Highly conserved salt bridge stabilizes a proteinase K subfamily enzyme, Aqualysin I, from Thermus aquaticus YT-1
© Sakaguchi et al.; licensee Springer 2014
Received: 16 June 2014
Accepted: 2 July 2014
Published: 13 August 2014
The proteinase K subfamily enzymes, thermophilic Aqualysin I (AQN) from Thermus aquaticus YT-1 and psychrophilic serine protease (VPR) from Vibrio sp. PA-44, have six and seven salt bridges, respectively. To understand the possible significance of salt bridges in the thermal stability of AQN, we prepared mutant proteins in which amino acid residues participating in salt bridges common to proteinase K subfamily members and intrinsic to AQN were replaced to disrupt the bridges one at a time. Disruption of a salt bridge common to proteinase K subfamily enzymes in the D183N mutant resulted in a significant reduction in thermal stability, and a massive change in the content of the secondary structure was observed, even at 70°C, in the circular dichroism (CD) analysis. These results indicate that the common salt bridge Asp183-Arg12 is important in maintaining the conformation of proteinase K subfamily enzymes and suggest the importance of proximity between the regions around Asp183 and the N-terminal region around Arg12. Of the three mutants that lack an AQN intrinsic salt bridge, D212N was more prone to unfolding at 80°C than the wild-type enzyme. Similarly, D17N and E237Q were less thermostable than the wild-type enzyme, although this may be partially due to increased autolysis. The AQN intrinsic salt bridges appear to confer additional thermal stability to this enzyme. These findings will further our understanding of the factors involved in stabilizing protein structure.
The molecular bases of protein adaptation to high and low temperatures are interesting from both basic and practical standpoints, as knowledge regarding these factors would enable the construction of genetically engineered proteins that could function under a variety of conditions. Psychrophilic and mesophilic enzymes are used in biotechnological applications requiring high activity at mild temperatures or quick heat-inactivation at moderate temperatures. Thermophilic and hyperthermophilic enzymes have major biotechnological advantages over mesophilic and psychrophilic enzymes because of their high activities at higher temperatures and substrate concentrations as well as their resistance to chemical denaturants. Thus far, various intramolecular interactions, including ionic interactions, hydrogen bonding and hydrophobic interactions, are assumed to make important contributions to the stability and maintenance of enzyme structure as well as the catalytic functions; however, their contributions have not been fully defined in individual cases. In addition, the comparative structural analysis of psychrophilic, mesophilic and thermophilic enzymes indicated that each protein family adopts a different structural strategy to adapt to different temperature ranges (Siezen & Leunissen ; Struvay & Feller ). To understand the thermal adaptation strategy of proteins, comparative studies among members of a protein family using site-directed mutagenesis as well as laboratory evolution via random mutagenesis using error-prone PCR will provide valuable information.
According to the MEROPS peptidase database (http://merops.sanger.ac.uk/), subtilisin-like protease (subtilase) superfamily members are classified as the S8 subfamily in the serine protease superfamily. These proteins exhibit a highly conserved arrangement of amino acids in the active site and have very similar overall structures consisting of an α/β protein scaffold. Nevertheless, their temperature stability profiles differ widely, and they may be psychrophilic, mesophilic, thermophilic or hyperthermophilic depending on the characteristics of the organisms from which they are derived. Because of these characteristics, they appear to be suitable for comparative studies to elucidate the basis of structure-function relationships. Aqualysin I (AQN) is an alkaline serine-protease produced by the Gram-negative thermophilic bacterium Thermus aquaticus YT-1 (Matsuzawa et al. ; Matsuzawa et al. ). Based on an analysis of sequence homology, AQN is classified into the proteinase K subfamily, which consists of a group of Gram-negative bacteria-derived proteinases within the subtilase superfamily (Siezen & Leunissen ). In our previous study, we demonstrated that Pro residues in the surface loops of AQN in the N-terminal region contribute significantly to its thermophilicity, and one of two disulfide bonds in AQN is more important for the catalytic activity and conformational stability of AQN than the other (Sakaguchi et al. ; Sakaguchi et al. [2008b]). These results are consistent with those reported for a subtilisin-like serine protease from Vibrio sp., VPR, which is a psychrophilic counterpart of AQN in the proteinase K subfamily (Kristjánsson et al. ; Arnórsdóttir et al. ). It was found that the introduction of Pro residues into VPR at positions corresponding to those in AQN could improve its thermal stability (Arnórsdóttir et al. ).
