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Crystal structure of Cry6Aa: A novel nematicidal ClyA-type α-pore-forming toxin from Bacillus thuringiensis
Accepted Manuscript Crystal structure of Cry6Aa: A novel nematicidal ClyA-type α-pore-forming toxin from Bacillus thuringiensis Jinbo Huang, Zeyuan Guan, Liting Wan, Tingting Zou, Ming Sun PII:
S0006-291X(16)31118-4
DOI:
10.1016/j.bbrc.2016.07.002
Reference:
YBBRC 36079
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 26 June 2016 Accepted Date: 1 July 2016
Please cite this article as: J. Huang, Z. Guan, L. Wan, T. Zou, M. Sun, Crystal structure of Cry6Aa: A novel nematicidal ClyA-type α-pore-forming toxin from Bacillus thuringiensis, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.07.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Crystal Structure of Cry6Aa: a novel nematicidal ClyA-type
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α-pore-forming toxin from Bacillus thuringiensis
3 Jinbo Huang1, Zeyuan Guan2, Liting Wan1,3, Tingting Zou1, and Ming Sun1,3,#
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College of Life Sciences and Technology, Huazhong Agricultural University, Wuhan 430070, China.
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National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan
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430070, China.
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State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070,
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China.
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Correspondence author. E-mail address: [email protected].
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ACCEPTED MANUSCRIPT ABSTRACT
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Crystal (Cry) proteins from Bacillus thuringiensis (Bt) are globally used in agriculture as
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proteinaceous insecticides . Numerous crystal structures have been determined, and most exhibit
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conserved three-dimensional architectures. Recently, we have identified a novel nematicidal
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mechanism by which Cry6Aa triggers cell death through a necrosis-signaling pathway via an
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interaction with the host protease ASP-1. However, we found little sequence conservation of
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Cry6Aa in our functional study. Here, we report the 1.90 angstrom (Å) resolution structure of
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the proteolytic form of Cry6Aa (1-396), determined by X-ray crystallography. The structure of
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Cry6Aa is highly similar to those of the pathogenic toxin family of ClyA-type α-pore-forming
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toxins (α-PFTs), which are characterized by a bipartite structure comprising a head domain and
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a tail domain, thus suggesting that Cry6Aa exhibits a previously undescribed nematicidal mode
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of action. This structure also provides a framework for the functional study of other nematicidal
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toxins.
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Keywords: Bacillus thuringiensis, Crystal structure, Nematicidal, Pore-forming toxin
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1. Introduction The Gram-positive soil bacterium Bacillus thuringiensis (Bt) produces parasporal crystal (Cry) toxins during the sporulation phase of growth [1]. On the basis of functional and
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bioinformatic analyses, more than 300 holotypes of Cry toxins have been distinguished and
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categorized into73 families [2,3]. Many of these proteins are toxic to many insect species and
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have been successfully used to directly kill insect pests as proteinaceous insecticides or to
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control insect growth on transgenic agricultural plants [4]. The mechanism by which these
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insecticidal toxins specifically target their hosts has been thoroughly studied. In the alkaline
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insect midgut [5], the monomeric Cry protoxin is first digested by the host protease and then
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binds to its receptors. This binding facilitates the proteolytic cleavage of the N-terminal α-helix,
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which in turn induces the oligomerization of the activated toxin and forms a pore via insertion
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into the membrane, thus eventually leading to osmotic cell death. Structural investigations of
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insecticidal Cry toxins have revealed that most share a distinct compact three-domain
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architecture [2,6]. Domain I comprises an N-terminal α-helix cluster and is responsible for toxin
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membrane penetration and pore formation. Domain II and Domain III are rich in β-sheets that
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resemble the β-prism fold and jelly-roll fold, respectively, and are involved in receptor
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recognition [2,7,8].
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A new group of Cry proteins has been identified as nematode parasite intoxicants,
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including the Cry5, Cry6, Cry14, Cry21 and Cry55 families [9,10]. In contrast to the
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well-studied insect model, the mode of activation of nematicidal Cry toxins has been
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investigated only in Caenorhabditis elegans (C. elegans) by using Cry5Ba [11]. Although
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Cry5Ba and Cry6Aa exhibit very little sequence homology with the classic cry toxins, they
ACCEPTED MANUSCRIPT represent two distinct families. Interestingly, the Cry5Ba crystal structure exhibits a conserved
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three-domain architecture, thus suggesting that Cry5Ba undergoes pore-forming activation
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reminiscent of that in insecticidal Cry proteins. The structural differences in Domain II between
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Cry5Ba and insecticidal Cry explain the varying selectivity for different hosts’ receptors
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[11,12,13].
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We have recently characterized a necrotic cell death pathway induced by Cry6Aa and
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identified the aspartic protease ASP-1, which primarily accumulates in the intestinal cells of C.
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elegans and acts as an activator in this pathway [14]. However, the molecular mechanism by
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which Cry6Aa is activated remains unknown.
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In this study, we determined the crystal structure of Cry6Aa at a resolution of 1.90 Å. Structural analysis demonstrated that Cry6Aa belongs to the ClyA-type α-pore-forming
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toxin (α-PFT) family and suggested that a membrane-insertion mechanism is responsible for its
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toxicity.
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2. Materials and methods
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2.1 . Cry6Aa protein preparation
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The Cry6Aa (GenBank Acc. No. AF499736) gene was amplified from Bt strain YBT-1518
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[15] using the primer pairs Cry6Aa-M1-F (5′- ATGATTATTGATAGTAAAACGAC-3′) and
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Cry6Aa-N475-R (5′- ATTATTATACCAATCCGAATTATTATAC-3′) and was subsequently
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cloned into the pQLinkH vector (GeneBank: EF025688) with a 7×His tag at the N-terminus
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[16]. To obtain Cry6Aa crystals, a series of gene boundaries were constructed in the same vector.
