Article pubs.acs.org/JPCB
Forces and Kinetics of the Bacillus subtilis Spore Coat Proteins CotY and CotX Binding to CotE Inspected by Single Molecule Force Spectroscopy Huiqing Liu,†,‡ Daniela Krajcikova,§ Nan Wang,†,‡ Zhe Zhang,† Hongda Wang,† Imrich Barak,§ and Jilin Tang*,† †
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, P. R. China ‡ University of Chinese Academy of Sciences, Beijing, 100049, P. R. China § Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, Bratislava 845 51, Slovakia S Supporting Information *
ABSTRACT: Spores are uniquely stable cell types that are produced when bacteria encounter nutrient limitations. Spores are encased in a complex multilayered coat, which provides protection against environmental insults. The spore coat of Bacillus subtilis is composed of around 70 individual proteins that are organized into four distinct layers. Here we explored how morphogenetic protein CotE guides formation of the outermost layer of the coat, the crust, around the forespore by focusing on three proteins: CotE, CotY, and CotX. Single molecule force spectroscopy (SMFS) was used to investigate the interactions among CotE, CotY, and CotX at the single-molecule level. Direct interactions among these three proteins were observed. Additionally, the dissociation kinetics was also studied by measuring the unbinding forces of the complexes at different loading rates. A series of kinetic data of these complexes were acquired. It was found that the interaction of CotE and CotY was stronger than that of CotE and CotX.
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INTRODUCTION In Bacillus subtilis, the sporulation is triggered by various environmental stresses, which produces metabolically dormant spores. The spores are able to withstand harsh environmental conditions, such as heat, lytic enzymes, and chemicals.1,2 These properties are conferred mainly by the spore coat that uses more than 70 different proteins to establish a complex multilayered structure.3−6 These proteins that make up the coat, are organized into four distinct layers: a diffuse undercoat, a lamellar inner coat, an electron-dense outer coat and a glycoprotein layer termed as a crust.5,7−9 For each layer, a specific morphogenetic protein was identified, which plays an especially crucial role in assembly of the coat.5,9 The coat assembly is a complex multistep process and a great many details about it have been determined.10−12 A critical earliest event in the coat assembly is the construction of a basement layer of the SpoIVA protein around the forespore surface.13 SpoIVA, anchored to the outer membrane of the developing forespore via SpoVM,14,15 is self-assembled into higher order filamentous structures and forms a layer which acts as a platform for the coat assembly.9 The correct localization of SpoIVA is required for the recruitment of other key morphogenetic proteins, such as SpoVID16,17 and SafA,18,19 to the forespore surface. The presence of SpoIVA and SpoVID in the coat is a prerequisite for the right assembly of another morphogenetic protein, CotE.20,21 This protein, located at the © XXXX American Chemical Society
interface of the inner and outer coat, is assembled in a ring-like manner around the forespore guiding the deposition of most of the outer coat proteins and also several inner coat proteins.4,7,22−24 Structure/function analysis of CotE demonstrated that the protein had a modular character. It possesses an N-terminal region responsible for homotypic CotE−CotE interactions, an interior portion connecting CotE with the surface of the forespore and a C-terminal region guiding assembly of the CotE-controlled proteins.23,25 The influence of CotE protein on the coat assembly is extended as far as the crust where assembly of CotX and CotZ is depended on CotE.24 It has been reported previously that CotE, together with CotX, CotY, and CotZ, is essential for assembly of the crust.9,26 CotX, CotY, and CotZ are not only the structural components of the crust, but they also are required for the crust formation.26 CotX is rich in hydrophobic residues but has a highly charged N terminus which may contribute to the anchoring protein to the outer coat.8 CotY and CotZ are cysteine-rich proteins. Their amino acid sequences are very similar and it also was observed that they are depended on each other to localize into the crust.26,27 CotX, CotY, and CotZ, individually or in combination, play morphogenetic roles in the Received: November 19, 2015 Revised: January 24, 2016
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DOI: 10.1021/acs.jpcb.5b11344 J. Phys. Chem. B XXXX, XXX, XXX−XXX
