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Zinc modulates self-assembly of Bacillus thermocatenulatus lipase Emel Timucin, and O Ugur Sezerman Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.5b00200 • Publication Date (Web): 09 Jun 2015 Downloaded from http://pubs.acs.org on June 15, 2015
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Zinc modulates self-assembly of Bacillus thermocatenulatus lipase This work is supported by Türkiye Bilimsel ve Teknolojik Arastirma Kurumu (TÜBĐTAK) 112T901. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Emel Timucin* and O. Ugur Sezerman Sabanci University, Faculty of Natural Sciences and Engineering, Molecular Biology, Genetics and Biotechnology, 34956, Istanbul, Turkey
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ABBREVIATIONS BTL2, Bacillus thermocatenulatus lipase 2; CD, circular dichorism; DLS, dynamic light scattering; MRE, Mean residue ellipticity; ThT, thioflavin T; Tm, melting temperature; TPEN, N,N,N',N'-Tetrakis(2-pyridylmethyl) ethylenediamine; Zn, zinc.
FOOTNOTES *
To whom correspondence should be addressed: email:
[email protected]; Tel: +90 216 483 9513.
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ABSTRACT Thermoalkalophilic lipases are prone to aggregation from their dimer interface to which structural zinc is very closely located. Structural zinc sites have been shown to induce protein aggregation, albeit the interaction between zinc and aggregation tendency in thermoalkalophilic lipases remains elusive. Here we delineate the interplay between zinc and aggregation of the lipase from Bacillus thermocatenulatus (BTL2) taken as a representative of thermoalkalophilic lipase. Results showed that zinc removal disrupted BTL2 dimer leading monomer formation and reduced thermostability manifesting a link between zinc and dimerization to achieve thermostability, while zinc addition induced aggregation. Biochemical and kinetic characterizations of zinc induced aggregates showed that the aggregates obtained from the early and late stages of aggregation had differential characteristics. At the early stages, the aggregates were soluble and possessed native-like structures, while at the late stages the aggregates became insoluble and showed fibrillar characteristics with binding affinities towards Congo red and thioflavin T (ThT). The impact of temperature on zinc induced aggregation were further investigated and it was found that the native-like early aggregates could completely dissociate into functional lipase forms at high temperatures, while dissociation of the late aggregates was limited. To this end, we report that the zinc induced aggregation of BTL2 is reversible by temperature switches and initiated by ordered aggregates at the early stages which gain fibrillar-like features in time. Insights revealed by this work contribute to the knowledge on aggregation mechanisms occur in thermophilic proteins, reflecting the potential use of metal addition/removal to fine-tune aggregation tendency.
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Bacterial thermoalkalophilic lipases grouped in the lipase family I.5 display 90% sequence identity with each other, while they share only 30% identity with other lipases. Despite the fact that these lipases offer robust alternatives to chemical transformations with ability to resist the harsh conditions of industrial processes,1 they are prone to aggregation2-5 which is a major concern for industrial enzymes. So far, numerous efforts have been devoted to understand aggregation processes which may decrease the solubility of potentially important bioproducts and thus result in loss of yield during industrial applications.6, 7 Hence identification of the factors leading to aggregation of thermoalkalophilic lipases is critical not only to design of novel lipase variants with optimal solubility, but also to the theoretical description of the main principles behind aggregation processes which have been also linked to neurodegenerative diseases affecting humans.8-10 Such efforts remain a recurrent issue concerning science, medicine, and biotechnological areas. Aggregation tendency is a well-documented feature of many lipases,11-13 for most of which high content of hydrophobic amino acids is attributed to the leading cause of aggregation.11, 14-16 On the other hand aggregation tendency of the thermoalkalophilic lipase originating from Bacillus thermocatenulatus (BTL2) cannot be explained by this criteria due to relatively less hydrophobic content of BTL2 compared to other aggregation-prone lipases.4 Along with this information, the evolutionary isolation of thermoalkalophilic lipases from other families reinforces the necessity of inspecting the factors contributing to the aggregation of thermoalkalophilic lipases with different rigor. Despite the fact that the underlying mechanism of aggregation in thermoalkalophilic lipases remains elusive, their natural aggregation tendency has been investigated thoroughly.4, 5, 