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The plasmids were transformed into Escherichia coli (E. coli) strain BL21(DE3). One liter of
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lysogeny broth (LB) medium supplemented with 100 µg ml-1 ampicillin was inoculated with a
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reached 1.0. After growing for 16 hours at 16 °C, the bacterial pellet was collected and
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homogenized in a buffer containing 25 mM Tris-HCl, pH 8.0, and 150 mM NaCl. After
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sonication and centrifugation at 23,000 g at 4 °C, the supernatant was loaded onto a column
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equipped with Ni2+ affinity resin (Ni-NTA, Qiagen); then washed with buffer containing 25 mM
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Tris-HCl, pH 8.0, 150 mM NaCl, and 15 mM imidazole; and eluted with buffer containing 25
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mM Tris-HCl, pH 8.0, and 250 mM imidazole. The protein was further purified using
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anion-exchange chromatography (Source 15Q, GE Healthcare). The purified Cry6Aa was
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concentrated to approximately 15 mg ml-1 (Amicon, 10 kDa cutoff, Millipore) and then
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subjected to size-exclusion chromatography (Superdex-200 Increase 10/300, GE Healthcare).
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The buffer used for the size-exclusion chromatography contained 25-mM Tris-HCl, pH 8.0,
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150-mM NaCl and 5-mM dithiothreitol (DTT).
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2.2. Purification of the Se-Met-labeled Cry6Aa
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A mutant carrying I125M/K228M was generated for the phase determination of the native structure of Cry6Aa. Mutagenesis of the Cry6Aa gene was introduced via two rounds of overlap
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polymerase chain reaction (PCR) and verified by sequencing. The plasmid was transformed into
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E. coli strain BL21(DE3), and the culture was grown in medium from a Seleno MetTM Medium
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Base* MD 12-501 Kit (Molecular Dimension Co., Ltd.). The expression and purification of the
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Se-Met-labeled mutant Cry6Aa were performed by using the same procedures as used for the
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native protein, except 10 mM DTT was added to the buffer after the protein was eluted from the
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Ni2+ affinity resin. The yield of the Se-Met-labeled mutant was estimated to be approximately
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10 mg of protein per liter of bacterial cell culture.
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2.3. Crystallization The crystallization experiments were performed using the sitting-drop vapor diffusion method at 18 °C by mixing 1 µl of sample with an equal volume of the reservoir solution.
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Needle-shaped crystals of native Cry6Aa (residues 1-395) appeared overnight and grew to
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maturity within seven days in drops containing 0.1 M Tris-HCl, pH 8.8, 15% PEG 1000 (Sigma),
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and 30 mM DTT. After several rounds of optimization, the Se-Met-labeled Cry6Aa (residues
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1-395, I125M/K228M) was crystallized in the reservoir solution of 100 mM Tris-HCl, pH 8.8, 6%
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PEG 1500, 10% PEG 400, 75 mM NaF, and 50 mM DTT and exhibited the same shape as that
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of the native Cry6Aa crystals, although they were smaller. The crystals were flash frozen in
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liquid nitrogen and cryoprotected via the addition of glycerol to a final concentration of 20%.
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The Se-Met-labeled Cry6Aa and native Cry6Aa crystals were diffracted beyond 1.90 Å at the
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SSRF beamline BL17U and BL19U.
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2.4. Data collection and structural determination
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All diffraction data were collected at the SSRF on beamlines BL17U and BL19U by using a charge-coupled device (CCD) detector cooled to 100 K. The data from the Cry6Aa crystals
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were processed with the HKL2000 program suite and XDS packages[17]. Further processing
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was performed using the programs from the CCP4 suite [18]. The Cry6Aa structure was solved
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via SAD of by Se-Met using the ShelxC/D/E program [19]. The model obtained from
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Se-Met-SAD was used for molecular replacement with the program Phaser into the native data
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for native structure determination [20]. Next, manual model building and refinement were
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performed iteratively with Coot and Phenix [21,22]. The data collection and structure
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refinement statistics are summarized in Extended Data Table 1. All figures representing the
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structures were prepared with PyMOL [23].
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3. Results and Discussion
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3.1. Overall Structure of Cry6Aa The Cry6Aa gene was cloned from Bt strain YBT-1518 [15] and expressed in E. coli. The
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full-length protein was purified to homogeneity and was easy to crystallized but yielded only
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small-needle type crystals with low resolution after numerous optimization trials. We speculated
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that Cry6Aa might be more stable after proteolytic activation. To test this possibility, we treated
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the Cry6Aa protein with increasing amounts of trypsin. A protease-resistant core domain
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(residues 1-396, identified by mass spectrometry) was observed and produced good
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diffraction-quality crystals. However, no homologous structure was available for structural
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determination. Furthermore, because only one methionine residue (Met 315) was found in this
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crystallized boundary, our identification of the phase using selenomethionine (Se-Met)-based
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single-wavelength anomalous diffraction (SAD) was hindered. After numerous
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methionine-substitution screenings, we finally collected the anomalous signal diffracted data by
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using the double mutant crystal (I125M/K228M) and determined the structure of the Cry6Aa
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core domain (Cry6Aa, residues 1-396) in the P65 space group at a refined resolution of 1.90 Å
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(Table 1). One molecule was observed in each asymmetric unit. Additionally, most of the
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residues were well built, except for residues 387–396 because of their limited electronic density.
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The overall structure of Cry6Aa exhibits a rod-like appearance with a diameter of
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approximately 25 Å and a height of approximately 95 Å (Fig. 1A). Cry6Aa comprises seven
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helices: αA (residues 10-68), αB (residues 78-142), αC (residues 148-215), αD (residues
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220-241), αE (residues 265-289), αF (residues 292-353), and αG (residues 357-383). The
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and αA3 (residues 61-68). The orientation of αA1 turns upward, and αA1 and αA3 are
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perpendicular to αA2 and connected to two flexible loops. αB, αC and αF are composed of
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many long helices that form the skeleton of the structure. Two outer loops, i.e., residues
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121-129 and 189-196, break the integrities of αB and αC, respectively, thus suggesting that
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they are flexible and play a role in pore formation. αD and αE are two short anti-parallel helices,
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which are separated by a long hydrophobic loop (residues 242-264). The C-terminal αG points
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to αA2 and inserts into the groove between αA and αB.
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3.2. Cry6Aa is structurally related to the ClyA α-PFT family.
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A structural similarity search on the Dali server [24] indicated that the structure of Cry6Aa
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is closely related to those of Hbl-B (PDB: 2nrj) [25] and NheA (PDB: 4k1p) [26], both of which
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are from Bacillus cereus (B. cereus), and the monomeric ClyA (PDB: 1qoy) [27,28] from E.