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The Journal of Physical Chemistry B crust assembly around the forespore.5 Ruthenium red staining of the spores showed that the cotX mutant had more loosely staining crust and that the crust was absent in cotXYZ mutant spores.9 Although the morphogenetic proteins play integral roles in the spore coat assembly by directing other coat proteins to their correct positions, the exact contribution of these proteins to the formation of the organized and functional coat is poorly understood. Besides the morphogenetic proteins, the order of assembly of the various coat components also depended on specific protein−protein interactions.8 To study these interactions is crucial for detailed understanding of the process of the coat assembly, its mechanical properties and its functions.24 The main focus of this study was on three proteins of the spore coat: CotE, CotY, and CotX. The binding behavior of CotE toward CotY and CotX was explored at the singlemolecule level by performing atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS), which is by now a well-established method to measure the intra- and intermolecular interaction forces and investigate the dynamics of the recognition process.28−31 The unbinding forces of the CotE−CotY and CotE−CotX interactions were measured. By stretching of CotE−CotY and CotE−CotX complexes, the kinetic parameters were acquired that are important for description of the strength of the interactions. These parameters also enable to further analyze the kinetic characteristics of the contacts between analyzed proteins. The presented data about the specific interaction among morphogenetic proteins CotE, CotY, and CotX expand our understanding of how CotE, along with CotY and CotX, controls assembly of the crust and guides it around the spore.
AFM Tip and Silicon Substrate Functionalization. AFM tips (MSCT, Veeco, USA) were cleaned in three changes of ethanol and chloroform, and dried under nitrogen stream. The cleaned AFM tips were functionalized with CotE through a heterobifunctional cross-linker as described before.32−36 By using this method only about one of the flexibly protein molecules were attached to the end of the tip, guaranteeing that the measurements can be performed at the single-molecule level. Shortly, the AFM tips were first treated with amine groups by a 2 h incubation with 50 μL of APTES and 15 μL of triethylamine as catalysts in the gas phase.37 “Curing” at room temperature for 2 days must be carried out to ensure fully strong binding of the generated APTES layer. Subsequent incubation of the tips in 0.5 mL of chloroform containing 2 mg/mL polyethylene glycol (PEG) linker (NHS-PEG18acetal33,38) and 0.5% (v/v) triethylamine for 2 h results in the stable attachment of the PEG linker NHS-ester to the amino-functionalized tips. The acetal-protecting group at the end of the PEG linker then was converted into aldehyde group by treating the tips in 1% citric acid (pH 2.2) for 10 min and rinsing with Milli-Q water. Lastly, for the protein immobilization, the activated tip was immersed into the mixture solution containing 100 μL of 100 μg/mL CotE protein and 2 μL of freshly prepared 20 mM NaCNBH3 and incubated for 1 h at room temperature. After that, 5 μL of 1 M ethanolamine hydrochloride (pH 9.6, preadjusted with NaOH) was added to inactivate the free aldehyde groups on the tip by incubating for 10 min. For control experiments, the tip was functionalized only with the PEG linker whose terminal aldehyde function was blocked with ethanolamine. The modification of silicon substrates which were cleaned as described before39 with CotY or CotX was the same as that of the AFM tips as described above. This method of functionalization is relatively simple, mild, and chemical modification of the protein usually does not alter its conformation.34,35 In addition, these Cot proteins seems to be very stable and robust when assembled either in vivo, in heterologous E. coli cells, or in vitro conditions in cryo TEM experiments as showed previously40 and importantly AFM and TEM structures are similar. So the proteins were still kept in their native states after the modification on the tips or silicon substrates. After washing with PBS buffer, the functionalized AFM tips and silicon substrates were stored in PBS at 4 °C for further use. AFM Measurements. A PicoSPM 5500 AFM (Agilent Technologies, Andover, USA) was used to carry out the force measurements in PBS buffer with the CotE-functionalized tips at room temperature. The spring constants of the CotEfunctionalized cantilevers, calibrated by the thermal noise method, were measured to be (22.56 ± 1.65) pN/nm.41 About 1000 force−distance curves for each protein pair were collected at randomly chosen five to eight locations on the same silicon surface to avoid position-dependent aberrances, and the experiment was repeated four times with different proteinmodified AFM tips. Quantitative analysis of the recorded curves was performed using Matlab Version 7 (MathWorks, Natick, MA) as previously described.42 Experimental probability density function (the distributions of the rupture forces of the last unbinding event) was measured as described earlier.43 The loading rate was calculated from the effective spring constant of the tip and retraction velocity. AFM imaging was performed on NanoScope Multimode 8 (Digital Instruments, Veeco, USA) in PBS at room temperature. The images were captured in Tapping Mode, using
