation of the loop3-5 is remarkedly different from the corresponding loop of the PomB, in the structure of the TssLCter, the loop3-5 is pointed away from the
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loop2-3 (the loop between β2-α3) and longer than that of the PomB, whereas loop3-5 is located close to loop2-
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3 in PomB. In the PomB, the loop3-5 is considered to contain PG binding site[31].
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3.3 The unique long loop of TssLCter provides structural basis for a functional related big conformational change
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Aside from PomB, in Pal, a PG-associated lipoprotein from Haemophilus influenzae, the corresponding loop3-
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5 also interacts with the peptidoglycan precursor[35]. Superposition of TssLCter with Pal (HiPal) bound to the PG precursor (PG-P, PDB code 2aiz) was shown in Fig. 4A. The PG binding site located at the cavity formed
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by the loop2-3, the loop3-5, and helix α5. Three residues (D71, R73 and D37) bind to PG, making PG and
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protein form a stable complex in this region (Fig 4B). Among these residues, arginine (R73) and aspartic acid (D71) are located on loop3-5, and aspartate (D37) is located on loop2-3, indicating that these two loops play a
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key role in PG binding. However, structural comparison of TssLCter with Pal-PG-P complex showed that the
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corresponding loop2-3 is similar, but the loop3-5 of TssLCter is remarkably different from the corresponding loop3-5 of Pal-PG-P complex. In Pal-PG-P complex, this loop is much shorter than that of TssLCter. In the complex of Pal-PG, D37 in Pal forms hydrogen bonds with the side chain oxygen atoms on m-DAP (mdiaminopimelic acid) in the PG through the amide group on the backbone. Residues D71 and R73 form hydrogen bonds through side-chain carboxyl group and the backbone oxygen, respectively, as shown in Figure 4B[35]. We found that only residue D71 is completely conserved, corresponding to D406 in TssLCter. But the side chain carboxyl group of D406 is approximately perpendicular to that of D71 in Pal, making the functional
ACCEPTED MANUSCRIPT bond deviates from the binding domain (Fig. 4C). In addition, R73 in Pal generates hydrogen bonds with peptidoglycan through the main chain carbonyl group, whereas the carbonyl group of the corresponding K408 in TssLCter points to the reverse direction and was away from peptidoglycan. The D37 of Pal is replaced by a glycine residue (G371) and locates away from peptidoglycan.
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Other than these two proteins, the difference of loop 3-5 exists in the comparison of TssLCter with other OmpA-
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like proteins. Structural superposition between TssLCter and YiaD (PDB ID 2k1s) [32] and YfiB (PDB ID 5eb0)
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[33], two other OmpA-like proteins from E. coli and P. aeruginosa, also revealed a remarked difference in the conformation of loop3-5 (Fig. 5). The loop3-5 in YiaD and YfiB are consistent in orientation that is close to
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loop2-3. This feature is a common characteristic of OmpA-like family protein which is consistent with its PG
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binding property for its anchoring function to the PG layers in bacteria. In TssLCter, the loop 3-5 flipped away from loop 2-3 makes it likely unable to bind PG with this conformation. All these differences between TssLCter
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and other OmpA-like proteins indicate that TssL may not bind to peptidoglycan in the periplasm or a big
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conformational change of loop 3-5 region must take place if TssLcter bind to PG. Another remarkable difference between TssLCter with other OmpA-like proteins is the unique long loop of the
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TssL between the helices α1 and α2, which is flexible in TssLCter. In PomB, the corresponding region is
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disordered before incorporation into the motor and was proposed changing its conformation to interact with the T-ring of the motor, and then the N-terminal two-thirds of α1 has to change its conformation to an extended form to anchor the PG layer[31]. Since PG layer is around 100 Å away from the inner membrane, TssL must undergo a big conformational change to reach the PG layer; this long loop region makes TssLCter structurally long enough to reach the PG layer (Fig. 1). TssL is a component of membrane complex in T6SS, which would have a big conformational change during the ejection of Hcp and effectors. To ensure this open-close conformational change, the protein must have enough structural flexibility; this long flexible loop region would
ACCEPTED MANUSCRIPT endow TssL to accomplish this process (Fig. 1). In conclusion, our study provides the first structural information of the TssL periplasmic domain which shares substantial similarities to other previously reported OmpA-like proteins. The structural analysis showed that the long unique flexible loop between the core domain and helix α1 endows TssL adaptation for its
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conformational changes during the ejection process of T6SS.