Amino acids that form salt bridges in AQN, VPR and SPRK
Intrinsic to VPR or SPRK
Intrinsic to AQN
Materials and methods
Strains and growth medium
E. coli TG1 was used as the expression host, and E. coli DH5α (TOYOBO, Osaka, Japan) and MV1184 (TAKARA BIO INC., Shiga, Japan) were used as the genetic engineering hosts. LB medium (1% Bacto-tryptone, 0.5% Bacto-yeast extract, 1% NaCl, pH 7.0) was used. The solid medium contained Bacto-agar (1.5%). Ampicillin (50 μg/ml) or kanamycin (50 μg/ml) was added to the medium as needed.
Genetic engineering and chemical reagents
Genetic engineering experiments were performed according to the procedure described by Sambrook and Russell (Sambrook & Russell ). The enzymes used for genetic engineering were purchased from TAKARA BIO and used according to the manufacturer’s instructions. Bacto-tryptone and Bacto-yeast extract were purchased from Becton Dickinson (Franklin Lakes, NJ, USA). Other reagents used were of the highest quality available and were obtained from Wako Pure Chemicals (Tokyo, Japan) and Sigma-Aldrich (St. Louis, MO, USA), unless otherwise specified.
The plasmid pMAQΔc2, which was designed to express wild-type AQN as a fusion protein with maltose binding protein (MBP), was constructed based on pAQNΔC105 and pMAL plasmids (New England Biolabs, Ipswich, MA, USA) as described previously (Sakaguchi et al. [2008a]).
Oligonucleotide primers used for site-directed mutagenesis
Purification and activity measurement of the wild-type enzyme and its mutants
After induction by isopropyl β-D-thiogalactopyranoside (IPTG) at OD660 = 0.8, the transformants were further cultivated overnight in LB medium. The cells were harvested by centrifugation and subsequently sonicated, and the crude extract was subjected to heat treatment, hydrophobic chromatography (Butyl Sepharose; GE Healthcare, Buckinghamshire, UK) and cation exchange chromatography (Resource S; GE Healthcare) as described previously (Sakaguchi et al. [2008a]). The enzymes were purified to homogeneity to yield a single band on SDS-polyacrylamide gel electrophoresis (PAGE) after staining with Coomassie Brilliant Blue R-250 (CBB R-250) (Laemmli ). Prior to SDS-PAGE analysis, the enzymes were treated with 25 mM phenylmethane sulfonyl fluoride (PMSF) dissolved in methanol for 30 min to prevent autolytic degradation. To minimize denaturation and autolysis which may occur at higher temperature, the enzyme activity was measured at 40°C with N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (N-suc-AAPF-pNA, Sigma-Aldrich) as a substrate in 50 mM 2-[4-(2-hydroxyethyl)-1-piperazynyl]ethanesulfonic acid (HEPES)-NaOH (pH 7.5) buffer containing 1 mM CaCl2. The change in absorbance at 410 nm was continuously monitored, and the activity was estimated using ε410 = 8,680 M−1 cm−1 as a molar absorption coefficient of p-nitroaniline (4-nitroaniline). One unit of enzyme was defined as the amount of enzyme that liberates 1 μmole of p-nitroaniline from the substrate in 1 minute. The protein concentration was measured using the micro-assay method (Bio-Rad Laboratories, Hercules, CA, USA), which is based on the Bradford method (Bradford ), using bovine serum albumin as a standard.
Determination of the temperature dependence of the proteolytic activity and heat stability of the wild-type enzyme and its mutants
To examine the temperature dependence of the enzyme activity, 480 μl of 1 mM N-suc-AAPF-pNA solution in 100 mM HEPES-NaOH (pH 7.5) containing 1 mM CaCl2 was preincubated at an appropriate temperature for 5 min; subsequently, 20 μl of enzyme solution (5 μg/ml) was added. The change in absorbance at 410 nm was continuously monitored, and the activity was estimated as described above based on the results of triplicate experiments.
To examine the heat stability, the enzymes were diluted with 20 mM 2-morpholinoethanesulfonic acid (MES)-NaOH buffer (pH 6.0) containing 1 mM CaCl2 to yield a 10 μg/ml solution. These experiments were performed at pH 6.0 to diminish the massive autolysis that would occur under more alkaline condition. This enabled us to observe the differential decrease of residual activity among mutants due to structural destabilization during the heat treatment processes. The enzyme solution was incubated for the appropriate time period at 70°C or 80°C and was subsequently cooled quickly. The remaining activity was determined based on the results of triplicate experiments using 1 mM N-suc-AAPF-pNA as a substrate at 40°C, as described above.
The initial rates of N-suc-AAPF-pNA hydrolysis induced by the wild-type enzyme and mutant enzymes were measured at 40°C in 50 mM HEPES-NaOH (pH 7.5) containing 1 mM CaCl2 as described above. The kinetic parameters Vmax and Km were estimated assuming a Michaelis-Menten kinetic model, and the graphics software package DeltaGraph version 6 (Nihon Poladigital K.K., Tokyo, Japan) was used with non-linear regression. The apparent values of kcat were estimated using a molecular mass of 28 kDa.
