1Department of Biochemistry and Molecular Biology, Life Science Institute, Beijing Normal University, Beijing, 100875, China and 2Department of Biochemistry, Queens University, Kingston, Ontario, K7L 3N6, Canada
3 To whom correspondence should be addressed. e-mail: weiq{at}bnu.edu.cn
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Abstract |
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Keywords: autoinhibition/calcineurin/calmodulin/loop 7/phosphatase activity
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Introduction |
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There are two subunits in CN, subunit A (CN-A, catalytic subunit, 61 kDa in size) and subunit B (CN-B, regulatory subunit,
19 kDa in size) (Frank and Pamela, 2000
). CN-A consists of four domains: a phosphatase domain, a CN-B binding domain, a calmodulin (CaM) binding domain and an autoinhibitory (AI) segment. CN shows no activity in the absence of CN-B and CaM, and the phosphatase activity is enhanced greatly by CN-B and/or CaM. CN-B and CaM regulate the activity of CN through specific association with CN-A and binding to the CaM binding domain, respectively. However, at the molecular and structural level the exact mechanism through which CN-B and CaM affect CN-A and, subsequently, its phosphatase activity, remain to be fully understood.
As revealed by crystal structures (Griffith et al., 1995; Kissinger et al., 1995
), the catalytic center in CN-A is formed by two ß-sheets, sheet 1 and sheet 2, making a ß-sandwich. In this ß-sandwich, there is an important loop (loop 7) between ß 12 (in sheet 1) and ß 13 (in sheet 2). Loop 7 contains approximately eight amino acids, from Y311 to K318. Previously we reported that the preliminary result demonstrated that the loop 7 single deletion mutant (V314) in CN-A exhibited phosphatase activity 10 times greater than that of wild-type CN (Wei and Lee, 1997
; Yan and Wei, 1999
). This was a rather surprising and unexpected observation. In the current study, in addition to V314 deletion we have generated other deletion mutants in loop 7 (single deletion mutants Y315 and N316, double deletion mutant V314Y315) and carried out comprehensive functional assays in an attempt to clarify the role of this loop in the regulation of CN. Compared with wild-type CN-A, all mutants exhibited markedly higher activity. Furthermore, the experimental results revealed that the activity of the two mutants containing the V314 deletion was no longer regulated by CN-B.
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Materials and methods |
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All chemicals were of reagent grade, unless otherwise stated. Single deletion mutants Y315, N316 and double deletion mutant V314Y315 were constructed using the Altered Sites mutagenesis system. The mutated cDNAs were cloned into the expression vector pET-21a and then transformed into Escherichia coli strain HMS174(DE3) cells. The sequences of all the mutated cDNAs were identified by DNA sequencing and confirmed by the expected pattern. Positive recombinant colonies were successfully demonstrated. The wild-type CN-A and all mutants were expressed and purified following the protocol previously established (Yan and Wei, 1999). Briefly, the transformed E.coli cells were cultured in TM media. IPTG (final concentration 16 µM) was added to the culture to induce expression. Four hours later, the cultures were centrifuged at 6000 g for 20 min at 4°C. The pellets were resuspended in buffer A (50 mM TrisHCl, pH 8.0, 1 mM EDTA, 2 mM 2-mercaptoethanol, 0.4 mM PMSF) and then disrupted using ultrasonic waves. The subsequent steps were all done at 4°C. The homogenate was centrifuged at 20 000 g for 30 min; the pellet was discarded and the supernatant was made up to 45% in (NH4)2SO4, stirred for 10 min and incubated on ice for 40 min. The precipitated proteins were collected by centrifugation at 11 000 g for 30 min and resuspended into 2 ml of buffer A, and combined with 10 ml of CaMSepharose resin (synthesized in our laboratory) equilibrated in buffer C (50 mM TrisHCl, pH 7.4, 1 mM EGTA, 1 mM DTT, 0.4 mM PMSF). The mixture was gently stirred for 20 min, and CaCl2 was added to a final concentration of 2 mM according to the total volume of sample plus resin; the mixture was stirred again for 30 min. The resin was transferred to a glass column (2x10 cm), washed with 10 volumes of buffer B (50 mM TrisHCl, pH 7.4, 0.5 mM CaCl2, 2 mM 2-mercaptoethanol, 0.4 mM PMSF). Wild-type CN-A and all mutant proteins were eluted with buffer C at a flow rate of 1 ml/min. The peak A280 fractions were pooled and dialyzed overnight against 50% glycerol. The purities of wild-type CN-A and mutants were assessed using SDS gels.