17, 18 In these studies, Rua et al.4 and Salameh et al.18 have found that detergent-like molecules that were able to dissolve aggregates enhance activity of thermoalkalophilic lipase aggregates by increasing the number of available active sites. Their findings, showing that the aggregates dissociate into functional lipase forms, essentially imply that thermoalkalophilic lipases are prone to aggregation at their native states. The particular capacity of these lipase aggregates to break into functional forms, analogous to true biochemically defined oligomers, accentuates the oligomeric structures of BTL2 for investigation of aggregation process. Inspection of the structures belonging to thermoalkalophilic lipases found in the protein databank (PDB) revealed that, when crystallization was performed in the absence of any substrate analog, thermoalkalophilic lipases adopted a homodimeric form (PDB IDs: 1KU019, 1JI320, 2DSN21, 3UMJ22). Upon investigation of these dimer structures, an identical subunit interface for all of the structures has been spotted. This unique interface was shown to be contoured by the asymmetric portions of the lipase monomers. The tendency of asymmetric subunit portions to contour the binding interface provides the homodimer with two additional faces for higher oligomer formation. Therefore, in theory the dimer can grow to higher oligomeric forms and/or native-like aggregates through the unique interface resolved in crystal structures. In line with this notion, a recent study has shown through mutagenesis studies that the disruption of the dimer interactions has been shown to inhibit aggregation of BTL2,17 rendering the dimer interface extremely important for deciphering the details of the aggregation mechanism allocated by thermoalkalophilic lipases. Although structural zinc sites are very rare in lipases,23 succinct investigation of the unique dimer interface reveals that thermoalkalophilic lipases possess a zinc coordination site at a very close proximity to the dimer interface. The close proximity of zinc to the dimer interface that has been shown to be employed in the aggregation of BTL2,17 implied that zinc might involve in mediating intermolecular interactions during aggregation. Moreover, considering that divalent metals such as zinc have been previously associated with native-like aggregation mechanisms,24-30 it is evident that identification of the interplay between zinc and aggregation tendency is crucial for a complete understanding of the self-assembly process of thermoalkalophilic lipases. Aggregation can be studied in various conditions such as incubation at acidic pH,31 altered proteolysis32 and divalent metal incubations.26-30 In this study we were particularly interested in zinc induced aggregation of the thermoalkalophilic lipase BTL2. We have investigated the impact of zinc presence and absence on the oligomeric state of BTL2 and explored the biochemical and biophysical characteristics of zinc induced aggregates. We also analyzed the effect of temperature on the zinc induced aggregates in CD experiments and enzymatic assays. Lastly we used previously identified structural data belonging to thermoalkalophilic lipases to address a molecular basis on zinc induced oligomerization. MATERIAL AND METHODS
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Lipase Expression and Purification. Escherichia coli SHuffle® cells (New England Biolabs) were transformed with the expression plasmid pMCSG7 (DNASU plasmid repository) carrying the 1,167-bp of DNA fragment corresponding to the mature BTL2 gene. BTL2 was expressed in E. coli cells that were cultivated in Luria-Bertani media (low salt formulation) supplemented with 100 µg/ml ampicillin and with 1 mM of isopropyl-β-dthiogalactopyranoside for induction. Expression was continued for 4 hours at 37°C and subsequent purification of BTL2 was carried out as described previously33 using HisTrap HP columns (GE Healthcare Life Sciences) by the availability of N-terminal polyhistidine tag. Protein concentration was determined using Bradford assay and purity of the recombinant lipase was tested by sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE). Preparation of Aggregates and Zinc-free BTL2. Zinc induced aggregates were prepared in a consistent manner for all of the experiments. The recombinant BTL2 was diluted to 10 µM (0.43 mg/ml) in 10 mM HEPES-KOH buffer at pH 7.4 unless otherwise stated and incubated at varying zinc concentrations for 2 hours at room temperature. The control sample was incubated under the same conditions without any zinc addition. Zinc-free BTL2 was prepared as described previously.34 100 µM lipase samples were incubated in 10 mM sodium phosphate buffer at pH 7.4 and 2 µM N,N,N',N'-Tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) for 12 hours at 4°C, then dialyzed against 10 mM sodium phosphate buffer at pH 7.4 to remove unbound TPEN and/or TPEN-Zn2+ complexes. The zinc-free BTL2 concentration was set to 10 µM. During