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coli (Table 2 and Fig. 2). Although none of these proteins share significant sequence homology
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with Cry6Aa (Table 3), their structures are highly similar. The closest entry was Hbl-B, with a
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Z-score of 24.5 and a root mean-square deviation (RMSD) of 2.8 Å between 338 α-carbon atom
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pairs. The second closest structural match was the NheA toxin with a Z-score of 21.4 and a
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RMSD of 3.5 Å between 338 α-carbon atom matches. The other important result is the
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monomeric ClyA toxin, which had a Z score of 12.1 and a RMSD of 4.3 Å over 303 aligned
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residues (Table 2).
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All of these proteins belong to the α-Pore-forming toxins (PFTs) family and are
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characterized by membrane-spanning α-helices. This family is mainly produced by pathogens,
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represents an essential component of the virulence arsenal [29], and can be grouped into many
ACCEPTED MANUSCRIPT subfamilies on the basis of the structure [30]. Most Cry insecticidal toxins have a typical
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three-domain structure [2,8], but the ClyA-subfamily exhibits a distinct structure containing two
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parts (a head domain and a tail domain, Fig. 2A, B and C) [25,26,27,28,29] . Close inspection of
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the structure revealed that Cry6Aa displays a canonic two-domain topology (Fig. 1). The head
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domain includes two anti-parallel helices, αD and αE, which are linked by a long loop
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(residues 242-264). However, in the structures of Hbl-B, ClyA and NheA, two β-strands
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connect the αD and αE helices. Additionally, parts of αA and αC can be regarded as building
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blocks of the head domain because of their unique spatial orientation. Similarly to the others
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toxins, the tail domain of Cry6Aa consists of five α-helices termed αA, αB, αC, αF, and αG
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(Fig. 1A). Nevertheless, αA in Cry6Aa is notably divided into three parts: αA1, αA2, and αA3.
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Moreover, αB and αC in Cry6Aa are not consecutive helices, as they are in the other structures;
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instead, αB and αC are disrupted by two small outer loops. Together, these results indicate that
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Cry6Aa contains a soluble monomer that is related to the ClyA family and appears to exhibit
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moderate conformational changes compared with those of the other three structures [27,28,31].
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3.3. Structural differences between Cry6Aa and ClyA
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Biochemical and structural studies revealed that the molecular mechanism by which ClyA
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is activated involves oligomerization and the formation of a pore for insertion into the
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membrane. Two sequential conformational changes of ClyA result in its pore assembly. First,
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the hydrophobic β-sheets in the head domain convert to an α-helical structure after membrane
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binding. Second, the long N-terminal helix αA undergoes a substantial rearrangement, thus
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generating a membrane-penetrating helix (Fig. 2C and D) [28,31,32,33].
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In the structure of Cry6Aa, instead of the long N-terminal helix, three discontinuous short
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tend to reassemble for membrane insertion. Coincidentally, the head domain in Cry6Aa contains
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a hydrophobic loop connecting αD and αE, and two β-strands localize in the same region in
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ClyA. Sequence analysis revealed that residues 254-268 are rich in bulky residues and exhibit
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the highest hydrophobicity (Fig. 3A and B); however, these residues are less conserved in
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Cry6Aa than in ClyA. These two notable differences suggest that the proteolytic Cry6Aa (1-396)
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is prone to conformational transit and that the structure of Cry6Aa captures an intermediate state
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between the soluble and oligomeric forms.
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Previously, we have reported that Cry6Aa triggers the necrotic cell death pathway in
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nematodes and that this process is mediated by the host aspartic protease ASP-1 [14]. The
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membrane environment is also essential for the oligomerization of pore-forming toxins
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[34,35,36]. Thus, the possibility that ASP-1 plays an enzymatic role in Cry6Aa cleavage or acts
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as an adaptor for the linkage of Cry6Aa to the cell membrane requires further investigation.
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3.4. Phylogenetic analysis of Cry6Aa
Phylogenetic analysis of Cry6Aa revealed the relationships between Cry6Aa and other
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proteins from 20 various species or subspecies (Fig. 4). The evolutionary tree is divergent and
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divides into two clades: The first contains closely related species, i.e., Bacillus, and the second
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contains distant species, such as Vibrio, Nocardia, and Pseudoalteromonas (Fig. 4). Surprisingly,
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the closest entry in the tree is the insecticidal delta-endotoxin Cry6Ba from Bt, thus indicating
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that this toxin has a toxicity mechanism similar to that of Cry6Aa. Furthermore, we also found
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that many pathogens in the phylogenetic tree exhibit a convergent evolutionary mechanism of
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membrane permeabilization among the nematicidal bacteria and pathogens.
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In summary, we presented a 1.90 Å resolution crystal structure of the proteolytic Cry6Aa toxin. This structure reveals a two-domain architecture similar to that of the ClyA toxin and
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other members of the α-PFT family. Although most of Cry6Aa shares nearly identical features
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with the monomeric state of the soluble ClyA, the membrane-penetration mechanisms of the
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two structural elements are significantly different, thus suggesting that Cry6Aa tends to exist in
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an intermediate state between the monomeric and oligomeric forms. This finding should provide
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a framework for the functional study of other phylogenetically related proteins.
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Conflicts of interest None
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Acknowledgments
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We thank J. He at the Shanghai Synchrotron Radiation Facility (SSRF) beamline BL17U and R. Zhang at the SSRF beamline BL19U for on-site assistance. We thank the
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research associates at the Center for Protein Research (CPR) of Huazhong Agricultural
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University for providing technical support. This work was supported by the Fundamental
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Research Funds for the Central Universities (program Nos. 2014BQ028). The authors have no
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conflicts of interest to declare. Correspondence should be addressed to M. Sun
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([email protected]).
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Accession Code The atomic coordinates and structure factors of the structure have been deposited in the
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Protein Data Bank (PDB) under accession codes 5ghe.
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E. coli hemolysin E (HlyE, ClyA, SheA): X-ray crystal structure of the toxin and
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observation of membrane pores by electron microscopy, Cell 100 (2000) 265-276.
300 301 302 303 304
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[28] M. Mueller, U. Grauschopf, T. Maier, R. Glockshuber, N. Ban, The structure of a cytolytic alpha-helical toxin pore reveals its assembly mechanism, Nature 459 (2009) 726-730. [29] M. Dal Peraro, F.G. van der Goot, Pore-forming toxins: ancient, but never really out of fashion, Nat Rev Microbiol 14 (2016) 77-92.