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MATERIALS AND METHODS Materials. Silicon wafers were purchased from Shengxu Electronic Technology Co., Ltd. (China). NHS-PEG18-acetal was purchased from Prof. Hermann J. Gruber (Johannes Kepler University, Austria). NaCNBH3 was supplied by J&K Scientific Ltd. (China). Triethylamine, ethanolamine and 3-aminopropyltriethoxysilane (APTES) were obtained from SigmaAldrich (USA). Anhydrous citric acid (content 99.5+%, based on anhydrous substance) was obtained from Alfa Aesar (USA). Analytical-grade reagents were used in all experiments. Protein Expression and Purification. The recombinant proteins were isolate and purified as described before by Krajcikova et al.25 Shortly, Escherichia coli BL21(DE3) strain which harbor the plasmids pETcotE, pETcotY, or pETDuetcotX were applied to the production of the protein. Bacteria were grown at 37 °C and the expression of the protein was induced by 1 mM IPTG. The recombinant proteins, Nterminally tagged with His-tag, were isolated and purified as was described previously25 with metal affinity chromatography on Ni2+ column in Tris buffer containing 8 M urea. Dialysis of CotE, CotY, and CotX. The three proteins (CotE, CotY, and CotX) at a concentration of 100 μg/mL were dialyzed against 3 L of water for 3 h at room temperature following the purification, during which the water was changed every hour. To eliminate the undissolved proteins and possible aggregates, the proteins then were centrifuged at 14000 rpm at 4 °C for 7 min. Ultimately, different concentrations of the proteins were set up by using phosphate-buffered saline (PBS) buffer (1.8 mM KH2PO4, 8.1 mM Na2HPO4, 2.7 mM KCl, 137 mM NaCl, pH 7.5). B
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Binding Behavior of CotE and CotY. The studies using genetic methods demonstrated that the CotY localization on the spore surface was controlled by CotE.4 However, no data of their direct contact or any quantitative information on the binding strength of CotE and CotY was derived from those experiments. Herein, AFM-based SMFS was used to investigate quantitatively the unbinding forces between CotE and CotY. For performing SMFS experiments, AFM tips and silicon substrates were modified with CotE and CotY via a flexible heterobifunctional PEG linker. The insertion of the PEG linker left CotE flexible enough to find and interact properly with CotY. A schematic representation of AFM tips and silicon substrates functionalization is depicted in Figure 1A. In SMFS, the unbinding forces of CotE−CotY protein interaction were measured in force−distance cycles by approaching the CotE-modified tip to the CotY-modified silicon substrate followed by its retraction (Figure 1B). During this cycle, the cantilever deflection (x) was continuously measured and converted into a force (f) according to Hook’s law (f = kx, where k is the cantilever spring constant). At the beginning of the tip−surface approach (Figure 1B, dot line), the cantilever deflection remained zero. Once the tip contacted with the surface and after its subsequent upward movement, the cantilever bent until a bending limit previously set was reached. After the tip was retracted from the surface (Figure 1B, solid line), the cantilever gradually bent downward, if a specific interaction between tip-carrying CotE and CotY immobilized on silicon substrates occurred. At a crucial unbinding force, CotE was detached from CotY and the cantilever jumped back again to its resting position at zero forces. The representative force−distance curve with a specific unbinding event between CotE and CotY is shown in Figure 3A. CotE−CotY binding is represented as a characteristic nonlinear parabola-like shape in the retraction curve owing to the elastic extension of the PEG linker before the tip−surface separation. This allows to distinguish clearly specific unbinding events from nonspecific forces.30 Probability density function (pdf) as the distribution of unbinding forces and maxima in the pdf reflect the most probable unbinding force. PDF for the CotE−CotY interaction obtained from about 1000 force−distance cycles is illustrated in
commercial Si3N4 tips (DNP-S10, 0.06 N/m, Veeco, USA). NanoScope Analysis (Veeco, USA) was carried out to analyze the acquired images.