Assay of phosphatase activity
The activities of wild-type CN-A and deletion mutants were assayed using p-nitrophenyl phosphate (pNPP) as the substrate, with Mn2+ as the activator. The experiment was carried out in assaying buffer (50 mM TrisHCl, pH 7.4, 1 mM MnCl2, 0.5 mM DTT, 0.2 mg/ml BSA and 20 mM pNPP. Reactions were performed in a 0.2 ml volume at 30°C for 20 min and terminated by the addition of 2.0 ml of 0.5 mM sodium carbonate. The absorbance was read at 410 nm. The units of activity were defined as nanomoles of pNPP hydrolyzed per minute. The CaM concentration was 2 µM and the CN-B versus CN-A ratio was 1:1 on a molar basis.
The assays were carried out with a number of variations, depending on the presence and absence of the two regulators, CN-B and CaM. Specifically, the assays were performed under four conditions: the absence of both regulators, the presence of CN-B, the presence of CaM, and finally, the presence of both CN-B and CaM.
Structure analysis
The structure of CN (Kissinger et al., 1995) (PDB code 1AUI) was used for structural analysis, which was carried out using graphics programs Turbo-Frodo (Jones, 1978
) and Sybyl (Tripos, St. Louis, MO, USA) on an SGI Octane graphic workstation.
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Results |
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Single deletion mutants V314, Y315, N316 and double deletion mutant V314Y315 in loop 7 of CN were constructed using the Altered Sites mutagenesis system. The mutated cDNAs were cloned into the expression vector pET-21a and then transformed into E.coli strain HMS174(DE3) cells. The sequences of all the mutated cDNAs were identified by DNA sequencing and confirmed by the expected pattern. Positive recombinant colonies were successfully demonstrated. Wild-type CN-A and all mutants were expressed. As seen in Figure 1A, the deletion mutants were successfully prepared and purified, demonstrated by clear protein bands at the positions corresponding to their estimated molecular masses of 61 kDa. Yields of the proteins were up to 20 mg/l of culture.
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The activity of the two mutants containing the V314 deletion was no longer regulated by CN-B
The results of comprehensive assays, in the absence or presence of CN-B, CaM or both, are listed in Table I. As is evident from the table, under all four conditions all mutants exhibited considerably higher phosphatase activity than wild-type CN-A. The activity of the V314 deletion mutant was the highest among all the mutants tested. Depending on the presence and absence of the regulators, the activity of V314 is 1042 times higher, Y315 is
726 times higher, N316 is
515 times higher and V314Y315 is
3.512.5 times higher than that of wild-type CN-A. Compared with wild-type CN-A, the activity enhancement of the mutants was highest in the absence of both regulators, the next highest was in the presence of CN-B, and the third highest was in the presence of CaM, followed by the condition in which both regulators were present.
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Structure analysis
Loop 7 in CN is located in CN-A but is also close to CN-B. It is sandwiched in the concave area (Figure 2). There is a long linker between the two globular domains, i.e. CN-A on the right and CN-B together with the association segment of CN-A on the left. The linker covers both domains and contains approximately 20 amino acids, N327M347. Residues of loop 7, for instance, D313, make a hydrogen-bonding interaction with H339 (2.96 Å), a residue in the middle of the linker. The CN structure containing part of the AI segment also shows that loop 7 is in direct contact with the AI segment. Among various contacts, for example, Y315 forms a strong hydrogen bond with D477 of AI segment (2.70 Å) (Figure 2). In addition, there are van der Waals contacts between V314 and A473 (3.88 Å), between L312 and D477 (3.14 Å) and aromaticaromatic interactions between Y315 and F470 (2.90 Å). Owing to the incomplete structure of the AI segment in the CN crystal structure determined (
30% missing), there are very likely to be other contacts that we cannot currently appreciate.