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4. Funding information
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This work was supported by grants from the National Natural Science Foundation of China (31770074, 31770050) and the Fundamental Research Funds for the Central Universities (KYZ201740).
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5. Acknowledgment
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We appreciate the staff of BL19U1 beamline at the National Center for Protein Sciences Shanghai, and the staff of beamline BL17U of Shanghai Synchrotron Radiation Facility Shanghai, People’s Republic of China,
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for the assistance during data collection.
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Weiwu Wang, Tingting Ran, Dongqing Xu and Jianhua He designed the experiments; Xiangbei Wang, Bo Sun, Mengxue Xu, Shenshen Qiu and Dongqing Xu conducted the experiments; all authors analyzed the data.
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Weiwu Wang, Xiangbei Wang, Tingting Ran, Dongqing Xu and Jianhua He wrote the manuscript. The authors
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FIGURE LEGENDS: Fig.1 Overall structure of MBP-TssLCter fusion protein in asymmetric unit. MBP: α-helix colored red, loop colored green and β strands colored yellow; TssLCter: α-helix colored cyan,
ACCEPTED MANUSCRIPT loop colored salmon and β strands colored magenta. Fig.2 Overall structure of the TssLCter. The protein is shown as ribbon, α-helices (α1- α6) are colored cyan, β-sheets (β1-β5) are colored magenta, loops are colored pink. Fig.3 Comparison of TssLCter with PomB. (A) Structural superimposition of TssLCter with PomB. (B). Comparison shows the different loops between
Fig.4 Structural comparison of the TssLCter with Pal-PG complex.
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TssLCter and PomB. TssLCter is colored magenta, PomB is colored gray.
(A) PG binding region in TssLCter and Pal. The structure of TssLCter is colored magenta, the structures of
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Pal is colored gray; PG is shown in sticks. (B) Interaction of PG with the residues in Pal. (C) Comparison of
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PG binding site in TssLCter and Pal. Residues in Pal are colored gray, corresponding residues in TssLCter are colored magenta.
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Fig.5 The structure superposition of TssLCter with YiaD and YfiB.
(A). Structure superimposition of TssLCter with the YiaD. (B). Structure superimposition of TssLCter with the
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YfiB. The structure of TssLCter is colored magenta, the structures of YiaD and YfiB are colored gray.
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Table 1 Data collection and structure refinement statistics Data collection 0.9785
Temperature (K)
100
Crystal-to-detector distance (mm)
350
Rotation range per image (o)
0.5
Total rotation range (o)
360
Space group
P212121
Unit-cell parameters (Å)
a=45.35Å, b=108.89Å, c=236.88Å
Resolution range (Å)
20-2.3（2.42-2.3）
Observed reflections
706361（102324）
Unique reflections
53243（7672）
0.051（0.271）
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Rpim (%)
99.7（100.0）
Completeness (%) I/σ(I)
11.0（2.8）
No. of reflections used Rwork(%)
Rmsd bonds (Å)
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Rmsd angles (o)
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Rfree(%)
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Total number of atoms
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Structure refinement
8913 53257 18.30 22.87 0.008 0.914
Ramachandran plot (%)
Allowed Outlier
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13.3（13.3）
Multiplicity
Favored
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Wavelength (Å)
97.58 2.42 0
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Table 2 Structure comparison between TssL-Cter and structurally similar proteins Z-score
RMSD
Residues
Indentity %
Protein
Reference
3wpw
19.0
2.0
149
31
[31]
2k1s
17.3
2.2
149
24
5eb0
17.1
2.5
135
28
3khn
16.8
2.2
162
24
3s0h
16.2
2.3
159
22
4rha
14.0
2.2
127
30
2n48
13.9
2.3
149
3td5
12.1
2.8
120
2aiz
9.6
3.3
134
PomB from Vibrio alginolyticus Innermembrane Lipoprotein YiaD from Escherichia coli YfiB from Pseudomonas aeruginosa MotB protein from Desulfovibrio vulgaris Motility protein B from Helicobacter pylori Outer-membrane protein A from Salmonella typhimurium Probable Lipoprotein YiaD from Escherichia coli Outer-membrane protein from Acinetobacter baumannii Outer membrane lipoprotein Pal from Haemophilus influenzae