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RESULTS AND DISCUSSION AFM Characterization of CotE, CotY, and CotX. To probe the interactions of CotE−CotY and CotE−CotX by SMFS, CotE, CotY and CotX were immobilized onto silicon substrates via the flexible heterobifunctional PEG linkers, respectively (Figure 1A). The functionalized surfaces were
Figure 1. (A) Schematic diagram of SMFS measurements between the CotE-functionalized AFM tip and CotY or CotX-modified silicon substrate. (B) Schematic representation of a force−distance cycle obtained by approaching the functionalized tip to the modified surface followed by its retraction.
characterized by AFM in PBS. Figure 2 shows that individual CotE, CotY, and CotX proteins are irregularly distributed on the silicon substrates, and some protein aggregates are also observed. The corresponding cross-section profiles indicate the heights are about 3−6 nm, 2−4.5 nm and 2.5−4 nm for CotE, CotY, and CotX, respectively.
Figure 2. AFM images of (A) CotE, (B) CotY, and (C) CotX covalently coupled to the silicon substrates via the PEG linker. Cross-section profiles corresponding to the white lines are displayed under the image revealing the height of the proteins. C
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Figure 3. AFM force measurements of the interaction between CotE and CotY. (A) Typical force−distance curve shows a CotE−CotY interaction. (Inset) Specific recognition of CotE and CotY is proved by using the PEG-modified tip and CotY-modified substrate. (B) Probability density function of the unbinding forces constructed from 265 out of 943 force−distance curves (solid line). In the control experiment, only 32 out of 987 force−distance curves show nonspecific adhesion forces (dot line).
Figure 4. SMFS study of the unbinding force distribution of CotE−CotY complex. (A) Most probable unbinding forces (f u) plotted against the logarithm of the loading rate. (B−F) Distribution of the f u at different loading rates: (B) 5.94 nN s−1, (C) 5.54 nN s−1, (D) 2.83 nN s−1, (E) 1.94 nN s−1, and (F) 1.71 nN s−1.
