Evidence that the amino acid residue Ile121 is involved in arginine kinase activity and structural stability

International Journal of Biological Macromolecules 51 (2012) 369–377

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Evidence that the amino acid residue Ile121 is involved in arginine kinase activity and structural stability Qing-Yun Wu a,b , Feng Li c , Xiao-Yun Wang d,∗ , Kai-Lin Xu a,∗∗ a

Department of Hematology, the Affiliated Hospital of Xuzhou Medical College, No. 99 West Huaihai Road, Xuzhou 221002, China Laboratory of Transplantation and Immunology, Xuzhou Medical College, No. 99 West Huaihai Road, Xuzhou 221002, China Department of Neurobiology, Xuzhou Medical College, 221002 Xuzhou, China d College of Life Sciences, State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai’an, Shandong 271018, China b c

a r t i c l e

i n f o

Article history: Received 10 April 2012 Received in revised form 17 May 2012 Accepted 21 May 2012 Available online 27 May 2012 Keywords: Arginine kinase Aggregation Conformational change Insecticides Structural stability

a b s t r a c t Arginine kinase (AK) catalyzes the reversible phosphorylation of arginine by ATP, yielding the phosphoarginine. Domain–domain interactions may be very important to the structure and functions of many multidomain proteins. However, little is known about the role of amino acid residues located in the linker between the N- and C-terminal domains in the structural stability and functions of multidomain proteins. In this research, A series mutation of conserved residue Ile121 located in the linker were mutated to explore its roles in the activity and structural stability of AK. The mutations I121D and I121K led to pronounced loss of activity and structural stability. Furthermore, these mutations also led to serious aggregation during heat-and GdnHCl-induced denaturation and refolding from the GdnHCl-denatured state. More importantly, all the mutantions except I121L could not successfully recover their activities by dilution-initiated refolding, and showed significant decreased rate constant during AK refolding. While the mutation I121L almost had no effect on AK activity and structural stability. These results suggested that mutations of the residue I121 in the linker might affect the correct positioning of the domains and thus disrupt the efficient recognition and interactions between the N- and C-terminal domains. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Arginine kinase (ATP: l-arginine phosphotransferase EC 2.7.3.3) (AK) catalyzes the reversible phosphorylation of arginine by ATP, yielding the phosphoarginine [1]. As an analogy of creatine kinase (CK) in vertebrates, AK is widely distributed in invertebrates playing a critical role as an energy reserve when energy is needed [1,2]. Due to its prominent role in energy metabolism and absence in vertebrates, AK could be chosen as a target to screen effective and harmless pesticide in agriculture [3,4]. Locust Migratoria manilensis is a noxious herbivorous insect in agriculture. Schneider et al. (1989) demonstrated that AK and arginine phosphate served as a temporal energy buffer system in locust femoral muscle [5]. Meanwhile, the high activities of this enzyme

Abbreviations: AK, arginine kinase; CK, creatine kinase; PK, phosphogen kinase; IPTG, isopropyl-d-thiogalactopyranoside; ANS, 1-anilinonaphtalene-8-sulfonate; SEC, size exclusion chromatography; GdnHCl, guanidine hydrochloride; Emax , emission maximum wavelength of the intrinsic fluorescence. ∗ Corresponding author. Tel.: +86 538 8242656 8430; fax: +86 538 8248696. ∗∗ Corresponding author. Tel.: +86 516 85802382; fax: +86 516 85601527. E-mail addresses: [email protected] (X.-Y. Wang), [email protected] (K.-L. Xu). 0141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2012.05.022

was also found in ion-transporting epithelia, such as the hindgut [6] and lepidopteran midgut [6,7]. In contrast to other phosphogen kinases (PK) which are mostly dimeric or octameric, AKs of insect are typically functional as monomers [8–11]. It has been demonstrated that insect AKs has allergenic potential contributing to allergies against Indianmeal moth [12] and cockroaches [13]. Due to its assumed prominent role in energy metabolism and its specific occurrence in invertebrates, AK has been proposed as a novel target structure for innovative insecticides to combat insect pest species [12–15]. So, investigation of the amino acids that play important role in keeping AK activity and structural stability will be useful in developing agents to control the insect population and its destructive role in agriculture. Protein folding is a process by which the amino acid sequence of a protein determines its three-dimensional conformation [16]. To achieve the native tertiary structures, adjacent domains need to recognize each other through domain–domain interactions [17,18]. The domains are connected sequentially by the linker in the primary structure. However, little is known about the role of the amino acid residues located in the linker in protein folding and structural stability. Previous studies suggested that the linker may participate in the folding or function of proteins [19,20]. The crystal structure of AK reveals that a small N-terminal domain and a large C-terminal