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PDB ID
25 29
17
[32] [33] [34] [36] [37] [38] [39]
[35]
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FIGURE 1
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FIGURE 2
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FIGURE 3
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FIGURE 4
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FIGURE 5
Xiangbei Wang, Bo Sun, Mengxue Xu, Shenshen Qiu, Dongqing Xu, Tingting Ran, Jianhua He, Weiwu Wang PII: DOI: Reference:
S0141-8130(18)34277-6 doi:10.1016/j.ijbiomac.2018.09.166 BIOMAC 10601
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
16 August 2018 15 September 2018 25 September 2018
Please cite this article as: Xiangbei Wang, Bo Sun, Mengxue Xu, Shenshen Qiu, Dongqing Xu, Tingting Ran, Jianhua He, Weiwu Wang , Crystal structure of the periplasmic domain of TssL, a key membrane component of Type VI secretion system. Biomac (2018), doi:10.1016/j.ijbiomac.2018.09.166
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Crystal structure of the periplasmic domain of TssL, a key membrane component of Type VI secretion system Xiangbei Wang1#, Bo Sun2#, Mengxue Xu1, Shenshen Qiu1, Dongqing Xu1, Tingting Ran1*, Jianhua He2*, Weiwu Wang1*
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1. Key Laboratory of Microbiological Engineering of Agricultural Environment of Ministry of Agriculture,
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Department of Microbiology, College of Life Sciences, Nanjing Agricultural University, Nanjing, China
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2. Shanghai Institute of Applied Physics, Chinese Academy of Sciences, 201204 Shanghai, PR China
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# These authors contribute equally.
Dr. Tingting Ran, E-mail: [email protected]
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*Correspondence authors:
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Prof. Dr. Jianhua He, E-mail: [email protected]
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Prof. Dr. Weiwu Wang, Tel,: +86-25-84395003; fax: +86-25-84396542; E-mail: [email protected]
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Abstract Type VI secretion system (T6SS), as a macromolecular system, is commonly found in Gram-negative bacteria and responsible for exporting effectors. T6SS consists of 13 core proteins. TssL is a component of the membrane complex and plays a pivotal role in T6SS. Here, we report the crystal structure of the C-terminal
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periplasmic domain of TssL (TssLCter) from Serratia marcescens FS14. The TssLCter (310-503) contain a five-
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stranded anti-parallel β-sheet flanked by five α-helices and a short N-terminal helix. Structural comparisons
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revealed that it belongs to the OmpA-like family with a remarked difference in the conformation of the loop35. In OmpA-like family, the corresponding loop is located close to loop2-3, forming a cavity with a small
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opening together with the longest α5, whereas in TssLCter, loop3-5 flipped away from this cavity region. In
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addition, significant differences in the peptidoglycan (PG) binding site suggest that big conformational change must take place to accomplish the PG binding for TssLCter. Furthermore, a long flexible loop between helices
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α1 and α2 is unique in TssL. TssL would have a big conformational change during the delivery of the Hcp
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needle and effectors. So we speculate that the long flexible endows TssL the adaptation of its evolutionary new function.
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Keywords: TssL; Type VI secretion system; Crystal structure; Peptidoglycan binding site
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1. Introduction T6SS is a new type of secretion system in Gram-negative bacterial, initially described in Vibrio cholerae in 2006[1]. T6SS is encoded by gene clusters of contiguous genes that often have a variable composition but typically contain at least 13 core genes, which encode the essential core components of the apparatus[2-4]. In
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addition to the core components, some of the gene clusters contain some regulatory components such as a
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Ser/Thr protein kinase (PpkA), phosphatase (PppA), TagF and some effector proteins[5-8].