Unfolding study of AQN proteins based on circular dichroism (CD) measurement
CD analysis was carried out to determine the transition temperature (Tm) and to monitor the unfolding of the wild-type and mutant enzymes. Prior to the CD measurements, purified enzymes were treated with 25 mM PMSF dissolved in methanol for 30 min to prevent autolytic degradation during the measurements. After complete inactivation of the protease activity was confirmed, the samples were dialyzed overnight against 20 mM MES-NaOH buffer (pH 6.0) containing 1 mM CaCl2 and filtered through a MILEX®-HV filter (0.45 μm pore size, Durapore (PVDF), Merck Millipore Ltd., Carrigtwohill, Ireland). CD measurements were conducted using a JASCO-725 circular dichroism spectropolarimeter equipped with PTC-348 Peltier type single cell holder, and the change in ellipticity at 220 nm was monitored under a constant heating rate (1°C/min) at temperatures ranging from 40 to 105°C. The melting curves were normalized according to the methods in the literature (Arnórsdóttir et al. ), and the melting temperature (Tm) values of the enzymes were estimated using a graphics software package, Delta graph. The experiments were performed in duplicate.
The unfolding of proteins as a function of time was observed at a constant temperature (70°C or 80°C) by CD measurement over a range of 200–250 nm. Measurements were performed with a JASCO-725 circular dichroism spectropolarimeter (JASCO, Tokyo, Japan) equipped with PTC-348 Peltier type single cell holder, and the change in ellipticity was monitored at every 5 min over a 30-min period at a constant temperature. Wavelength scans in the range of 200–250 nm were performed in rectangular quartz cells (JASCO model: T-11-ES-1) with a path length of 0.1 cm.
Mutagenesis of salt bridge-forming residues in AQN and purification of the wild-type enzyme and its mutants
Thermal stability of the wild-type enzyme and its mutants
Disruption of the salt bridges common to AQN and VPR did not yield a common outcome. For example, the residual activity of D183N declined rapidly, with a half-life of approximately 30 min at both 70°C and 80°C, whereas the activities of D58N and D138N were almost indistinguishable from that of the wild-type enzyme at both temperatures (Figure 2c and d). The above results indicate that the Arg12-Asp183 salt bridge is important for conferring structural stability to proteinase K subfamily enzymes, although the other two common salt bridges are not significantly involved in thermal stabilization. Additional salt bridges were introduced at the positions where VPR-intrinsic salt bridges are located in mutants G61D, G262D and S277D. The inactivation time courses of the three mutants were similar to that of the wild-type enzyme at 70°C and 80°C. In fact, S277D was slightly less stable than the wild-type enzyme at both 70°C and 80°C (Figure 2e and f).
Temperature dependence of wild-type and mutant enzyme activity
Kinetic analysis of wild-type and mutant enzymes
Kinetic parameters of the wild-type and mutant enzymes 1
91.6 ± 2.75
0.79 ± 0.04
110 ± 4.48
0.98 ± 0.05
75.2 ± 3.13
1.10 ± 0.10
96.1 ± 0.98
0.91 ± 0.03
68.5 ± 1.37
0.99 ± 0.07
59.5 ± 6.14
0.77 ± 0.01
53.0 ± 3.03
0.74 ± 0.05
47.2 ± 1.77
0.82 ± 0.13
61.3 ± 1.86
0.93 ± 0.15
73.6 ± 5.51
0.80 ± 0.12
Unfolding study on AQN proteins based on CD measurements
To analyze the unfolding process of active AQN mutants at a fixed temperature, changes in the CD spectra of the wild-type enzyme, D17N, E237Q, D212N or D183N as a function of time were recorded at 70°C and 80°C. Figure 4a and b show the CD spectra of the wild-type enzyme during the 30-min incubations at 70°C and 80°C, respectively. The spectrum was not significantly changed during the 30-min incubation at 70°C. However, at 80°C, the ellipticity at 222 nm was slightly but significantly decreased in the first 5 min, and it subsequently remained unchanged for up to 30 min. Figure 4c and d show the CD profiles of D17N during the 30-min incubations at 70°C and 80°C, respectively. The change in ellipticity at 222 nm at 70°C followed a time course that was very similar to that of the wild-type enzyme at 80°C. However, at 80°C, the initial change at 5 min was slightly more pronounced and there was a gradual decrease in ellipticity that continued for up to 30 min in the D17N mutant. The change in the CD spectrum of E237Q was similar to that of D17N during incubation at both 70°C and 80°C (Figure 4e and f). These results indicated that the secondary structure contents of D17N and E237Q did not show major decreases for 30 min at 70°C. The change in the CD profile of D212N was very similar to that of D17N at 70°C (Figure 4g). However, at 80°C, the ellipticity of D212N in the 200–240 nm range continued to decrease for up to 30 min in parallel with the rapid inactivation of this mutant at 80°C (Figures 4h and 2b). The change in the CD profile of D183N at 70°C is shown in Figure 4i. The peak at 222 nm gradually decreased as a function of time down to 40% of the value before treatment, indicating that more than half of the secondary structures of D183N were destroyed. A comparable change in the ellipticity of D212N occurred only at 80°C. These results suggest that a salt bridge involving Asp183 plays a significant role in maintaining the structure of AQN. This may also be true of other members of the proteinase K subfamily, as the salt bridge involving Asp183 is conserved among these enzymes.