product of retraction velocity and the effective spring constant.29,44 In order to explore the relation between unbinding forces and loading rates, the unbinding force was measured at different loading rates. By varying the pulling velocities, the loading rates ranging from 1.5 × 103 to 6.6 × 103 pN/s were obtained. It was found that the most probable unbinding force scaled linearly with the logarithm of the loading rate (Figure 4), which can be described by the BellEvans model.45,46 The function of Bell-Evans model is given in eq 1:
Figure 3B, solid line. The most probable unbinding force of CotE−CotY interaction was about 92 pN for a loading rate of 5.4 × 103 pN/s. The binding probability which represents frequency for observing specific interaction events in force− distance cycles, was 28.1% (265 unbinding events in 943 curves). To demonstrate that the measured unbinding forces were indeed a consequence of CotE−CotY interaction, the control experiments were performed by using the PEGmodified tips and CotY-modified substrate. As a consequence, the specific recognition events were completely absent in the retraction curves (Figure 3A, insert), and the binding probability dropped remarkably to 3.2% (Figure 3B, dot line). The values of the forces measured in the experiments show the strength of the studied protein−protein interactions. Althouth the specific forces and nonspecific ones were fallen into the same distribution, we still can confirm the specificity by the control experiment. These results show at the singlemolecule level that CotE can interact directly with CotY and that this interaction is specific. The nature of measured forces between CotE and CotY was further investigated by varying the dynamics of the unbinding experiments. Unbinding forces are not uniform values but depend on the loading rate, which was derived from the
⎛ k T ⎞ ⎛ rxβ ⎞ fu = ⎜⎜ B ⎟⎟ ln⎜ ⎟ ⎝ xβ ⎠ ⎝ kBTkoff ⎠
(1)
Here f u is the most probable unbinding force, r is the loading rate, xβ is the width of the energy barrier from the free equilibrium position, koff is the dissociation rate constant at zero force, kB is the Boltzmann constant, and T is the absolute temperature. From fitting the plot of unbinding force versus the loading rate shown in Figure 4A to eq 1 the value for koff, acquired from the intercept, is 0.11 s−1 and xβ, acquired from the slope, is 0.37 nm. Additionally, the lifetime of the CotE− CotY complex at zero force τ, calculated by the inverse of the D
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Figure 5. AFM force measurements of the interaction between CotE and CotX. (A) Typical force−distance curve shows a CotE−CotX interaction. (Inset) The specific recognition of CotE and CotX is proved by using the PEG-modified tip and CotX-modified substrate. (B) Probability density function of the unbinding forces constructed from 247 out of 925 force−distance curves (solid line). In the control experiment, only 15 out of 994 force−distance curves show nonspecific adhesion forces (dot line).
Figure 6. SMFS study of the unbinding force distribution of CotE−CotX complex. (A) Most probable unbinding forces ( f u) plotted against the logarithm of the loading rate. (B−F) Distribution of the f u at different loading rates: (B) 2.14 nN s−1, (C) 3.30 nN s−1, (D) 4.21 nN s−1, (E) 5.28 nN s−1, and (F) 7.06 nN s−1.
kinetic off-rate constant, was 9.35 s. These kinetic parameters characterizing the CotE−CotY interaction indicate that CotE has a binding affinity for CotY to form a stable complex. The existence of such stable protein complex, though measured in vitro, indicates that this complex can be likely formed also on the spore surface and that the localization of CotY is directly controlled by CotE. Binding Behavior of CotE and CotX. CotX is one of the proteins important for the crust formation and previous studies showed that its assembly onto the spore is dependent on CotE.24,26 In order to investigate if these proteins associate directly and eventually extract quantitative information on the binding affinity between CotE and CotX, SMFS was carried out to study the interaction of CotE and CotX. For this purpose, CotE and CotX were covalently attached to AFM tip and silicon substrate via the heterobifunctional PEG linker, respectively (Figure 1A). A representative force−distance cycle with specific recognition event is plotted in Figure 5A. The unbinding force and binding probability were assessed by analyzing the recorded force−distance curves. The results showed that the most probable unbinding force for CotE− CotX interaction was about 72 pN at a loading rate of 2.4 × 103