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The core structure of T6SS consists of 13 core components (TssA-TssM), which assemble a macromolecular complex[9]. Based on their proposed functions, the core components of T6SS can be divided into two parts:
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phage-like subunits and trans-membrane complex [6, 9, 10]. The membrane complex consists of TssJ, TssL
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and TssM[2]. TssJ is an outer-membrane lipoprotein[11, 12]. TssM consists of an N-terminal region, three transmembrane helices and a C-terminal region that connects the inner and outer membranes[13, 14]. The
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cytoplasm and periplasmic domain of TssM interact with TssL and TssJ, respectively[2, 15, 16]. TssL is an
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integral membrane protein anchored to the inner-membrane through its single transmembrane fragment[17]. The crystal structure of TssL cytoplasmic domain (TssLCyto) has been determined in different species, such as
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Escherichia coli, Francisella tularensis, and Vibrio cholerae[18-20]. The crystal structure revealed that
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TssLCyto consists of two three-helix bundles and showed a hook-like structure in the cytoplasm. Two helices of TssLCyto associate to form a functional complex, which interacts with the cytoplasmic portion of TssM, TssE and TssK baseplate components[13, 18, 21, 22]. But the structural information of the C-terminal domain of TssL (TssLCter) is still lacking. In this study, we cloned and expressed the gene of the TssLCter (residues 310 to 503) from S. marcescens FS14. We report the crystal structure of the TssLCter at 2.3Å, the structure exhibits a mixed α/β-fold with five-stranded anti-parallel β-sheet flanked by five short α-helices and a short N-terminal helix. Structural comparisons
ACCEPTED MANUSCRIPT revealed that TssLCter belongs to the OmpA-like family but with remarked differences in the conformation of the loop between β3-α5 (loop 3-5) and the peptidoglycan (PG) binding residues, and a long flexible loop between helices α1 and α2 of TssLCter is unique which provides structural basis for the big conformational change during the ejection process of T6SS.
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2. Materials and Methods
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2.1 Cloning, protein expression, and protein purification
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The C-terminal domain of TssL (TssLCter) gene encoding amino-acid residues 310-503 was amplified through PCR method with the chromosomal DNA of serratia sp. FS14 as template. PCR was carried out with pfu DNA
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polymerase with the forward primer 5’-atacatatgGATCAGTCCCGCGCGCT-3’ and the reverse primer 5’-
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tctaagcttGTTATCCTCGGGGACCTCG-3’. The restriction sites, Nde I and Hind III, in the primers are underlined, respectively. The PCR product was digested with Nde I and Hind III, and then cloned into the
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expression vector pET-28a with a maltose-binding protein (MBP) gene, which is digested with the same
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restriction enzymes to create the expression construct. This plasmid contains the TssLCter gene with 6×Histagged at the C-terminus. The construct was verified by restriction-enzyme digestion and DNA sequencing.
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The verified plasmid was then transformed into E. coli C43 (DE3) for protein expression.