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Fig. 1. The modeling structure of WT AK (A) and the alignment of the amino acid sequence of AKs (B). (A) represents the structure of WT AK. In (A), the linker, N-terminal and C-terminal domains of AK are indicated by red, green and cyanine, respectively. (B), Alignment of amino acid sequences of AKs in Limulus polyphemus (LPU09809), Locusta migratoria manilensis (DQ513322), Artemia franciscana (AF426741), Bombyx mori (DQ272299), Periplaneta Americana (AAT77152), Schistosoma japonicum (CAX73626). Alignment was produced using DNAMAN software and the conserved hydrophobic residues indicated by red color. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

domain are connected by a linker with the active site located in the cleft between the two domains. However, recent analysis suggests that both AK and CK consists of four dynamic domains [21]. Moreover, the importance of the interactions among the four domains has been proven in AK and CK [22]. A close inspection of the structure of AK indicates that the linker does not form any regular secondary structures and seems to be flexible (Fig. 1A) [23]. The flexible structure of the linker might play a key role in sustaining the correct positioning of the domains and contributing to the efficient recognition and interactions between the two domains. Furthermore, the linker is mainly composed of charged residues and only contains six hydrophobic residues: F104, V107, L110, L113, V120 and I121 (Fig. 1B). These hydrophobic residues are not conserved in all AKs, but fully or highly conserved in monomer AKs. Interestingly, the side-chains of almost all hydrophobic residues are exposed to solvent, whereas those of L113 and I121 residues especially the I121 residue is buried and interact with the hydrophobic residues in the C-terminal domain. This suggest that the amino acid residue I121 which located in the linker might play important roles in keeping AK activity and structural stability. However, whether this amino acid residue involved in AK activity and structural stability is still unclear. In this study, the hydrophobic amino acid I121, was mutated to the hydrophobic, non-typical nonpolar, positively and negatively charged hydrophilic amino acids of Leu (L), Gly (G), Lys (K) and Asp (D), respectively, to investigate its role in AK substrate synergism, structural stability and activity. Our study implied that the mutations I121D and I121K led to pronounced loss of activity and structural stability. More importantly, all the mutations except I121L could not successfully recover their activities by dilutioninitiated refolding, and showed significant decreased rate constant during AK refolding. These results suggested that mutations of the residue I121 might affect the correct positioning of the domains and thus disrupt the efficient recognition and interactions between the two domains. This study may provide a clue for the development of insecticides specific to agricultural herbivorous and the indoor allergens insects.

2. Materials and methods 2.1. Cloning, site-directed mutagenesis and expression of the mutant AK pMD-18T simpler plasmid with locust AK cDNA (pMD-Locust AK) inserted was used as a template for mutagenesis [4]. The gene encodes 355 amino acids with a molecular weight of about 40 kDa. Four mutations (I121L, I121D, I121K and I121G) were introduced into the template of WT AK by overlap PCR. Then the mutants was cloned into expression vector pET-28a, sequenced and transformed into the Escherichia coli BL21 (DE3) codon plus. The WT and mutant AKs fusion proteins were expressed in E. coli BL21 and purified as described previously [8–10]. The purity was checked by SDS-PAGE. Protein concentration was determined according to Bradford’s method [24]. All protein samples were prepared by dissolving the proteins in the standard buffer (10 mM glycine-NaOH, 1 mM DTT at pH 8.6). The final concentrations of the enzymes were 3.2 ␮M for most experiments unless otherwise indicated.