[30] I. Iacovache, F.G. van der Goot, L. Pernot, Pore formation: an ancient yet complex form of attack, Biochim Biophys Acta 1778 (2008) 1611-1623.
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[31] S. Benke, D. Roderer, B. Wunderlich, D. Nettels, R. Glockshuber, B. Schuler, The assembly dynamics of the cytolytic pore toxin ClyA, Nat Commun 6 (2015) 6198.
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[32] N. Eifler, M. Vetsch, M. Gregorini, P. Ringler, M. Chami, A. Philippsen, A. Fritz, S.A.
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Muller, R. Glockshuber, A. Engel, U. Grauschopf, Cytotoxin ClyA from Escherichia
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coli assembles to a 13-meric pore independent of its redox-state, EMBO J 25 (2006)
309 310 311 312
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2652-2661.
[33] D. Roderer, S. Benke, M. Muller, H. Fah-Rechsteiner, N. Ban, B. Schuler, R. Glockshuber, Characterization of variants of the pore-forming toxin ClyA from Escherichia coli controlled by a redox switch, Biochemistry 53 (2014) 6357-6369.
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[34] N. Poklar, J. Volker, G. Anderluh, P. Macek, T.V. Chalikia, Acid- and base-induced
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conformational transitions of equinatoxin II, Biophys Chem 90 (2001) 103-121.
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[35] K. Tiewsiri, W.B. Fischer, C. Angsuthanasombat, Lipid-induced conformation of helix 7
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from the pore-forming domain of the Bacillus thuringiensis Cry4Ba toxin: implications
317
for toxicity mechanism, Arch Biochem Biophys 482 (2009) 17-24.
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[36] M. Fahie, F.B. Romano, C. Chisholm, A.P. Heuck, M. Zbinden, M. Chen, A non-classical
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assembly pathway of Escherichia coli pore-forming toxin cytolysin A, J Biol Chem 288
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(2013) 31042-31051.
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ACCEPTED MANUSCRIPT Figure legends
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Fig. 1. Structural overview of Cry6Aa. The structure is displayed in ribbon (A) and schematic
324
(B) representations. (A) The overall structure of Cry6Aa displayed in a ribbon representation.
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The secondary structural elements are highlighted in different colors from the N terminus (light
326
blue) to the C terminus (brown). (B) The overall structure of Cry6Aa displayed in a schematic
327
representation. Each secondary structural element is colored as in (A). The extents of the
328
secondary structural elements are indicated by the residue numbers in the schematic
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representations. All structure figures were prepared using PyMOL and Photoshop.
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Fig. 2. Structural contrast of Cry6Aa, its structural homologs and overviews of the head and tail
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domains. The overall structures of the proteins from left to right. (A) Hbl-B (PDB ID: 2nrj)
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from B. cereus, (B) NheA (PDB ID: 4k1p) from B. cereus, (C) and (D) are the monomers (PDB
334
ID: 1qoy) and protomers (PDB ID: 2wcd) of ClyA from E. coli. , respectively. All of the
335
proteins are presented in the same orientation, and the head and tail domains are labeled on the
336
basis of their functions and structures. (C) and (D) display the conformational changes between
337
the soluble and protomeric forms of ClyA.
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Fig. 3. (A) The view of the hydrophobic loop in the head domain and the α-helix in the tail
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domain in the structure of Cry6Aa. The top, green square shows details of the hydrophobic loop
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in the head domain. The loop is labeled in purple, and the key residues are highlighted. The
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lower yellow square presents details of the α-helix in the tail domain, and αA1 and αA2 are
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labeled in brown. A schematic model demonstrating a hypothetical conformational change of
ACCEPTED MANUSCRIPT the αΑ1 helix in the tail domain. (B) Hydrophobicity analyses of Cry6Aa. Hydrophobicity plots
345
of the Cry6Aa sequence. The hydrophobicity score was calculated by using DNAMAN with a
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window size of 10 residues. The vertical axis represents increasing hydrophobicity; i.e., more
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positive scores indicate greater hydrophobicity. The red dashed rectangle indicates the extent of
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the head domain loop region (residues 254-268) of Cry6Aa.
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Fig. 4. Phylogenetic relationships among Cry6Aa gene sequences from 20 species. The tree was
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generated with the distance-based neighbor-joining method in the MEGA 6.0 program. The
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reliability of the tree was assessed by bootstrap analysis with 1,000 replications. The horizontal
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distances are proportional to the genetic distances. Cry6Aa is labeled with a red triangle. The
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phylogenetic tree shows that these proteins are evolutionarily homologous with Cry6Aa.
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Table 1
Data collection and refinement statistics for the Cry6Aa structure
Crystal Form Space Group Unit cell dimensions a, b, c (Å) α, β, γ (°) Number of molecules in ASU Wavelength (Å) Resolution range(Å)
Se-Cry6Aa(I125M, K228M) P65
101.64, 101.64, 78.90 90, 90, 120 1 0.9200 45-1.90 (1.94-1.90) 12.8(102.1) 5.9(49.3) 17.2(2.6) 11.3(10.2) 99.8(97.8) 36,545 411,712 18.46/19.58
101.47, 101.47, 79.05 90, 90, 120 1 0.9793 45-2.60 (2.69-2.60) 31.3(122.3) 4.7(18.5) 56.4(35.4) 44.5(43.4) 99.9(99.8) 14,216 2,472,556
0.011 0.888 1544 1523 3067 368
358 359 360 361 362
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Rmerge (%) Rpim (%) Ι /σ Redundancy Completeness (%) No. of unique reflections No. of measured reflections Rwork /Rfree (%) RMSD bonds (Å) angle (°) Number of protein atoms main chain side chain all atoms water molecules Ramachandran plot statistics (%) most favorable additionally allowed generously allowed disallowed Wilson B factor (Å2) Average B value (Å2) main chain side chain all atoms water molecules
Cry6Aa(native) P65
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95.2 4.5 0.3 0 12.5
22.7 30.9 26.7 38.4
22.0
ACCEPTED MANUSCRIPT Table 2. Dali search results for Cry6Aa Chain
Gene
Z-score
name
RMSD
Residue
Identity
(Å)
number
(%)
Description
2nrj-A
Hbl-B
24.5
2.8
338
11
Hemolysin-binding component from B. cereus
4k1p-A
NheA
21.4
3.5
338
13
Nhe toxin from B. cereus
1qoy-A
ClyA
12.1
4.3
303
12
2wcd-V
ClyA
11.1
3.8
285
6
Monomer of E. coli hemolysin E (HlyE, ClyA, SheA)
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Protomer of E. coli hemolysin E (HlyE, ClyA, SheA)
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365 366
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Table 3. Sequence alignment results for Cry6Aa with its structural homology. The results were
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analyzed with the Needleman-Wunsch Global Align Protein Sequences method of the National
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Center for Biotechnology Information (NCBI). Bt-Cry6Aa
B. cereus-Hbl-B
15
B. cereus-NheA
17
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E. coli-ClyA
B. cereus-Hbl-B
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B. cereus-NheA
20
20
17
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Figure 1
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374 375 Figure 2
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380 381 Figure 4
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ACCEPTED MANUSCRIPT Highlights: 1. The structure of nematicidal Cry6Aa toxin core domain was determined. 2. Cry6Aa is structurally related to the ClyA α-PFT family.