preparation of zinc-free BTL2, all of the buffers were treated with 0.2 g/ml of Chelex (Sigma) at room temperature for 2 hours. Dynamic Light Scattering. Samples containing 10 µM of lipases in 10 mM HEPES-KOH buffer at pH 7.4 were incubated at varying concentrations of zinc for 2 hours and filtered (0.22 µm) prior to measurements. Dynamic light scattering (DLS) was conducted using Zetasizer NanoXS (Malvern Instruments) at 25°C and repeated three times. Although the size distribution generated by DLS is an intensity distribution, it was converted to volume distribution using Mie’s theory.35 This conversion was performed via the Zetasizer software. Circular Dichorism Spectroscopy. Far-UV circular dichorism (CD) spectra of the same samples were collected with 1.0 mm path length using J-815 spectropolarimeter (Jasco) in N2 atmosphere equipped with thermostatically controlled cuvette at a scanning speed of 100 nm/min. Three scans were averaged to obtain the final spectra which were corrected for the background. Mean residue ellipticity (MRE) [θ] is calculated by the equation, [θ] = θ × M/(10 × × l × n ) where is the observed ellipticity in mdeg, M is the molecular mass in g mol-1, c is the concentration in mg ml-1, l is the cell length in cm, and is the number of residues. Thermal denaturation profiles were determined by tracing 222 nm signal at a 6°C/min heating rate from 30°C to 90°C. Melting temperature (Tm) values were calculated from the midpoint of the transition curves between folded and unfolded states of the lipases. Time-course measurements were conducted at every second for 2 hours. CD data were not corrected for the zinc induced aggregates, but given in ellipticity due to excess light scattering from the aggregates. Congo Red Binding Assays. 100 µl of 10 µM lipase in 10 mM HEPES-KOH buffer at pH 7.4 were mixed with 400 µl of 20 µM Congo red in 5 mM phosphate buffer, 150 mM NaCl at pH 7.4.36 The absorption spectra were obtained from 425 to 700 nm using UV-3600 UV-visible spectrophotometer (Shimadzu). The differential spectra were obtained by subtracting only Congo red and only lipase spectra from the spectrum of the Congo red with lipase preincubated with zinc. 5 mM phosphate buffer, 150 mM NaCl at pH 7.4 is used as reference for all measurements. Thioflavin T Fluorescence Assay. The aggregates formed upon 2 hours of incubation of 10 µM BTL2 at the indicated zinc concentrations were mixed with 400 µl of 20 µM Thioflavin T (ThT), 10 mM phosphate buffer at pH 7.4. The fluorescence of the resulting samples was measured at 25 °C using Gemini XS (Molecular Devices) spectrofluorimeter. The excitation and emission wavelengths were 440 and 485 nm, respectively.37 Lipase Activity Assays. Enzyme assays were performed with 4-methylumberrylferyl (4-MU) caprylate as substrate in 10 mM phosphate buffer at pH 7.4 in a kinetic fashion. Gemini XS (Molecular Devices) was used to measure 4MU fluorescence with excitation at 355 nm and emission at 460 nm. The assays were carried out at room temperature using 5 nm of lipase in the final reaction mixture. Percent residual activity was calculated by setting the rate of the control sample without any incubation to 100%. All of the assays were repeated at least three times to test for reproducibility. Gel Electrophoresis. Aggregation was induced by either 20 or 50 µM zinc and at either 25°C or 60°C. The mixtures were sampled before zinc addition and at every 30 minutes for 2 hours after zinc addition. The soluble fractions were
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obtained by centrifugation at 13,000 rpm for 30 minutes. Before gel loading, total and soluble fractions were mixed with SDS sample buffer and boiled for 5 minutes. The samples were run on 12% polyacrylamide gel for 2 hours at 100 V and stained with coomassie blue. Structural Investigations. We selected the thermoalkalophilic lipase from Bacillus stearothermophilus (PDB ID: 1KU0) for structural investigations because there was not any structure in the absence of substrate analog belonging to BTL2. The illustrations were obtained by using visual molecular dynamics (VMD)38 with special focus on the zinc site and the subunit interface of the dimer. RESULTS Effect of Zinc on the Size of BTL2. BTL2 was incubated at zinc concentrations ranging from 0 to 100 µM and analyzed by DLS. Fig. 1 shows the size distributions by volume. The recombinant BTL2 yielded a 7 nm peak which became aggregated with sizes that grew gradually larger upon incubation with increasing zinc concentration (Fig. 1). On the other hand the zinc-free BTL2 had a radius of ~3.5 nm (Fig. 1). Effect of Zinc on the Secondary Structure. The far-UV CD spectra of the samples used in DLS experiments were shown in Fig. 2. The native BTL2 yielded two minimal peaks at 208 and 222 nm (Fig. 2). Although incubation of BTL2 in 5 µM zinc displayed similar far-UV CD spectrum with the native BTL2, incubation conditions of 10, 50 and 100 µM zinc resulted in loss of the native far-UV