2.2. Enzyme assay and determination of kinetic parameters AK activity (phosphoarginine synthesis) was assayed as previously described with some modification [8,25]. The assay mixture for AK determination consist of 100 mM Tris, pH 8.0, 10 mM larginine, 8 mM ATP-Na and 10 mM mercapto-ethanol. The reaction was carried out with 10 ␮L of 0.01 mM enzyme solution added to assay mixture. After 2 min, the reaction was stopped by the addition of 2.5% TCA and the mixture heated for 2 min at 100 ◦ C. The sample was immediately cooled to 25 ◦ C for 1 min and then incubated at 30 ◦ C for 5 min. The absorbance at 660 nm was measured at 30 ◦ C using an Ultrospec4300 pro UV/Visible spectrophotometer. The activity of AK in all text is defined as micromoles of phosphate mL−1 min−1 and the specific activity as the activity per milligram of proteins.

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The two-substrate graphical method was used to obtain the kinetic parameters [4]. Exactly, to estimate kinetic constants (Km and Vmax ), a Lineweaver–Burk plot was fitted using GRAF4WIN softwares. As to the parameters Kd , the dissociation constant in the absence of one substrate, is obtained by fitting data directly according to the method of Cleland [26], using the software written by Dr. Viola. The activity assays were carried out at the optimum pH (pH 8.6) and temperature (30 ◦ C) with different concentrations of ATP and arginine. All the reactions were carried out at least three times. 2.3. Heat- and GdnHCl-induced AK inactivation Thermal inactivation was performed by incubating the samples for 10 min at given temperatures varying from 25 to 60 ◦ C, then cooled on ice and the activity was measured at 30 ◦ C. For GdnHCl-induced inactivation, the protein solutions were incubated in the standard buffer containing various concentrations of GdnHCl (0–0.8 M) at 25 ◦ C overnight, then the activity was determined at 30 ◦ C. 2.4. Spectroscopic experiments The temperature for all spectroscopic experiments was controlled by a thermoelectrically controlled cell holder. The aggregation of the proteins was monitored by recording the turbidity at 400 nm with an Ultraspec4300 pro UV/Visible spectrophotometer. Far-UV circular dichroism (CD) spectra were measured on a Jasco 715 spectrophotometer with a cell path-length of 0.1 cm. The intrinsic fluorescence emission spectra were collected on a Hitachi F-4500 spectrofluorimeter using 1-cm path-length cuvettes. For ANS-fluorescence measurements, 10-fold molar excess of ANS was added to the samples. The samples were equilibrated for 30 min in the dark, and then the extrinsic fluorescence was measured. All spectroscopic experiments were carried out at 25 ◦ C. 2.5. Unfolding and refolding experiments The thermal unfolding was carried out by incubating the samples in a thermoelectrically controlled cell holder of the CD spectrophotometer or fluorescence spectrofluorimeter. The temperature range was from 25 to 80 ◦ C and the CD or Trp fluorescence spectra were recorded at intervals of 2.5 ◦ C with an equilibration time of 2 min. The time-course thermal aggregation was detected by incubating the samples continuously at 48 ◦ C for 20 min and the turbidity at 400 nm was recorded. The GdnHCl-induced unfolding was investigated by denaturing the proteins in stranded buffer with GdnHCl concentrations ranging from 0 to 3 M at 25 ◦ C overnight. Then CD, intrinsic and ANS fluorescence, and the turbidity at 400 nm were recorded using the same set of samples at 25 ◦ C. In the dilution refolding process, the aggregation of the proteins was monitored by recording the turbidity at 400 nm. For the kinetic refolding of dimer AKs, proteins with a final concentration of 200 ␮M were completely denatured in 3 M GdnHCl overnight at 25 ◦ C. The refolding was initiated by a 100-fold dilution of the denatured AK into standard buffer containing 0.1 M GdnHCl. The kinetics data was derived by monitoring the changes of the intrinsic fluorescence intensity at 350 nm on an F-4500 spectrofluorimeter with an excitation wavelength of 280 nm. The refolding rate constants k1 (fast phase rate constant) and k2 (slow phase rate constant) were calculated by non-linear fit using Origin 6.0 software. The reactivation course was studied using the kinetic method of the substrate reaction as previously described [27,28]. In brief, the reactivation was started by a 400-fold dilution of the denatured AKs into the buffer used for activity assay. Then the changes at 575 nm were monitored by UV/visible spectrophotometer for 10 min. The apparent reactivation rate constants (A) were calculated according