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3. The N-terminal helices of Cry6Aa adopts pre-pore-forming conformations.
S0006-291X(16)31118-4
DOI:
10.1016/j.bbrc.2016.07.002
Reference:
YBBRC 36079
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 26 June 2016 Accepted Date: 1 July 2016
Please cite this article as: J. Huang, Z. Guan, L. Wan, T. Zou, M. Sun, Crystal structure of Cry6Aa: A novel nematicidal ClyA-type α-pore-forming toxin from Bacillus thuringiensis, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.07.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Crystal Structure of Cry6Aa: a novel nematicidal ClyA-type
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α-pore-forming toxin from Bacillus thuringiensis
3 Jinbo Huang1, Zeyuan Guan2, Liting Wan1,3, Tingting Zou1, and Ming Sun1,3,#
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College of Life Sciences and Technology, Huazhong Agricultural University, Wuhan 430070, China.
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2
National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan
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430070, China.
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State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070,
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China.
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#
Correspondence author. E-mail address: [email protected].
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ACCEPTED MANUSCRIPT ABSTRACT
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Crystal (Cry) proteins from Bacillus thuringiensis (Bt) are globally used in agriculture as
15
proteinaceous insecticides . Numerous crystal structures have been determined, and most exhibit
16
conserved three-dimensional architectures. Recently, we have identified a novel nematicidal
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mechanism by which Cry6Aa triggers cell death through a necrosis-signaling pathway via an
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interaction with the host protease ASP-1. However, we found little sequence conservation of
19
Cry6Aa in our functional study. Here, we report the 1.90 angstrom (Å) resolution structure of
20
the proteolytic form of Cry6Aa (1-396), determined by X-ray crystallography. The structure of
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Cry6Aa is highly similar to those of the pathogenic toxin family of ClyA-type α-pore-forming
22
toxins (α-PFTs), which are characterized by a bipartite structure comprising a head domain and
23
a tail domain, thus suggesting that Cry6Aa exhibits a previously undescribed nematicidal mode
24
of action. This structure also provides a framework for the functional study of other nematicidal
25
toxins.
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Keywords: Bacillus thuringiensis, Crystal structure, Nematicidal, Pore-forming toxin
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1. Introduction The Gram-positive soil bacterium Bacillus thuringiensis (Bt) produces parasporal crystal (Cry) toxins during the sporulation phase of growth [1]. On the basis of functional and
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bioinformatic analyses, more than 300 holotypes of Cry toxins have been distinguished and
32
categorized into73 families [2,3]. Many of these proteins are toxic to many insect species and
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have been successfully used to directly kill insect pests as proteinaceous insecticides or to
34
control insect growth on transgenic agricultural plants [4]. The mechanism by which these
35
insecticidal toxins specifically target their hosts has been thoroughly studied. In the alkaline
36
insect midgut [5], the monomeric Cry protoxin is first digested by the host protease and then
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binds to its receptors. This binding facilitates the proteolytic cleavage of the N-terminal α-helix,
38
which in turn induces the oligomerization of the activated toxin and forms a pore via insertion
39
into the membrane, thus eventually leading to osmotic cell death. Structural investigations of
40
insecticidal Cry toxins have revealed that most share a distinct compact three-domain
41
architecture [2,6]. Domain I comprises an N-terminal α-helix cluster and is responsible for toxin
42
membrane penetration and pore formation. Domain II and Domain III are rich in β-sheets that
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resemble the β-prism fold and jelly-roll fold, respectively, and are involved in receptor
44
recognition [2,7,8].
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A new group of Cry proteins has been identified as nematode parasite intoxicants,
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including the Cry5, Cry6, Cry14, Cry21 and Cry55 families [9,10]. In contrast to the
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well-studied insect model, the mode of activation of nematicidal Cry toxins has been
48
investigated only in Caenorhabditis elegans (C. elegans) by using Cry5Ba [11]. Although
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Cry5Ba and Cry6Aa exhibit very little sequence homology with the classic cry toxins, they
ACCEPTED MANUSCRIPT represent two distinct families. Interestingly, the Cry5Ba crystal structure exhibits a conserved
51
three-domain architecture, thus suggesting that Cry5Ba undergoes pore-forming activation
52
reminiscent of that in insecticidal Cry proteins. The structural differences in Domain II between
53
Cry5Ba and insecticidal Cry explain the varying selectivity for different hosts’ receptors
54
[11,12,13].
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We have recently characterized a necrotic cell death pathway induced by Cry6Aa and
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identified the aspartic protease ASP-1, which primarily accumulates in the intestinal cells of C.
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elegans and acts as an activator in this pathway [14]. However, the molecular mechanism by
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which Cry6Aa is activated remains unknown.
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In this study, we determined the crystal structure of Cry6Aa at a resolution of 1.90 Å. Structural analysis demonstrated that Cry6Aa belongs to the ClyA-type α-pore-forming
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toxin (α-PFT) family and suggested that a membrane-insertion mechanism is responsible for its
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toxicity.
