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Identification of critical arginine residues in the functioning of Rubisco activase
ABB Archives of Biochemistry and Biophysics 450 (2006) 176–182 www.elsevier.com/locate/yabbi
Identification of critical arginine residues in the functioning of Rubisco activase Cishan Li a, Dafu Wang a, Archie R. Portis Jr. b
a,b,*
a Department of Plant Biology, University of Illinois, 190 ERML, 1201 W Gregory Ave., Urbana IL 61801, USA United States Department of Agriculture, Agricultural Research Service, Photosynthesis Research Unit, 1201 W Gregory Ave., Urbana IL 61801, USA
Received 9 February 2006, and in revised form 30 March 2006 Available online 2 May 2006
Abstract Rubisco activase is a member of the AAA+ family in which arginines located in the Box VII and Sensor 2 domains are a recurrent feature and typically contribute to ATP-binding/hydrolysis or an inter-subunit interface. Replacement of R241 or R244 in Box VII or R294 or R296 in Sensor 2 with alanine in tobacco activase did not greatly alter the binding of ATP or ADP. However, ATP hydrolysis was minimal (R241A and R244A) or greatly diminished (R296A) and none of these mutants were able to activate Rubisco. R241, R244 and R296 were also required for nucleotide-dependent conformational changes detected by intrinsic fluorescence and limited proteolysis. ATP-induced oligomerization, monitored by gel filtration, was not observed with the wild type and mutant tobacco activases in contrast to spinach activase and a R239A mutant (corresponding to R244A in tobacco). Thus, there is not a strict correlation of oligomerization with ATP hydrolysis and intrinsic fluorescence. Published by Elsevier Inc. Keywords: ATP binding/hydrolysis; Arginine finger; AAA+ protein; Conformational change; Oligomerization
Rubisco activase facilitates the activation of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco;1 EC 4.1.1.39) by the removal of sugar phosphate inhibitors from various inactive forms of Rubisco [1–3]. This process requires the hydrolysis of ATP by activase [1,4–6], which is inhibited by the presence of ADP [4,5]. The hydrolysis of ATP by activase is considered to proceed in three steps: first, ATP and Mg2+ bind to activase, which can be detected by fluorescence quenching of 1-anilinonaphthalene-8-sulfonic acid [6] or photoaffinity labeling [7,8]. Then activase undergoes a conformational change which can be detected by an increase in its intrinsic fluorescence [9] and which has been associated with aggregation into high-ordered oligomers, as detected by gel filtration [9]. Finally, ATP hydrolysis occurs [1,9]. *
Corresponding author. Fax: +1 217 244 4419. E-mail address: [email protected] (A.R. Portis Jr.). 1 Abbreviations used: Rubisco, ribulose 1,5-bisphosphate carboxylase/ oxygenase; RuBP, ribulose 1,5-bisphosphate. 0003-9861/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.abb.2006.04.002
Rubisco activase belongs to an AAA+ superfamily whose members share a common two-domain nucleotidebinding fold consisting of a Rossmann fold (a b-sheet flanked by a-helices) and a separate C-terminal, a-helical domain. A nucleotide-binding pocket is formed from residues in the Walker A, Walker B, Sensor 1, Box VII and Sensor 2 domains [10–12]. The involvement of residues in the Walker A, Walker B and Sensor 1 domains has been demonstrated by either site-directed mutagenesis [13–15] or ATP analog photoaffinity labeling [7,8]. Critical roles for residues in the other two domains, Box VII and Sensor 2, have not yet been demonstrated with activase although highly conserved arginines in these domains in other AAA+ proteins were reported to play important roles in ATP binding/hydrolysis [11]. Structural studies of several AAA+ proteins indicate that conserved arginines in Box VII, upon oligomerization, come close to the c-phosphate of ATP from an adjacent subunit and facilitate the hydrolysis of ATP in a manner resembling the ‘‘arginine finger’’ in GTPase [16], while an arginine in Sensor 2 may mediate
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Fig. 1. (A) Alignment of tobacco activase with E. coli RuvB and E. coli FtsH to highlight some conserved subdomains in the AAA domains related to nucleotide binding and hydrolysis. Critical regions in the domains are colored pink and conserved arginine residues are colored blue. (B) Predicted 3D structure of the activase AAA+ domains [25] using Modbase to highlight the ATP binding pocket (http://alto.compbio.ucsf.edu/modbase-cgi/index.cgi). ADP is colored red and Walker A, Walker B, Sensor 1, Box VII and Sensor 2 are colored orange, yellow, light green, dark green, and blue respectively. The numbered residues in (A) are labeled. For clarity, some regions surrounding the core b-sheets are omitted.