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to the reactivation kinetics model of AK as previously described [27,28]. 2.6. Modeling the structure of mutant AKs In order to analyze the effect of the mutations on the AKs structures, both the SPDBV software (http://swissmodel.expasy.org/) and the VMD modeling procedures (version 1.8.7, http://www.ks.uiuc.edu/Research/vmd/) [29] were used to model the structure of mutated AKs based on PDB files (PDB ID:1RL9) for Limulus AK from the Protein Data Bank. 3. Results and discussion 3.1. Mutations decreased AK activity All the recombinant enzymes were successfully expressed as soluble fusion proteins. The recombinant WT AK showed similar enzymatic characteristics to native AK, indicating that the His6 -tag portion had no effects on its activity (Table 1). As shown in Table 1, all the mutated AKs retained 37.8–94.8% of WT AK activity (kcat ) and displayed decreased substrate affinity. The Arg Km values of I121G, I121K and I121D mutant AKs (1.74–3.04 mM) was 1.8 to 3.2 fold higher than that of recombinant WT AK Arg (0.951 mM), while their Kd values (3.91–5.02 mM) were 1.46 to 1.88 fold higher than that of WT AK (2.67 mM), resulting in a Kd /Km value of 1.65–2.24, indicating that the synergism in substrate binding had been decreased. Furthermore, the substrate synergism Arg for the mutation I121D had almost lost and its kcat /Km value −1 −1 (19.9 s mM ) only maintained 11.9% in enzyme catalysis efficiency, compared with that (167.6 s−1 mM−1 ) of WT AK. When I121 was replaced by non-typical polar and hydrophilic positively charged amino acid (I121G and I121K), the substrate synergism also decreased, less than that of I121D. More importantly, the mutation I121L showed similar characteristics to WT AK. Thus, the changes of synergism may be caused by the characteristics of the amino acids. To sum up, when I121 was replaced by the nonpolar amino acid (L) it almost did not lead to changes, while when it was replaced by the polar amino acid (positively charged K or negatively charged D), it led to significant changes of the substrate synergism. Glycine is special in polarity, between nonpolar and polar amino acids. The replacement of the residues in the mutants (D and K) might lead to much looser tertiary structure than that of WT AK. Since the active site of AK is located in the cleft between the N- and C-terminal domains [23], these results indicated that mutations in the linker might alter domain–domain interactions. Previous studies implied that mutations of the amino acid residues near the active sites may decrease the substrate synergism by affecting the correct active sites conformation [4,23,30]. Our studies also suggested that the substrate synergism decrease was related to the conformational changes in the active sites [9–11]. 3.2. Mutations destroyed the secondary and tertiary structure of AK In order to explore the effects of mutations on AK secondary and tertiary structures, the spectroscopic spectra were determined. The mean residue ellipticity of CD spectra at 222 nm of I121K, I121G and I121D mutant AKs was smaller than that of WT AK at the same concentration (Fig. 2A). This result suggested that these mutations impaired the secondary structures of WT AK. While the I121L mutation showed similar characteristic to the WT AK. Meanwhile the intrinsic fluorescence spectra of I121K, I121G and I121D displayed red shift, compared to WT AK (Fig. 2B). Since all the two Trp residues (position 214 and 221) are located on the C-terminal of AK, the

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Table 1 Comparison of kinetic parameters for the forward reaction and apparent reactivation rate of recombinant WT and mutants AKs. Mutations Native AKa Recombinant WT AK I121L I121G I121K I121D

Vmax (␮mol Pi· min−1 mg−1 ) 239.16 226.88 164.66 106.40 90.44

± ± ± ± ±

7.65 8.83 7.82 4.33 1.54

Arg

kcat (s−1 ) 163 159.43 ± 151.22 ± 109.77 ± 70.93 ± 60.36 ±

6.22 3.06 3.06 3.56 0.89

Arg

Km (mM)

Kd

0.94 0.951 ± 0.98 ± 1.74 ± 2.53 ± 3.04 ±

2.67 2.53 3.91 4.76 5.02

0.08 0.06 0.08 0.34 0.54

(mM)

± ± ± ± ±

0.22 0.11 0.32 0.48 0.88

ATP Km (mM)

KdATP (mM)