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2. Materials and methods
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2.1 . Cry6Aa protein preparation
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The Cry6Aa (GenBank Acc. No. AF499736) gene was amplified from Bt strain YBT-1518
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[15] using the primer pairs Cry6Aa-M1-F (5′- ATGATTATTGATAGTAAAACGAC-3′) and
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Cry6Aa-N475-R (5′- ATTATTATACCAATCCGAATTATTATAC-3′) and was subsequently
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cloned into the pQLinkH vector (GeneBank: EF025688) with a 7×His tag at the N-terminus
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[16]. To obtain Cry6Aa crystals, a series of gene boundaries were constructed in the same vector.
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The plasmids were transformed into Escherichia coli (E. coli) strain BL21(DE3). One liter of
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lysogeny broth (LB) medium supplemented with 100 µg ml-1 ampicillin was inoculated with a
ACCEPTED MANUSCRIPT transformed bacterial pre-culture and shaken at 37 °C until the optical density at 600 nm
73
reached 1.0. After growing for 16 hours at 16 °C, the bacterial pellet was collected and
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homogenized in a buffer containing 25 mM Tris-HCl, pH 8.0, and 150 mM NaCl. After
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sonication and centrifugation at 23,000 g at 4 °C, the supernatant was loaded onto a column
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equipped with Ni2+ affinity resin (Ni-NTA, Qiagen); then washed with buffer containing 25 mM
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Tris-HCl, pH 8.0, 150 mM NaCl, and 15 mM imidazole; and eluted with buffer containing 25
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mM Tris-HCl, pH 8.0, and 250 mM imidazole. The protein was further purified using
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anion-exchange chromatography (Source 15Q, GE Healthcare). The purified Cry6Aa was
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concentrated to approximately 15 mg ml-1 (Amicon, 10 kDa cutoff, Millipore) and then
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subjected to size-exclusion chromatography (Superdex-200 Increase 10/300, GE Healthcare).
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The buffer used for the size-exclusion chromatography contained 25-mM Tris-HCl, pH 8.0,
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150-mM NaCl and 5-mM dithiothreitol (DTT).
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2.2. Purification of the Se-Met-labeled Cry6Aa
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A mutant carrying I125M/K228M was generated for the phase determination of the native structure of Cry6Aa. Mutagenesis of the Cry6Aa gene was introduced via two rounds of overlap
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polymerase chain reaction (PCR) and verified by sequencing. The plasmid was transformed into
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E. coli strain BL21(DE3), and the culture was grown in medium from a Seleno MetTM Medium
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Base* MD 12-501 Kit (Molecular Dimension Co., Ltd.). The expression and purification of the
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Se-Met-labeled mutant Cry6Aa were performed by using the same procedures as used for the
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native protein, except 10 mM DTT was added to the buffer after the protein was eluted from the
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Ni2+ affinity resin. The yield of the Se-Met-labeled mutant was estimated to be approximately
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10 mg of protein per liter of bacterial cell culture.
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2.3. Crystallization The crystallization experiments were performed using the sitting-drop vapor diffusion method at 18 °C by mixing 1 µl of sample with an equal volume of the reservoir solution.
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Needle-shaped crystals of native Cry6Aa (residues 1-395) appeared overnight and grew to
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maturity within seven days in drops containing 0.1 M Tris-HCl, pH 8.8, 15% PEG 1000 (Sigma),
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and 30 mM DTT. After several rounds of optimization, the Se-Met-labeled Cry6Aa (residues
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1-395, I125M/K228M) was crystallized in the reservoir solution of 100 mM Tris-HCl, pH 8.8, 6%
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PEG 1500, 10% PEG 400, 75 mM NaF, and 50 mM DTT and exhibited the same shape as that
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of the native Cry6Aa crystals, although they were smaller. The crystals were flash frozen in
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liquid nitrogen and cryoprotected via the addition of glycerol to a final concentration of 20%.
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The Se-Met-labeled Cry6Aa and native Cry6Aa crystals were diffracted beyond 1.90 Å at the
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SSRF beamline BL17U and BL19U.
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2.4. Data collection and structural determination
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All diffraction data were collected at the SSRF on beamlines BL17U and BL19U by using a charge-coupled device (CCD) detector cooled to 100 K. The data from the Cry6Aa crystals
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were processed with the HKL2000 program suite and XDS packages[17]. Further processing
110
was performed using the programs from the CCP4 suite [18]. The Cry6Aa structure was solved
111
via SAD of by Se-Met using the ShelxC/D/E program [19]. The model obtained from
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Se-Met-SAD was used for molecular replacement with the program Phaser into the native data
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for native structure determination [20]. Next, manual model building and refinement were
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performed iteratively with Coot and Phenix [21,22]. The data collection and structure
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refinement statistics are summarized in Extended Data Table 1. All figures representing the
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structures were prepared with PyMOL [23].
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3. Results and Discussion
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3.1. Overall Structure of Cry6Aa The Cry6Aa gene was cloned from Bt strain YBT-1518 [15] and expressed in E. coli. The
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full-length protein was purified to homogeneity and was easy to crystallized but yielded only
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small-needle type crystals with low resolution after numerous optimization trials. We speculated
122
that Cry6Aa might be more stable after proteolytic activation. To test this possibility, we treated
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the Cry6Aa protein with increasing amounts of trypsin. A protease-resistant core domain
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(residues 1-396, identified by mass spectrometry) was observed and produced good
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diffraction-quality crystals. However, no homologous structure was available for structural
126
determination. Furthermore, because only one methionine residue (Met 315) was found in this
127
crystallized boundary, our identification of the phase using selenomethionine (Se-Met)-based
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single-wavelength anomalous diffraction (SAD) was hindered. After numerous
129
methionine-substitution screenings, we finally collected the anomalous signal diffracted data by
130
using the double mutant crystal (I125M/K228M) and determined the structure of the Cry6Aa
131
core domain (Cry6Aa, residues 1-396) in the P65 space group at a refined resolution of 1.90 Å
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(Table 1). One molecule was observed in each asymmetric unit. Additionally, most of the
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residues were well built, except for residues 387–396 because of their limited electronic density.