signals, with the one around 208 nm being the most significant (Fig. 2). When zinc was chelated, the far-UV spectrum of the zinc-free BTL2 only slightly differed from the native BTL2 (Fig. 2). Dye Binding Characteristics of the Zinc Induced Aggregates. The samples incubated at various zinc concentrations were analyzed through binding assays of specific dyes as indicators of ordered aggregates.36, 39 The dye, Congo red, gave a maximum absorption at 490 nm (Fig. 3A). Although the absorption maximum of Congo red with BTL2 was also located at 490 nm, it gave higher absorption for every wavelength compared with the spectrum of only Congo red (Fig. 3A), a highly likely result to be due to the presence of protein. After incubation of BTL2 at 20 µM or higher zinc, we observed a peak around 540 nm (Fig. 3B), suggesting ordered aggregates in those samples. The same samples were also stained with ThT to further assess the presence of ordered aggregates (Fig. 3C). The samples incubated in the presence of low zinc concentrations did not show any significant increase in ThT fluorescence, while samples incubated at zinc concentrations in the range of 50-100 µM produced 4-fold increase in the ThT fluorescence (Fig. 3C). The aggregates formed at 50 µM zinc were also examined under fluorescence microscope and Fig. S1 shows the micrographs of the aggregates emitting ThT fluorescence. Overall dye binding analyses suggested that the zinc induced aggregates formed at 20 µM or higher zinc concentrations possessed binding affinities towards both Congo red and ThT. Kinetic Analysis of Zinc Induced Aggregation. The far-UV CD spectra confirmed that zinc induced aggregation led to reduction in ellipticity at 208 nm (Fig. 2). To probe the kinetics of zinc induced aggregation, we relied on this CD spectroscopic change and measured the changes in the CD signal at 208 nm for 2 hours upon zinc addition. Despite the observation that the zinc induced aggregates also led a reduction in the CD signal at 222 nm (Fig. 2), CD spectroscopic changes at 222 nm was not sufficient to unveil the impacts of varying zinc concentrations on the rate of aggregation. Hence for kinetic analysis we used the 208 m signal for which we observed a larger scattering, i.e. a larger reduction in the CD intensity, than that at 222 nm enabling us to monitor aggregation rates at varying zinc concentrations. Table SI lists the rate constants of the process that were calculated using linear or exponential fits of the CD data. BTL2 in the presence of 0 µM zinc did not show any changes in ellipticity at 25°C (Fig. 4A). Although 10 µM zinc addition did not make any noticeable changes for 2 hours (Fig. S2), we observed gradual loss of ellipticity at both wavelengths for 20, 50 and 100 µM zinc additions (Fig. 4A), in which the rate of aggregation was increased with increasing zinc concentration (Table SI). Parallel to the above observation suggesting that increasing zinc concentration hastens aggregation, higher zinc (50 µM) led to a profound decrease in the soluble protein amount at 25°C, yielding fainter protein bands on the SDS-PAGE (Fig. 5B) compared with the condition at lower zinc (20 µM) (Fig. 5A). Effect of Temperature on the Zinc Induced Aggregation. To assess the impact of temperature on the secondary structure of zinc induced aggregates, we collected the far-UV CD spectra for every 10°C intervals in the range of 3090°C (Fig. 6A). The control BTL2 showed a gradual destabilization in the far-UV CD signals at 208 and 222 nm
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(Figs. 6A, S3). Particularly the signals at 208 (Fig. S3) and 222 nm (Fig. 6A) attained to the highest levels at 30°C, gradually decreased from 30-to-60°C and reached to the lowest level at 60°C before denaturation. The sample preincubated at 5 µM zinc produced very similar results to the control BTL2. However, in contrast to the control and the 5 µM zinc preincubated samples, the samples incubated in the presence of 10 µM or higher zinc did not display gradual destabilization in the CD signals such that the 60°C spectrum showed the strongest CD signals at 208 nm and 222 nm for BTL2 preincubated in 50 and 100 µM zinc (Figs. 6A, S3). Thermal denaturation of the native BTL2 resulted in a smooth profile with two distinct plateaus before and after denaturation at 222 nm, and similarly BTL2 incubated at 5 µM zinc had the same denaturation curve with the native (Fig. 6B). However, when BTL2 was incubated at 10 µM or higher zinc concentrations, we observed instead of a flat line, a distinct reduction in the CD intensity near denaturation region (Fig. 6B). Specifically the denaturation curve of 10 µM zinc incubation resulted in a curve overlapping with the native sample after 45°C and similarly 50 µM zinc preincubated sample yielded overlapped 222 nm signals with the native BTL2 at 65°C (Fig. 6B). On the other hand, denaturation curve of the aggregates formed at 100 µM zinc fell lagged behind overlapping with the native curve and