1.29 1.270 ± 1.36 ± 2.38 ± 3.56 ± 4.06 ±

3.56 3.71 5.33 6.69 6.72

0.23 0.12 0.45 0.78 0.66

± ± ± ± ±

0.32 0.35 0.65 0.48 0.78

Kd /Km

2.80 2.72 2.24 1.88 1.65

± ± ± ± ±

Apparent reactivation rateb (×103 s−1 ) 0.66 0.26 0.42 0.23 0.25

1.78 1.69 1.22 0.94 0.85

± ± ± ± ±

0.13 0.08 0.08 0.04 0.03

Note: kinetic parameters were obtained from at least three runs of the reaction. a Kinetic parameters cited from the article [7]. b The apparent reactivation rate was obtained from the data in Fig. 5B according to the reactivation kinetics model of AK described previously [31,32].

red-shift of Trp fluorescence spectra of I121K, I121G and I121D suggested that mutations might induce microenvironmental changes of the Trp residues and affect the structure of C-terminal domain. Furthermore, the ANS fluorescence spectra also confirmed above results which impiled that the mutations had more hydrophobic exposure than that of WT AK to allow binding of ANS molecules (Fig. 2C). These spectroscopic experiments indicated that the mutations I121K, I121G and I121D may impair the secondary, tertiary structures of AK. 3.3. Effects of the mutations on AK thermal inactivation and unfolding

10

Relative flourescence intensity (100%)

Previous studies had shown that the inactivation of AK occurred prior to its unfolding, which suggested that the active site unfolded before the conformational changes of the overall structure

A

5

[ ] MRN

0 -5

WT I121L I121K I121D I121G

-10 -15 -20 180

200 220 240 Wavelength (nm)

ANS Fluorescence intensity

50

[9–11,31,32]. As shown in Fig. 3A, WT AK could retain its activity well at temperatures lower than 48 ◦ C, and then its activity showed a steep decrease from 45 to 60 ◦ C and completely lost its activity at temperatures above 65 ◦ C. This result was consistent with those reported in the literature [9,31]. The thermal inactivation of mutant AKs were inactivated at relatively low temperature. Consistent with spectroscopic experiments, thermal inactivation result also implied that the mutations I121K, I121G and I121D affected the stability of the active site. This conclusion is consistent with the fact that amino acid residues in the linker may play key roles in the structural stability of AK [19,20]. The thermal unfolding and aggregation of AKs were also detected to further investigate the effects of mutations on the overall structural stability of AK. The data presented in Fig. 3B and C could be well-fitted to a two-state model, and the midpoints of the thermal denaturation (Tm ) are summarized in Table 2. The Tm

260

120

B

100 WT I121L I121K I121D I121G

80 60 40 20 0 300

320

340 360 380 Wavelength (nm)

400

C

40

WT I121L I121K I121D I121G ANS control

30 20 10 0 400

440

480 520 560 Wavelength (nm)

600

Fig. 2. Effect of mutations on AK structures detected by CD (A), intrinsic fluorescence spectra (B) and ANS fluorescence spectra (C). The CD data were presented as the mean residue ellipticity ([]MRW ) expressed in [103 deg cm2 dmol−1 ]. The final protein concentration was 3.2 ␮M. All experiments were carried out at 25 ◦ C.

Q.-Y. Wu et al. / International Journal of Biological Macromolecules 51 (2012) 369–377

A

0

B

-4

] MRW

80

222

60 WT I121G L121K

40 20

WT

-8

I121G I121K I121D I121L

[

Relative activity (100%)

100

373

-12

I121D I121L

-16

0 30

2.0

40 50 60 Temperature ( ºC)

20

70

0.50

Turbidity (A 400)

I320/I 365

1.6 WT I121G I121K I121D

0.8

40

50

60

70

Temperature ( ºC)

C

1.2

30

I121L

D

WT

I121K

I121G

I121D

I121L

0.40 0.30 0.20 0.10 0.00

0.4 20

30

40

50

60

70

80

0

10

Temperature ( ºC)

20 30 40 50 Time (min)

60

70

Fig. 3. Thermal inactivation (A), thermal unfolding monitored by CD (B) and intrinsic fluorescence (C), and thermal aggregation at 48 ◦ C (D) of the WT and mutant AKs. In panel (C), thermal melting curves were obtained by monitoring the ratio of I320 /I365 . The data in (A–C) were fitted to a two-state model, and the parameters are presented in Table 2.