pN/s and the binding event probability was found to be 26.7% (247 unbinding events in 925 curves, Figure 5B, solid line). The specificity of the measured interaction between CotE and CotX was verified by control experiments, in which the tip and silicon substrate were modified with the PEG liker and CotX, respectively. The data demonstrated that no specific unbinding event was observed in the force−distance curve (Figure 5A, insert) and the binding probability dropped significantly to 1.5% (Figure 5B, dot line), indicating the specific binding of CotE to CotX. Furthermore, to acquire dynamic information about the dissociation of CotE−CotX complex, the unbinding force was plotted against the logarithm of the loading rate (Figure 6). From fitting into the unbinding force-loading rate correlation, the kinetic parameters, koff, xβ, and τ were obtained to be 1.56 s−1, 0.29 nm and 0.64 s for CotE−CotX complex, respectively. The acquired data clearly show that CotE and CotX directly and specifically interact with each other. Taken together, the interaction forces determined between CotE, CotY, and CotX by SMFS experiments characterize the strength and specificity of the studied protein−protein interactions. On the basis of the experimental data acquired here, it was shown that at the same loading rate the larger E
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unbinding forces were needed to detach CotE from CotY than CotE from CotX (Figure S1, Supporting Information). These results indicate that the CotE−CotY interaction is stronger than CotE−CotX. Moreover, the dynamic force spectroscopy offers a way to assess dissociation rates and lifetimes of protein complexes, which are used to describe affinity of protein pairs. The CotE−CotX complex show larger dissociation rate and shorter lifetime compared to the CotE−CotY complex, probably attributing to the lower stability of CotE−CotX complex. All these results illustrate the stronger binding affinity between CotE and CotY than between CotE and CotX. The stability of the CotE−CotY complex raises the possibility that CotE and CotY as morphogenetic proteins are included together in creation of a scaffold base for the assembly of other coat proteins. However, to speculate how association of CotY and CotE is actually pursued is difficult, considering that CotE forms extended two-dimensional nets assemblies and CotY forms also extremely stable and resistant two-dimensional supramolecular structure.40 Nevertheless the formation of the stable CotE−CotY protein complex could be one of the prerequisite for performing their morphogenetic role during the crust formation. The other protein which cannot be omitted in any hypothesis concerning the crust formation is CotZ. We have detected the strong interaction between CotY and CotZ25,39 by several techniques, thus a possibility that in fact these three proteins make the crust skeleton should not be avoided. In the case of CotE−CotX association one could hypothesize that CotX is placed in the opening of the CotE ring encircling the forespore. Nonetheless, it seems that the role of this protein in crust is less critical. The genetic studies indicated that for the assembly of the crust the presence of CotYXZ is crucial. Further experiments of the crust proteins obtained by TEM or AFM would be extremely useful for understanding the assembly of the crust. Still it is rather plausible that hierarchical interactions among individual proteins exist within the crust layer. The stronger binding affinity of CotE to CotY than that of CotE to CotX indicates that CotY can play a more dominant role in the crust assembly. However, we cannot exclude the possibility that all three proteins, CotY, CotZ, and CotX, can cooperate to assemble the crust properly.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.5b11344. Overlapped dynamic force spectra of CotE/CotY and CotE/CotX (PDF)
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AUTHOR INFORMATION
Corresponding Author
*(J.T.) E-mail:
[email protected]. Telephone and Fax: +86 43185262734. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Science Foundation of China (21275140, 21405152), the Science and Technology Development Plan of Jilin Province (20130101126JC), a grant from the Slovak Research and Development Agency (APVV14-0181) to I.B. and VEGA Grant 2/0131/14 from the Slovak Academy of Sciences to D.K. We are indebted to Dr. Christian Rankl for analyzing the data.
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REFERENCES
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CONCLUSION
The binding activities such as interaction forces and binding probability, between CotE−CotY and CotE−CotX were probed by SMFS at the single-molecule level. The protein CotE interacts directly with CotY and CotX, respectively. Furthermore, kinetic parameters of the interactions that describe the affinity between two proteins were obtained. The smaller dissociation rate and longer lifetime for CotE− CotY complex were observed compared to CotE−CotX complex, suggesting that CotE displayed the stronger binding affinity to CotY forming a more stable complex. Singlemolecule force study investigates the kinetic aspect of the interactions that are not accessible by other conventional methods thus providing data which could significantly influence our perception about how the interactions among CotE, CotY and CotX contribute to the crust formation. This study describes additional details about how CotE can control the crust formation around the forespore and how the individual proteins interact within the crust. F
DOI: 10.1021/acs.jpcb.5b11344 J. Phys. Chem. B XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.jpcb.5b11344 J. Phys. Chem. B XXXX, XXX, XXX−XXX