The denaturation curve of the PMSF-treated D183N mutant as monitored by the change in the ellipticity at 220 nm is shown in Additional file1: Figure S1. The Tm value of D183N was estimated to be 74°C. This is consistent with the extensive decrease of 222 nm peak intensity observed in Figure 4i. Denaturation of other mutants as well as the wild type enzyme apparently occurred at much higher temperature range than D183N mutant, but we could not obtain reliable estimates for their Tm values. It should be noted that the change in CD spectrum gradually proceeded over 30 min at constant temperature as shown in Figure 4, while measurement of Tm was carried out at a heating rate of 1°C/min. It is possible that the rate of protein unfolding did not catch up with the elevation of the temperature, and this might give apparent Tm higher than true Tm.
Subtilases, a group of serine proteases in the subtilisin superfamily, are composed of approximately 275 amino acid residues. Mutation studies on more than 50% of the amino acid residues in their primary structure have been described in the literature (Bryan ). Various factors appear to make complex contributions to subtilase stability. There are six subfamilies in the subtilisin superfamily, and it is possible that the mechanism by which thermal stability is conferred may differ from one subfamily to another. In this report, we investigated the roles of salt bridges in the thermal stabilization of AQN, a proteinase K subfamily member.
Regarding the salt bridges intrinsic to AQN (Asp17-Arg259, Arg31-Glu237 and Arg43-Asp212), the kcat values of D17N and E237Q at 40°C and the activities at elevated temperatures toward a synthetic substrate were slightly increased compared to that of the wild-type enzyme; however, these mutants were apparently less thermostable than the wild-type enzyme during heat treatment experiments. These results suggest that the decline of the residual activities of the mutant enzymes during prolonged incubation at high temperatures might be caused in part by extensive autolysis under conditions in which these enzymes exhibit higher activity than the wild-type enzyme. However, it should also be noted that examination by CD spectrometry indicated that D17N and E237Q showed a small change in ellipticity at 222 nm in the first 5 min at 70°C, although the ellipticity remained unchanged over the course of the subsequent incubation for up to 30 min (Figure 4c and e). These results suggest that incubation at 70°C may affect regions of the D17N and E237Q mutants that are not rich in secondary structures, including loop regions, but that the structural perturbation due to the D17N or E237Q mutation may destabilize the active site conformation during a prolonged incubation at 70°C and above. This result agrees with a previous report indicating that the D17N mutant exhibited reduced thermal stability compared to the wild-type enzyme (Arnórsdóttir et al. ).
Recently, Jakob et al. reported an intensive analysis of the roles of charged amino acid residues in a Bacillus gibsonii subtilisin protease, BgAP, using site-directed mutagenesis. BgAP Q230E showed increased thermal resistance compared to wild-type BgAP (Jakob et al. ). This result is consistent with our data on E237Q. Glu237 of AQN corresponds to Gln230 of BgAP, and disruption of a salt bridge in E237Q resulted in a rapid decrease of activity during incubation at 70°C and 80°C.
The stability of D212N was similar to that of the wild-type enzyme at 70°C; however, it was inactivated rapidly at 80°C (Figure 2a and b). This result is consistent with the results of the CD spectrometry analysis showing that the secondary structure content was rapidly decreased as a function of time at 80°C. The inactivation mechanism of D212N at 80°C may be different from that of D17N and E237Q at 70°C.
Introducing additional salt bridges at positions mimicking VPR-intrinsic salt bridges in G61D, G262D and S277D resulted in kcat values lower than that of the wild-type enzyme; however, this did not affect the thermal stability.
In conclusion, mutation of a salt bridge-forming amino acid that is highly conserved among members of the proteinase K subfamily, Asp183 of AQN, destabilized the protein structure, indicating that this salt bridge plays the most important role in the stability of AQN compared with the other salt bridges. Furthermore, AQN-intrinsic salt bridges confer additional thermal stability to AQN.
This study was supported in part by a grant of Strategic Research Foundation Grant-aided Project for Private Universities from Ministry of Education, Culture, Sport, Science, and Technology, Japan (MEXT), 2014-2018 (S1411005).
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