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The overall structure of Cry6Aa exhibits a rod-like appearance with a diameter of
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approximately 25 Å and a height of approximately 95 Å (Fig. 1A). Cry6Aa comprises seven
136
helices: αA (residues 10-68), αB (residues 78-142), αC (residues 148-215), αD (residues
137
220-241), αE (residues 265-289), αF (residues 292-353), and αG (residues 357-383). The
ACCEPTED MANUSCRIPT N-terminal αA helix is divided into three parts: αA1 (residues 10-21), αA2 (residues 41-56),
139
and αA3 (residues 61-68). The orientation of αA1 turns upward, and αA1 and αA3 are
140
perpendicular to αA2 and connected to two flexible loops. αB, αC and αF are composed of
141
many long helices that form the skeleton of the structure. Two outer loops, i.e., residues
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121-129 and 189-196, break the integrities of αB and αC, respectively, thus suggesting that
143
they are flexible and play a role in pore formation. αD and αE are two short anti-parallel helices,
144
which are separated by a long hydrophobic loop (residues 242-264). The C-terminal αG points
145
to αA2 and inserts into the groove between αA and αB.
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3.2. Cry6Aa is structurally related to the ClyA α-PFT family.
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A structural similarity search on the Dali server [24] indicated that the structure of Cry6Aa
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is closely related to those of Hbl-B (PDB: 2nrj) [25] and NheA (PDB: 4k1p) [26], both of which
149
are from Bacillus cereus (B. cereus), and the monomeric ClyA (PDB: 1qoy) [27,28] from E.
150
coli (Table 2 and Fig. 2). Although none of these proteins share significant sequence homology
151
with Cry6Aa (Table 3), their structures are highly similar. The closest entry was Hbl-B, with a
152
Z-score of 24.5 and a root mean-square deviation (RMSD) of 2.8 Å between 338 α-carbon atom
153
pairs. The second closest structural match was the NheA toxin with a Z-score of 21.4 and a
154
RMSD of 3.5 Å between 338 α-carbon atom matches. The other important result is the
155
monomeric ClyA toxin, which had a Z score of 12.1 and a RMSD of 4.3 Å over 303 aligned
156
residues (Table 2).
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All of these proteins belong to the α-Pore-forming toxins (PFTs) family and are
158
characterized by membrane-spanning α-helices. This family is mainly produced by pathogens,
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represents an essential component of the virulence arsenal [29], and can be grouped into many
ACCEPTED MANUSCRIPT subfamilies on the basis of the structure [30]. Most Cry insecticidal toxins have a typical
161
three-domain structure [2,8], but the ClyA-subfamily exhibits a distinct structure containing two
162
parts (a head domain and a tail domain, Fig. 2A, B and C) [25,26,27,28,29] . Close inspection of
163
the structure revealed that Cry6Aa displays a canonic two-domain topology (Fig. 1). The head
164
domain includes two anti-parallel helices, αD and αE, which are linked by a long loop
165
(residues 242-264). However, in the structures of Hbl-B, ClyA and NheA, two β-strands
166
connect the αD and αE helices. Additionally, parts of αA and αC can be regarded as building
167
blocks of the head domain because of their unique spatial orientation. Similarly to the others
168
toxins, the tail domain of Cry6Aa consists of five α-helices termed αA, αB, αC, αF, and αG
169
(Fig. 1A). Nevertheless, αA in Cry6Aa is notably divided into three parts: αA1, αA2, and αA3.
170
Moreover, αB and αC in Cry6Aa are not consecutive helices, as they are in the other structures;
171
instead, αB and αC are disrupted by two small outer loops. Together, these results indicate that
172
Cry6Aa contains a soluble monomer that is related to the ClyA family and appears to exhibit
173
moderate conformational changes compared with those of the other three structures [27,28,31].
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3.3. Structural differences between Cry6Aa and ClyA
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is activated involves oligomerization and the formation of a pore for insertion into the
177
membrane. Two sequential conformational changes of ClyA result in its pore assembly. First,
178
the hydrophobic β-sheets in the head domain convert to an α-helical structure after membrane
179
binding. Second, the long N-terminal helix αA undergoes a substantial rearrangement, thus
180
generating a membrane-penetrating helix (Fig. 2C and D) [28,31,32,33].
181
In the structure of Cry6Aa, instead of the long N-terminal helix, three discontinuous short
ACCEPTED MANUSCRIPT helices that are perpendicular to each other enable relatively open conformations that probably
183
tend to reassemble for membrane insertion. Coincidentally, the head domain in Cry6Aa contains
184
a hydrophobic loop connecting αD and αE, and two β-strands localize in the same region in
185
ClyA. Sequence analysis revealed that residues 254-268 are rich in bulky residues and exhibit
186
the highest hydrophobicity (Fig. 3A and B); however, these residues are less conserved in
187
Cry6Aa than in ClyA. These two notable differences suggest that the proteolytic Cry6Aa (1-396)
188
is prone to conformational transit and that the structure of Cry6Aa captures an intermediate state
189
between the soluble and oligomeric forms.
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Previously, we have reported that Cry6Aa triggers the necrotic cell death pathway in
191
nematodes and that this process is mediated by the host aspartic protease ASP-1 [14]. The
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membrane environment is also essential for the oligomerization of pore-forming toxins
193
[34,35,36]. Thus, the possibility that ASP-1 plays an enzymatic role in Cry6Aa cleavage or acts
194
as an adaptor for the linkage of Cry6Aa to the cell membrane requires further investigation.
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3.4. Phylogenetic analysis of Cry6Aa
Phylogenetic analysis of Cry6Aa revealed the relationships between Cry6Aa and other
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proteins from 20 various species or subspecies (Fig. 4). The evolutionary tree is divergent and
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divides into two clades: The first contains closely related species, i.e., Bacillus, and the second
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contains distant species, such as Vibrio, Nocardia, and Pseudoalteromonas (Fig. 4). Surprisingly,
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the closest entry in the tree is the insecticidal delta-endotoxin Cry6Ba from Bt, thus indicating
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that this toxin has a toxicity mechanism similar to that of Cry6Aa. Furthermore, we also found
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that many pathogens in the phylogenetic tree exhibit a convergent evolutionary mechanism of
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membrane permeabilization among the nematicidal bacteria and pathogens.
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In summary, we presented a 1.90 Å resolution crystal structure of the proteolytic Cry6Aa toxin. This structure reveals a two-domain architecture similar to that of the ClyA toxin and
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other members of the α-PFT family. Although most of Cry6Aa shares nearly identical features
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with the monomeric state of the soluble ClyA, the membrane-penetration mechanisms of the
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two structural elements are significantly different, thus suggesting that Cry6Aa tends to exist in
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an intermediate state between the monomeric and oligomeric forms. This finding should provide
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a framework for the functional study of other phylogenetically related proteins.