values of the WT AK were almost similar for the two techniques: 55.8 ± 0.5 ◦ C from the change of [ 222 ]MRW and 56.6 ± 0.5 ◦ C from that of fluorescence. Furthermore, both the CD and the intrinsic fluorescence data indicated that the Tm values of the I121K, I121G and I121D mutant AKs were smaller than that of WT AK. This result suggested that mutations I121K, I121G and I121D decreased the structural stability of AK. Previous studies indicated that the thermal denaturation of AK was an irreversible process accompanied with serious aggregation [9,10,31,32]. To detect the effects of mutations on the thermal aggregation, the aggregation of the WT AK and mutant AKs at

48 ◦ C was monitored by the turbidity at 400 nm (Fig. 3D). The thermal aggregation of the WT AK had an obvious lag time about 20 min, while the lag time of the mutants was much shorter (<1 min). Meanwhile, the aggregation amounts of the I121K, I121G and I121D mutations were much more than that of WT AK. The shorter lag time and the faster aggregation rate indicated that during heating, those mutations were more prone to aggregate than that of WT AK. Analyzing the reason, mutation of the residue located in the linker may affect the correct direction of C- and N-terminal domains which resulted in looser structure of WT AK.

Table 2 The structural stability of WT and mutant AKs in the folding process. Parameters

WT AK

T0.5 (◦ C)a Tm , CD (◦ C)b Tm , Trp fluorescence (◦ C)b c0.5 (M)c Cm , NI (M)d Cm , IU (M)d k1 (×103 s−1 )e k2 (×103 s−1 )e

53.6 55.8 56.6 0.48 0. 42 1.39 18.6 1.7

a

± ± ± ± ± ± ± ±

I121L 0.5 0.5 0.5 0.02 0.02 0.11 2.1 0.1

51.8 54.2 54.8 0.44 0.38 1.36 16.4 1.3

± ± ± ± ± ± ± ±

I121G 0.5 0.5 0.5 0.02 0.02 0.12 1.8 0.1

46.6 ± 40.6 ± 51.5 ± 0.32 ± 0.30 ± 1.29 ± 9.8 ± –

I121K 0.5 0.5 0.5 0.01 0.01 0.12 1.4

43.6 36.2 50.6 0.26 0.27 1.25 7.2

± ± ± ± ± ± ±

I121D 0.5 0.5 0.5 0.01 0.01 0.12 0.9

42.4 ± 34.6 ± 49.4 ± 0.24 ± 0.25 ± 1.23 ± 6.2 ± –

0.5 0.5 0.5 0.01 0.02 0.13 0.5

Midpoint temperature of thermal inactivation. Midpoint temperature of thermal unfolding. Midpoint concentration of inactivation induced by GdnHCl. d Midpoint concentrations of denaturation by GdnHCl for native to intermediate (Cm , NI) and for intermediate to unfolded states (Cm , IU), respectively. e Rate constants for the fast phase (k1 ) and slow phase (k2 ) of AK refolding from the GdnHCl-denatured state. The kinetic data of the WT AK was fit by a biphasic process, while that of the mutant AKs were fit by a monophasic process. b c

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360

A

100

B

355 WT

80

I121G I121K

60

I121D I121L

40

350

E max (nm)

Relative activity (100%)

120

340

20

335

0

330

0.00 80

0.20 0.40 0.60 [GdnHCl] (M)

WT I121G I121K I121D I121L

345

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 [GdnHCl] (M)

0.80

C

D 0

] MRW 222

I121D

40

-4

I121L

-8

WT I121G

-12

I121K

[

ANS intensity

WT I121G I121K

60

20

I121D I121L

-16 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 [GdnHCl] (M) 1.60

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 [GdnHCl] (M) 0.10

E

WT

WT

0.08

Turbidity (A 400)

I 320 /I 365

1.20

I121G I121K I121D

0.80

F

I121L

0.40

I121G I121K

0.06

I121D I121L

0.04 0.02 0.00

0.00 0.0

1.0

2.0 3.0 4.0 [GdnHCl] (M)

5.0

6.0

0.0

0.5

1.0 1.5 2.0 [GdnHCl] (M)

2.5

3.0

Fig. 4. Inactivation (A) and unfolding monitored by CD (B), Emax (C) and ANS fluorescence(D), I320 /I365 (E) values of intrinsic fluorescence, and turbidity at 400 nm (F) of the WT and mutant AKs induced by GdnHCl. The data in panel A were fitted to a two-state model, and the parameters are presented in Table 2.