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Discussion |
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Loop 7 contains approximately eight amino acids, from Y311 to K318. At the tip of the loop there is a tight turn that is stabilized by many intra-loop interactions. Single deletion would certainly disrupt the conformation of loop 7 and the tight turn structure would be altered. Likewise, site-directed mutagenesis could also have an impact on the conformation of the tight turn, influencing its interactions with neighboring residues.
The regulation of CN is complex and involves multiple factors. Ca2+-dependent CN-B is one of the most important factors. Although the exact structural basis by which Ca2+ binding CN-B regulates the activity of CN remains elusive, the peptide segment connecting the catalytic core and the region where CN-A is in direct association with CN-B is important as it is the only peptide that fully spans the two regions. Because linker residue H339 forms an interaction with loop 7 residue D313, it is plausible that the conformational changes resulting from loop 7 mutation could have an impact on the regulatory effect of CN-B, thereby altering the phosphatase activity of CN and its regulation.
The AI segment is also in close contact with loop 7. There are several hydrogen bonds, and van der Waals and aromaticaromatic interactions. These interactions play a crucial role in stabilizing the AI segment in the binding pocket of CN. Disruption of these contacts would therefore facilitate the removal or release of the AI segment, producing less inhibition or higher activity. It appears that the suppression of CN activity is by, at least, a dual inhibition mechanism, i.e. the effects of the AI segment and the constrained state (inactive state) of the catalytic core regulated by Ca2+-dependent CN-B. However, truncation of the C-terminus of CN-A, which completely removes the entire AI segment, does not lead to significant activation of the free CN-A (Wei and Lee, 1997; Perrino, 1999
). Therefore, it is the latter control mechanism that has a much more profound effect on the whole process. In the light of this possibility, it is then perhaps not surprising that, in some cases where interaction between loop 7 and the linker may be severely impaired (such as mutations involving V314), the regulatory effect of CN-B could no longer be felt by CN-A.
In conclusion, in this work we have shown the functional and regulatory significance of loop 7. Mutations of loop 7 residues show an increased phosphatase activity, which in some cases is no longer regulated by CN-B. Based on the structural analysis, we have proposed possible structure-based mechanisms through which loop 7 may mediate the regulation of CN.
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Acknowledgements |
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References |
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Bonnefoy-Berard,N., Genestier,L., Flacher,M. and Revillard,J.P. (1994) Eur. J. Immunol., 24, 325329.[ISI][Medline]
Frank,R. and Pamela,M. (2000) Physiol. Rev., 80, 14831521.
Griffith,J.P., Kim,J.L., Kim,E.E., Sintchak,M.D., Thomson,J.A., Fitzgibbon,M.J., Fleming,M.A., Caron,P.R., Hsiao,K. and Navia,M.A. (1995) Cell, 82, 507522.[ISI][Medline]
Jones,T.A. (1978) J. Appl. Crystallogr., 11, 268272.[CrossRef][ISI]
Kissinger,C.R., Parge,H.E., Knighton,D.R., Lewis,C.T., Pelletier,L.A., Tempczyk,A., Kalish,V.J., Tucker,K.D., Showalter,R.E. and Moomaw,E.W. (1995) Nature, 378, 641644.[CrossRef][ISI][Medline]
Maho,M.K., Masato,H., Koji,T., Masami,S., Hirotaka,Y., Atsushi,W., Koiti,T. and Yasuo,I. (1995) Neurobiol. Aging, 16, 365380.[CrossRef][ISI][Medline]
Perrino,B.A. (1999) Arch. Biochem. Biophys., 372, 159165.[CrossRef][ISI][Medline]
Perrino,B.A. and Soderling,T.R. (1998) In Van Eldik,L.J. and Watterson,D.M. (eds), Calmodulin and Signal Transduction. Academic Press, New York, pp. 170234.
Thomas,C.F., Keith,M.S., James,R.M., Christopher,M.N. and Ashok,K. (2001) J. Neurosci., 21, 40664073.
Wei,Q. and Lee,E.Y. (1997) Biochemistry, 36, 74187424.[CrossRef][ISI][Medline]
Yan,L.J. and Wei,Q. (1999) Biol. Chem., 380, 12811285.[ISI][Medline]
Zhao,Y., Tozawa,Y., Iseki,R., Mukai,M. and Iwata,M. (1995) J. Immunol., 154, 63466354.
Received April 30, 2003; revised September 9, 2003; accepted September 12, 2003.