showed only subtle reduction in the ellipticity at 65°C (Figs. 6B). To assess the impact of temperature on the kinetics of zinc induced aggregation, time-course CD measurements were performed at different temperatures. The ellipticity at 208 nm did not show any changes at 25°C and 60°C for 2 hours (Figs. 4B), suggesting a stable structure for the control BTL2 throughout these measurements. Compared with the condition at 25°C, the rates were reduced with increasing temperature regardless of zinc concentration used (Figs. 4B and Table SI). The particular reduction in the aggregation rate at 60°C was also inferred from the SDSPAGE analysis in which the soluble protein amount was increased gradually upon incubation at 60°C (Figs. 5A and 5B), clarifying the outcome that increased kinetic energy impedes with zinc induced aggregation. Impacts of Zinc on Thermal Denaturation. Thermal denaturation was profiled by monitoring the CD signals at 222 nm (Fig. 6B). The melting temperatures (Tm) are listed in Table SII. Similar to the previous result,17 the Tm of the native BTL2 was found 73°C at 222 nm, while the zinc induced aggregates did not have any significant impact on the Tm. We have also performed thermal denaturation at a slower rate (2°C/min) for the control and the sample preincubated at 5 µM zinc (Fig. S4). Although the Tm of these conditions were slightly lower than those measured at the heating speed of 6°C/min, the Tm values obtained from slow heating did not differed from those obtained from fast heating in both the control and the zinc preincubated sample, suggesting that zinc induced aggregation does not affect thermodynamic stability of BTL2. We also monitored the CD signals at 222 nm upon cooling of the same samples at 2°C/min. Yet none of the conditions was able to restore native secondary structure upon cooling, suggesting that thermal unfolding of control BTL2 and zinc induced BTL2 aggregates is irreversible. Lastly the zincfree BTL2 showed a drastic decrease (~10°C) in Tm (Fig. S5 and Table SII), a result which agrees with the previous observations regarding zinc’s involvement in structural stability of thermoalkalophilic lipases.34, 40 Functional Analysis of the Zinc Induced Aggregates. To assess the lipase activity during zinc induced aggregation, the samples analyzed in the kinetic analysis (Figs. 4, 5A and 5B) were used in lipase assays. The control BTL2 did not show any changes in the activity regardless of the incubation temperature (Fig. S6), showing that BTL2 preserved its function at 25°C and 60°C upon 2 hours of incubation. At 25°C, 20 and 50 µM zinc additions significantly reduced the lipase activity to 25% and 15% respectively (Fig. 5C) which was restored to 75% and to 40% upon incubation at 60°C respectively (Fig. 5D). We also assayed the residual lipase activity of the zinc induced aggregates after 1 hour of incubation at 60°C. At 10 µM, BTL2 aggregates were able to restore almost all of the total activity upon thermal incubation for 1 hour at 60°C (Fig. 7), similar to what we have observed during thermal denaturation of these aggregates which produced overlapping signals with the native structure at 60°C (Fig. 6B). On the other hand, the aggregates formed at high zinc supplies (50 or 100 µM) showed only a slight activity increase upon incubation at 60°C (Fig. 7). We lastly recorded the residual activity of the zinc-free BTL2 and reported that zinc removal significantly reduced the lipase’s stability at 60°C by reducing the residual activity more than 50% (Fig. 7). DISCUSSION Zinc is required for dimerization and thermostability. Initially a DLS analysis was performed to assess the sizes of BTL2 molecules in response to zinc addition and removal. Intensity distribution is the fundamental size distribution that can be obtained from a DLS measurement, albeit a realistic size description was not possible from our intensity distributions (Fig. S7). Although the samples were filtered prior to analysis, we could not eliminate the presence of large aggregates in the control BTL2, which interfered with the accurate determination of the sizes from
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intensity distributions. Such large aggregated particles, even if they are at negligible quantities, may comprise significant portion of the scattered intensity because they scatter light proportional to the sixth-power of their sizes. Hence even at small portions the presence of large particles may lead to inconclusive results in intensity size 41 distributions (Fig. S7). This phenomenon has been previously encountered for another aggregation-prone protein, 35 in which the intensity distribution was converted to a volume distribution by the application of Mie’s theory. In line with this example, we also relied on the volume size distributions (Fig. 1) by which we were able to obtain a fair description of BTL2 sizes showing that the large aggregated particles correspond to only a negligible portion of the total particles (