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The fresh colony of E. coli C43 (DE3) with the pET28a-MBP-TssLCter was inoculated overnight in LB medium supplemented with 30 µg/ml kanamycin with shaking (180 rev/min) at 37 ℃. The overnight cultures of the transformant were diluted 1:50 and grown to an OD600 of ~1.0 at 37 ℃, then the cells were induced with 0.5 mM IPTG (isopropyl β-D-thiogalactopyranoside) and grown for another 3 h at 28 ℃. The cells were harvested by centrifugation at 5000 rev/min (Hitachi Rotor R20A2) for 5 min and the cell pellet was then resuspended in binding buffer (50 mM K2HPO4/KH2PO4 pH 7.6, 300 mM NaCl and 5 mM imidazole), supplemented with 1% PMSF, After sonication, and the cell debris was clarified by centrifugation at 12000 rev/min for 30 min
ACCEPTED MANUSCRIPT (Hitachi Rotor R20A2), the recombinant protein was purified by immobilized metal-affinity chromatography (IMAC) on Ni-NTA Superflow (Qiagen). The supernatant was loaded onto a 2 ml bed volume Ni-NTA column pre-equilibrated with 6 bed volumes of binding buffer. Then the column was washed with binding buffer containing 5 mM, 20 mM, 50 mM, 100 mM
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and 250 mM imidazole (6 bed volumes each) at 4 ℃, respectively, the target protein was mainly eluted in the
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buffer with 50 mM imidazole. Peak fractions were pooled and concentrated to approximately 1 ml by
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centrifugation with an Amicon Ultra-15 filter (Millipore). The concentrated protein sample was then loaded onto a Superdex G-200 (Pharmacia Biotech) column pre-equilibrated with 20 mM Tris-HCl pH 8.8, 300 mM
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NaCl. The column was then eluted with 1.2 bed volumes of the same buffer. The elution pattern shows a single
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monodisperse peak. The sample was analyzed by SDS-PAGE to check the protein purity by gel staining with Coomassie Brilliant Blue R250.
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2.2 Crystallization, Date collection, and Structure determination
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The protein was concentrated to 60 mg/ml in 20 mM Tris-HCl pH 8.8, 300 mM NaCl using an Amicon Ultra15 filter (Millipore). Initial crystallization screens were performed using screening kits from Hampton
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Research and Microlytic company employing the sitting-drop vapour-diffusion method. All trials were stored
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at 4 ℃ and 22 ℃, respectively. The first crystal was obtained at 4 ℃ by mixing the protein solution in a 1:1 ratio with reservoir solution consisting of 0.1 M KSCN, 30% (w/v) PEG MME 2000. The initial obtained crystals were not suitable for data collection. So the condition was optimized by grid screening of the precipitate concentration, the pH of the crystallization condition, different concentrations of the protein sample combined with commercial additive and detergent screens (Hampton Research). After around three weeks, single crystals suitable for diffraction were obtained by adding 2.0 M NDSB-221 [3-(1-Methylpiperidinio)-1-propanesulfonate] as additive into the crystallization condition. Before data
ACCEPTED MANUSCRIPT collection, the crystals were looped out from the crystallization drop and quickly transferred into fresh mother liquor with 10% glycerol for a few seconds, and then the crystal was flash-cooled directly in liquid nitrogen. The X-ray diffraction data-sets were collected on beam line BL19U1 of Shanghai Synchrotron Radiation Facility (Shanghai, People’s Republic of China), using a Pilatus 6 M detector. The data were processed using
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the XDS to a resolution of 2.3 Å[23]. The structure of MBP-TssLCter fusion protein was solved by molecular
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replacement method with program Phaser using MBP as the search model[24] The final model of TssLCter was
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then manually built and adjusted with the program coot[25]. The structure was refined using REFMAC5 and Phenix[26, 27]. The data-collection and processing statistics are summarized in Table 1.