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Conflicts of interest None
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Acknowledgments
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We thank J. He at the Shanghai Synchrotron Radiation Facility (SSRF) beamline BL17U and R. Zhang at the SSRF beamline BL19U for on-site assistance. We thank the
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research associates at the Center for Protein Research (CPR) of Huazhong Agricultural
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University for providing technical support. This work was supported by the Fundamental
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Research Funds for the Central Universities (program Nos. 2014BQ028). The authors have no
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conflicts of interest to declare. Correspondence should be addressed to M. Sun
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([email protected]).
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Accession Code The atomic coordinates and structure factors of the structure have been deposited in the
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Protein Data Bank (PDB) under accession codes 5ghe.
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Fig. 1. Structural overview of Cry6Aa. The structure is displayed in ribbon (A) and schematic
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(B) representations. (A) The overall structure of Cry6Aa displayed in a ribbon representation.
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The secondary structural elements are highlighted in different colors from the N terminus (light
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blue) to the C terminus (brown). (B) The overall structure of Cry6Aa displayed in a schematic
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representation. Each secondary structural element is colored as in (A). The extents of the
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secondary structural elements are indicated by the residue numbers in the schematic
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representations. All structure figures were prepared using PyMOL and Photoshop.
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Fig. 2. Structural contrast of Cry6Aa, its structural homologs and overviews of the head and tail
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domains. The overall structures of the proteins from left to right. (A) Hbl-B (PDB ID: 2nrj)
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from B. cereus, (B) NheA (PDB ID: 4k1p) from B. cereus, (C) and (D) are the monomers (PDB
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ID: 1qoy) and protomers (PDB ID: 2wcd) of ClyA from E. coli. , respectively. All of the
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proteins are presented in the same orientation, and the head and tail domains are labeled on the
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basis of their functions and structures. (C) and (D) display the conformational changes between
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the soluble and protomeric forms of ClyA.
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Fig. 3. (A) The view of the hydrophobic loop in the head domain and the α-helix in the tail
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domain in the structure of Cry6Aa. The top, green square shows details of the hydrophobic loop
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in the head domain. The loop is labeled in purple, and the key residues are highlighted. The
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lower yellow square presents details of the α-helix in the tail domain, and αA1 and αA2 are
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labeled in brown. A schematic model demonstrating a hypothetical conformational change of
ACCEPTED MANUSCRIPT the αΑ1 helix in the tail domain. (B) Hydrophobicity analyses of Cry6Aa. Hydrophobicity plots
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of the Cry6Aa sequence. The hydrophobicity score was calculated by using DNAMAN with a
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window size of 10 residues. The vertical axis represents increasing hydrophobicity; i.e., more
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positive scores indicate greater hydrophobicity. The red dashed rectangle indicates the extent of
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the head domain loop region (residues 254-268) of Cry6Aa.
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Fig. 4. Phylogenetic relationships among Cry6Aa gene sequences from 20 species. The tree was
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generated with the distance-based neighbor-joining method in the MEGA 6.0 program. The
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reliability of the tree was assessed by bootstrap analysis with 1,000 replications. The horizontal
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distances are proportional to the genetic distances. Cry6Aa is labeled with a red triangle. The
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phylogenetic tree shows that these proteins are evolutionarily homologous with Cry6Aa.
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Table 1
Data collection and refinement statistics for the Cry6Aa structure
Crystal Form Space Group Unit cell dimensions a, b, c (Å) α, β, γ (°) Number of molecules in ASU Wavelength (Å) Resolution range(Å)
Se-Cry6Aa(I125M, K228M) P65
101.64, 101.64, 78.90 90, 90, 120 1 0.9200 45-1.90 (1.94-1.90) 12.8(102.1) 5.9(49.3) 17.2(2.6) 11.3(10.2) 99.8(97.8) 36,545 411,712 18.46/19.58
101.47, 101.47, 79.05 90, 90, 120 1 0.9793 45-2.60 (2.69-2.60) 31.3(122.3) 4.7(18.5) 56.4(35.4) 44.5(43.4) 99.9(99.8) 14,216 2,472,556
0.011 0.888 1544 1523 3067 368
358 359 360 361 362
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Rmerge (%) Rpim (%) Ι /σ Redundancy Completeness (%) No. of unique reflections No. of measured reflections Rwork /Rfree (%) RMSD bonds (Å) angle (°) Number of protein atoms main chain side chain all atoms water molecules Ramachandran plot statistics (%) most favorable additionally allowed generously allowed disallowed Wilson B factor (Å2) Average B value (Å2) main chain side chain all atoms water molecules
Cry6Aa(native) P65
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95.2 4.5 0.3 0 12.5
22.7 30.9 26.7 38.4
22.0
ACCEPTED MANUSCRIPT Table 2. Dali search results for Cry6Aa Chain
Gene
Z-score
name
RMSD
Residue
Identity
(Å)
number
(%)
Description
2nrj-A
Hbl-B
24.5
2.8
338
11
Hemolysin-binding component from B. cereus
4k1p-A
NheA
21.4
3.5
338
13
Nhe toxin from B. cereus
1qoy-A
ClyA
12.1
4.3
303
12
2wcd-V
ClyA
11.1
3.8
285
6
Monomer of E. coli hemolysin E (HlyE, ClyA, SheA)
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Protomer of E. coli hemolysin E (HlyE, ClyA, SheA)
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Table 3. Sequence alignment results for Cry6Aa with its structural homology. The results were
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analyzed with the Needleman-Wunsch Global Align Protein Sequences method of the National
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Center for Biotechnology Information (NCBI). Bt-Cry6Aa
B. cereus-Hbl-B
15
B. cereus-NheA
17
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E. coli-ClyA
B. cereus-Hbl-B
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Identity (%)
B. cereus-NheA
20
20
17
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Figure 1
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ACCEPTED MANUSCRIPT Highlights: 1. The structure of nematicidal Cry6Aa toxin core domain was determined. 2. Cry6Aa is structurally related to the ClyA α-PFT family.
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3. The N-terminal helices of Cry6Aa adopts pre-pore-forming conformations.