3.4. Effects of mutations on GdnHCl induced AK inactivation and unfolding A similar result was obtained for AK inactivation induced by GdnHCl (Fig. 4A). The I121K, I121G and I121D were more unstable than that of WT AK, suggesting that this residue may be more important for sustaining the conformation of active sites. In agreement with previous studies [31,32], this study also impiled that the active site unfolded before the conformational changes of the AK overall structure. Thus AK inactivation may be caused by the changes of the active site which located in the cleft between the N- and C-terminal domains [23,31,32]. It is more likely that these mutations might affect the correct direction of C- and the

N-terminal domains and destroy the domain–domain interactions. In the unfolding process, the Emax of WT AK and mutant AKs showed a sharp red shift in the ranges of 0.2–0.6 M and 1.0–1.4 M GdnHCl concentrations, while the Emax of mutant AKs was at 0.1–0.4 M and 1.0–1.4 M GdnHCl concentrations (Fig. 4B). Meanwhile the ANS intensity reached peaks at 0.4 M and 1.2 M GdnHCl for the WT AK, while that of the mutant AKs was at 0.3 M GdnHCl and 1.2 M GdnHCl concentrations (Fig. 4C). Furthermore, in the CD spectra, the [ 222 ]MRW showed similar transition, the [ 222 ]MRW of WT decreased dramatically as GdnHCl concentration increased from 0.1 to 0.5 M, while the [ 222 ]MRW of mutant AKs was from 0 to 0.3 M GdnHCl. The ellipticity began to increase and reached a

Q.-Y. Wu et al. / International Journal of Biological Macromolecules 51 (2012) 369–377

0.50

A

I121K

I121G

I121D

0.50

I121L

B WT

0.40

0.30

P (A 575 )

Turbidity (A 400)

0.40

WT

375

0.20

I121G I121K I121D I121L

0.30 0.20

0.10

0.10 0.00

0.00 0

0

100 200 300 400 500 600

100 200 300 400 500 600

Time (s)

Time (s)

C Normalized intensity

1.00

0.80 WT

0.60

I121G I121K I121D

0.40

I121L

0.20 0

400

800

1200 1600 2000

Time (s) Fig. 5. Aggregation (A), reactivation (B) and refolding (C) kinetics of the WT and the mutant AKs. The kinetics was monitored by the changes of UV absorbance at 400 nm (panel A) or the intrinsic fluorescence intensity at 350 nm (panel C). The data in panel C were fitted to a biphasic process. In panel B, the reactivation was started by a 1000-fold dilution of the fully denatured enzyme into the buffer used for activity assay. Then the changes at 575 nm were monitored.

maximum at 1.0 M GdnHCl, with 59% secondary structure of WT AK, indicating that an intermediate with more secondary structure was induced (Fig. 4D). These results suggested that two equilibrium intermediate states existed in the unfolding process of WT and mutant AKs. The first intermediate, which exists at about 0.4 M GdnHCl for WT AK and 0.3 M for mutant AKs, while the second intermediate, forming at 1.2 M GdnHCl which showed similar characteristics to the globular folding intermediates as described in previous literature [9,10,31,32]. The refolding transitions of WT and mutant AKs from the GdnHCl-denatured state were similar to those unfolding at GdnHCl concentrations above 0.5 M when monitored by all the three techniques (data not shown). The deviation between the unfolding and refolding at GdnHCl concentrations below 0.5 M indicated that the folding of WT and mutant AKs was not fully reversible under our conditions. Thus irreversibility might be caused by the formation of soluble non-native oligomers from the aggregation-prone intermediates [9,10,31,32]. Moreover, the transition curves of the mutant AKs were almost identical to that of WT AK at GdnHCl concentrations above 0.5 M, which suggested that the mutations did not affect the MG ↔ U transitions. The I320 /I365 values of intrinsic Trp fluorescence also showed the same trends as to the effects of mutations on the unfolding transition as above three techniques (Fig. 4E). The turbidity experiment further confirmed that the mutations I121K, I121G and I121D impaired the structural stability of AK, as the aggregation amounts of the mutations was higher than that of WT AK and I121L mutant AK at the