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3. Results and Discussion
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3.1 Overall structure of TssLCter
The TssLCter was crystallized in the space groups P212121 with unit-cell dimension parameters a=45.35 Å,
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b=108.89 Å, c=236.88 Å. The crystal structure of the MBP-TssLCter fusion protein was solved using the
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molecular replacement method with the fusing tag MBP as template[28]. The structure was refined to 2.3 Å resolution. Each asymmetric unit contains two molecules of the fusion protein MBP-TssLCter. The final model
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was refined to Rwork values of 18.30% (Rfree of 22.87%). The Ramachandran plot shows that 97.58% of the
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residues were in the favored region, 2.42% were in the allowed region. The superposition of TssLCter two monomers (monomer A and monomer B) shows a backbone rmsd (root mean square deviation) of 0.30 Å indicating that the two TssLCter monomers are almost identical. The data collection and structure refinement statistics are presented in Table 1. The final model has been deposited in the worldwide Protein Data Bank under the PDB ID code 6AEO. The overall structure of the MBP-TssLCter fusion protein is shown in Figure 1. In monomer A, no electron density is visible for the fragment from residues Pro327 to Lys329 in loop between helices α1-α2, in contrast, that loop is clearly resolved in monomer B. The loop between the β3-α5 is clearly
ACCEPTED MANUSCRIPT seen in monomer A, but no electron density is visible for residues from Arg411 to Ser414 in monomer B. These results suggest that these two loops are flexible in the TssLCter. The structure of the TssLCter (310-503 aa) consists of a five-strand β-sheet (β1-2-5-3-4) and six α-helices (α16). TssLCter is composed of a core domain and an N-terminal helix (α1) which is the extensional portion of the
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transmembrane helix. Helix α1 (311-317 aa) locates at the N-terminal and connects with the core domain by a
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long loop (318-339 aa). In the core domain, helices α2-α5 packs against the β–sheet on one side, helix α6
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locates just before the N-terminal of strand β5 and in the same surface level of the β-sheet. Helix α2 (340-346 aa) runs approximately parallel to α4 (384-393 aa); the shortest helix α3 (377-382 aa), which is separated only
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by an arginine residue with α4, runs parallel to the longest helix α5 (416-438 aa). These two parallel groups
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form an angle about 135º. In the β-sheet, β1 and β5 are parallel with each other and be approximately antiparallel to β2, β3 and β4. In the core domain, β2-α3, β3-α5 and β4-α6 are connected by long loops containing
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overall, a question mark shape (Fig. 2).
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13 residues (loop2-3), 11 residues (loop3-5) and 12 residues (loop4-6), respectively (Fig.2). TssLCter presents,
3.2 Structural comparisons indicate TssLCter belong to OmpA family
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The structure similarity search with the Dali server[29] revealed that TssLCter belongs to the OmpA-like family,
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which is conserved in many other Gram-negative organisms and contains the PGB domain[30]. The search retrieved the following structures with backbone rmsd from 2.0 to 3.3 Å: PomB (PDB code 3wpw)[31], YiaD (PDB code 2k1s)[32], YfiB (PDB code 5eb0)[33], and MotB (PDB code 3khn)[34] et al, with relative high Z scores (9.6-19.0) (Table 2). Despite their low sequence identity (≤31%), the core backbone fold of the TssLCter is similar to these proteins. TssLCter shares the highest sequence identity (31%) with PomB, TssLCter superposed very well with PomB (Fig. 3A), but remarked differences are found in the N-terminal helices α1, α3-4 and the loop between β3-α5 (loop3-
ACCEPTED MANUSCRIPT 5) (Fig.3A, 3B). α1 of TssLCter is an extra helix which is not presented in other similar structures and is the extensional portion of the transmembrane helix. A long loop connected the α1 helix with the core domain; this long loop and the α1 helix are unique in TssLCter. The orientation of the loop3-5 is remarkedly different from the corresponding loop of the PomB, in the structure of the TssLCter, the loop3-5 is pointed away from the
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loop2-3 (the loop between β2-α3) and longer than that of the PomB, whereas loop3-5 is located close to loop2-
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3 in PomB. In the PomB, the loop3-5 is considered to contain PG binding site[31].
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3.3 The unique long loop of TssLCter provides structural basis for a functional related big conformational change
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Aside from PomB, in Pal, a PG-associated lipoprotein from Haemophilus influenzae, the corresponding loop3-
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5 also interacts with the peptidoglycan precursor[35]. Superposition of TssLCter with Pal (HiPal) bound to the PG precursor (PG-P, PDB code 2aiz) was shown in Fig. 4A. The PG binding site located at the cavity formed
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by the loop2-3, the loop3-5, and helix α5. Three residues (D71, R73 and D37) bind to PG, making PG and
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protein form a stable complex in this region (Fig 4B). Among these residues, arginine (R73) and aspartic acid (D71) are located on loop3-5, and aspartate (D37) is located on loop2-3, indicating that these two loops play a
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key role in PG binding. However, structural comparison of TssLCter with Pal-PG-P complex showed that the
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corresponding loop2-3 is similar, but the loop3-5 of TssLCter is remarkably different from the corresponding loop3-5 of Pal-PG-P complex. In Pal-PG-P complex, this loop is much shorter than that of TssLCter. In the complex of Pal-PG, D37 in Pal forms hydrogen bonds with the side chain oxygen atoms on m-DAP (mdiaminopimelic acid) in the PG through the amide group on the backbone. Residues D71 and R73 form hydrogen bonds through side-chain carboxyl group and the backbone oxygen, respectively, as shown in Figure 4B[35]. We found that only residue D71 is completely conserved, corresponding to D406 in TssLCter. But the side chain carboxyl group of D406 is approximately perpendicular to that of D71 in Pal, making the functional
ACCEPTED MANUSCRIPT bond deviates from the binding domain (Fig. 4C). In addition, R73 in Pal generates hydrogen bonds with peptidoglycan through the main chain carbonyl group, whereas the carbonyl group of the corresponding K408 in TssLCter points to the reverse direction and was away from peptidoglycan. The D37 of Pal is replaced by a glycine residue (G371) and locates away from peptidoglycan.
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Other than these two proteins, the difference of loop 3-5 exists in the comparison of TssLCter with other OmpA-
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like proteins. Structural superposition between TssLCter and YiaD (PDB ID 2k1s) [32] and YfiB (PDB ID 5eb0)
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[33], two other OmpA-like proteins from E. coli and P. aeruginosa, also revealed a remarked difference in the conformation of loop3-5 (Fig. 5). The loop3-5 in YiaD and YfiB are consistent in orientation that is close to
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loop2-3. This feature is a common characteristic of OmpA-like family protein which is consistent with its PG
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binding property for its anchoring function to the PG layers in bacteria. In TssLCter, the loop 3-5 flipped away from loop 2-3 makes it likely unable to bind PG with this conformation. All these differences between TssLCter
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and other OmpA-like proteins indicate that TssL may not bind to peptidoglycan in the periplasm or a big
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conformational change of loop 3-5 region must take place if TssLcter bind to PG. Another remarkable difference between TssLCter with other OmpA-like proteins is the unique long loop of the
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TssL between the helices α1 and α2, which is flexible in TssLCter. In PomB, the corresponding region is
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disordered before incorporation into the motor and was proposed changing its conformation to interact with the T-ring of the motor, and then the N-terminal two-thirds of α1 has to change its conformation to an extended form to anchor the PG layer[31]. Since PG layer is around 100 Å away from the inner membrane, TssL must undergo a big conformational change to reach the PG layer; this long loop region makes TssLCter structurally long enough to reach the PG layer (Fig. 1). TssL is a component of membrane complex in T6SS, which would have a big conformational change during the ejection of Hcp and effectors. To ensure this open-close conformational change, the protein must have enough structural flexibility; this long flexible loop region would
ACCEPTED MANUSCRIPT endow TssL to accomplish this process (Fig. 1). In conclusion, our study provides the first structural information of the TssL periplasmic domain which shares substantial similarities to other previously reported OmpA-like proteins. The structural analysis showed that the long unique flexible loop between the core domain and helix α1 endows TssL adaptation for its
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conformational changes during the ejection process of T6SS.
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4. Funding information
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This work was supported by grants from the National Natural Science Foundation of China (31770074, 31770050) and the Fundamental Research Funds for the Central Universities (KYZ201740).
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5. Acknowledgment
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We appreciate the staff of BL19U1 beamline at the National Center for Protein Sciences Shanghai, and the staff of beamline BL17U of Shanghai Synchrotron Radiation Facility Shanghai, People’s Republic of China,
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for the assistance during data collection.
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Weiwu Wang, Tingting Ran, Dongqing Xu and Jianhua He designed the experiments; Xiangbei Wang, Bo Sun, Mengxue Xu, Shenshen Qiu and Dongqing Xu conducted the experiments; all authors analyzed the data.
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Weiwu Wang, Xiangbei Wang, Tingting Ran, Dongqing Xu and Jianhua He wrote the manuscript. The authors
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declare that they have no conflict of interest.