GdnHCl concentrations below 1.75 M (Fig. 4F). These results suggested that amino acid residues in the linker may play key roles in the structural stability of AK. 3.5. Effects of mutations on AK reactivation and refolding kinetics Similar to previous studies [9,10,31,32], when the refolding of GdnHCl denatured AK was initiated by dilution, aggregation appeared immediately (Fig. 5A). All the mutations showed higher absorbance at 400 nm, which suggested that the mutations promoted the formation of off-pathway aggregates during refolding. Consistent with aggregation results, only WT AK and the I121L mutant AK could efficiently reactivate after dilution for 10 min, while other mutants could not (Fig. 5B). To further elucidate the effect of mutations on the refolding of AK, refolding kinetics of WT and mutant AKs was determined. The refolding of the WT AK was best fit by a biphasic process (Fig. 5C). And the rate constant for the fast phase (k1 ) and the slow phase (k2 ) were 18.6 ± 2.1 × 10−3 and 1.7 ± 0.1 × 10−3 s−1 , respectively, which is consistent with those reported in previous studies [31,32]. As for the refolding of most mutants, the data were best fit by a one-stage process, which indicated that the samples might be trapped by off-pathway misfolding and could not refold to their native states. Previous studies implied that the fast phase of AK refolding involved the transition from MG state to the native like intermediate, while the slow phase involved the transition from the native like intermediate state to

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the native state [31,32]. Thus the significant difference in the refolding kinetics might be due to the failure to generate native states from the native like intermediate [9,10,31,32]. To further confirm this deduction, the reactivation kinetics was determined as previously described [27,28,31,32]. As shown in Fig. 5B and Table 1, the reactivation kinetic courses of the substrate reaction of the mutations showed that the mutations I121K, I121G and I121D gradually decreased the reactivation rate of AK. Since the reactivation rate is mainly limited by the slow phase of the refolding, the results in Fig. 5B also suggested that the mutations I121K, I121G and I121D affected the transition from the native like AK to native AK by destroying the correct position of the N-and C- terminal domains. Although the folding pathway of AK has been thoroughly studied [9,10,31,32], as a two-domain protein (Fig. 1A), the refolding of AK not only involves the folding of the two domains, but also the recognition and assembly of the two domains to form the active state. Although the linker was presumed to play a crucial role in the AK activity and structural stability [9,10,31,32], little is known about the roles of the amino acid residues located at the linker in its activity and structural stability. Since the folding of the individual domains is mostly completed during the burst phase of AK refolding, the observed kinetic parameters in Table 2 mainly reflected the reorganization of the two domains. Interestingly, the folding of the linker might be an essential process for AK refolding and reactivation, as the mutations in the linker decreased the rate constants of AK refolding. Combined with the conclusion that the mutations in the linker also affected the properties of the equilibrium unfolding intermediate (Fig. 5), it is safe to deduce that well-folded linker might further help the correct positioning and interactions between the N- and C-terminal domains. The crystal structure of AK indicated that the I121 was close to E353. In the I121 mutant AKs except I121L, the electronic repulsion between these residues might break the hydrophobic interactions between the linker and the C-terminal domain and thus affected the structural stability of AK. The decreased activity of AK can be understood by the fact that the mutations in the linker led to improper positioning of the domains and resulted in looser structure (Fig. 3). Furthermore, the reactivation experiments indicated that none of the mutants I121D, I121L and I121K could successfully recover their activity by dilution- initiated refolding (Fig. 5). The refolding kinetics indicated that the mutations might affect the refolding of AK by decreasing the rate constant of the slow phase. As a result, more off-pathway aggregates could be observed for the mutant AKs (Fig. 5A). All those results suggested that mutants I121K, I121G and I121D impaired the domain–domain interactions. In conclusion, mutations of the residue I121 in the linker led to pronounced loss of activity and structural stability. The mutations also led to serious aggregation and all the mutants could not successfully recover their activities by dilution-initiated refolding, and showed significant decreased rate constant during AK refolding. These results implied that the linker was involved in multidomain proteins folding and structural stability. Considering the linker important roles in AK activity and structural stability, it may be a target to screen specific inhibitor to AKs. Acknowledgements The present investigation was supported by grants from the Natural Science Foundation of China (30971281, 81070447, 81000210 and 81100349) and the National Key Scientific Program of China (2007CB914504). Program for Changjiang Scholar and Innovative Research Team in University (IRT0635)

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