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Zoued, A., et al., TssK Is a Trimeric Cytoplasmic Protein Interacting with Components of Both Phage-like and Membrane Anchoring Complexes of the Type VI Secretion System. Journal of Biological Chemistry, 2013. 288(38): p. 27031-27041.
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Zhang, F., et al., Crystal structure of MBP-PigG fusion protein and the essential function of PigG in the prodigiosin biosynthetic pathway in Serratia marcescens FS14. International Journal of Biological Macromolecules, 2017. 99: p. 394-400. Holm, L. and P. Rosenström, Dali server: conservation mapping in 3D. Nucleic Acids Research, 2010. 38(Web
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Ramagopal, U.A., et al., Crystal structure of putative MotB like protein DVU_2228 from Desulfovibrio vulgaris.www.pdb.org; PDB ID:3khn.
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Parsons, L.M., F. Lin, and J. Orban, Peptidoglycan recognition by Pal, an outer membrane lipoprotein.
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FIGURE LEGENDS: Fig.1 Overall structure of MBP-TssLCter fusion protein in asymmetric unit. MBP: α-helix colored red, loop colored green and β strands colored yellow; TssLCter: α-helix colored cyan,
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Fig.4 Structural comparison of the TssLCter with Pal-PG complex.
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TssLCter and PomB. TssLCter is colored magenta, PomB is colored gray.
(A) PG binding region in TssLCter and Pal. The structure of TssLCter is colored magenta, the structures of
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Pal is colored gray; PG is shown in sticks. (B) Interaction of PG with the residues in Pal. (C) Comparison of
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PG binding site in TssLCter and Pal. Residues in Pal are colored gray, corresponding residues in TssLCter are colored magenta.
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Fig.5 The structure superposition of TssLCter with YiaD and YfiB.
(A). Structure superimposition of TssLCter with the YiaD. (B). Structure superimposition of TssLCter with the
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YfiB. The structure of TssLCter is colored magenta, the structures of YiaD and YfiB are colored gray.
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Table 1 Data collection and structure refinement statistics Data collection 0.9785
Temperature (K)
100
Crystal-to-detector distance (mm)
350
Rotation range per image (o)
0.5
Total rotation range (o)
360
Space group
P212121
Unit-cell parameters (Å)
a=45.35Å, b=108.89Å, c=236.88Å
Resolution range (Å)
20-2.3（2.42-2.3）
Observed reflections
706361（102324）
Unique reflections
53243（7672）
0.051（0.271）
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Rpim (%)
99.7（100.0）
Completeness (%) I/σ(I)
11.0（2.8）
No. of reflections used Rwork(%)
Rmsd bonds (Å)
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Rmsd angles (o)
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Rfree(%)
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Total number of atoms
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Structure refinement
8913 53257 18.30 22.87 0.008 0.914
Ramachandran plot (%)
Allowed Outlier
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Multiplicity
Favored
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Wavelength (Å)
97.58 2.42 0
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Table 2 Structure comparison between TssL-Cter and structurally similar proteins Z-score
RMSD
Residues
Indentity %
Protein
Reference
3wpw
19.0
2.0
149
31
[31]
2k1s
17.3
2.2
149
24
5eb0
17.1
2.5
135
28
3khn
16.8
2.2
162
24
3s0h
16.2
2.3
159
22
4rha
14.0
2.2
127
30
2n48
13.9
2.3
149
3td5
12.1
2.8
120
2aiz
9.6
3.3
134
PomB from Vibrio alginolyticus Innermembrane Lipoprotein YiaD from Escherichia coli YfiB from Pseudomonas aeruginosa MotB protein from Desulfovibrio vulgaris Motility protein B from Helicobacter pylori Outer-membrane protein A from Salmonella typhimurium Probable Lipoprotein YiaD from Escherichia coli Outer-membrane protein from Acinetobacter baumannii Outer membrane lipoprotein Pal from Haemophilus influenzae
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PDB ID
25 29
17
[32] [33] [34] [36] [37] [38] [39]
[35]
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FIGURE 5