a conformational change of the C-terminal a-helix bundle in response to the presence of ATP [11]. Similar to its role in several AAA+ proteins, Sensor 2 has been shown to be a substrate recognition domain in activase and a conformational change of this domain has been suggested to be important in Rubisco activation [17]. Comparisons of the domains that contribute residues to the ATP-binding pocket in tobacco activase and two representatives of AAA+ proteins, E. coli RuvB and E. coli FtsH, which have 21% sequence identity and 53% to 55% similarity within the AAA module to tobacco activase, indicate that activase has corresponding arginine residues at several positions (Fig. 1). In this report, we examined the effects of mutating the three matching residues (R241, R244, and R296) to alanine, along with two more arginines (R181 and R294), on several properties of activase. R181 is near an arginine residue in FtsH that is critical for ATP hydrolysis [18] and R294 was of interest because of its close proximity to R296.
Materials and methods Materials Wild type Rubiscos from spinach/tobacco were isolated as previously reported [19]. Site-directed mutagenesis primers were obtained from Integrated DNA Technologies.2 A QuikChange site-directed mutagenesis kit was purchased from Stratagene. Chymotrypsin was purchased from Sigma–Aldrich. Trypsin was purchased from Promega. ATP-c-S was purchased from Boehringer–Mannheim. All other reagents were of the highest purity readily available.
2 Mention of a trademark, proprietary product or vendor does not constitute a guarantee or warranty of the product by the US Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.
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Site-directed mutagenesis, protein expression, and purification The desired activase mutations were created according to protocols provided in the QuikChange kit. Six single mutants were generated from the wild type tobacco activase (WT) construct: K116A, R181A, R241A, R244A, R294A, and R296A. A mutant in spinach activase, R239A corresponding to R244A in tobacco, was generated from the spinach activase small isoform (SA). All mutations were confirmed by DNA sequencing. All verified constructions/mutations were introduced into BL21 (DE3) cells (Novagen). The procedures for expression and purification were the same as previously reported [19,20]. ATP hydrolysis and Rubisco activation assays ATP hydrolysis activity of activase (50 lg/ml) was assayed spectrophotometrically at 25 C by coupling ADP production to NADH oxidation as reported previously [19]. Rubisco activation by activase was also assayed spectrophotometrically as previously reported [21] using 5 lg/ml inactive Rubisco-RuBP (ER) complex and 80 lg/ ml activase. Fluorescence nucleotide binding assays and intrinsic fluorescence
20 mM KCl, 0.2 mM EDTA, and 0.2 mM ATP (or 0.1 mM ADP). The column was run with an FPLC system at 4 C with a flow rate of 0.25 ml/min. Results Enzymatic activities All mutants were successfully expressed and purified, but only R181A retained an ATP hydrolysis activity comparable to wild type (Fig. 2). R241A and R244A (in Box VII domain) exhibited essentially no ATP hydrolysis activity which indicated that they have a direct role in ATP hydrolysis. Both R294A and R296A (in Sensor 2 domain) exhibited low ATP hydrolysis activity, similar to a K247R mutant previously reported [24]. Thus the Sensor 2 arginines play important but not critical roles in ATP hydrolysis. Rubisco activation by the arginine mutants was also examined (Fig. 3). R241A and R244A were not assayed, because it is has been repeatedly found that significant ATP hydrolysis activity is required for measurable rates of Rubisco activation [1,14,21,24]. Rubisco activation by R181A was similar to the recombinant wild type, whereas both R294A and R296A were unable to effectively activate Rubisco. Nucleotide binding
Changes in the fluorescence of 1-anilinonaphthalene-8sulfonic acid after the addition of ADP or ATP were determined to estimate nucleotide affinity as previously described [6,15]. Apparent dissociation constants (Kd) and goodness of fit were calculated as previously described [6]. Intrinsic fluorescence measurements with activase (40 lg/ml) were performed as described previously [9,22] with the exception that all measurements were conducted at room temperature.
The ability of the mutants to bind ADP or ATP was assayed by changes in 1-anilinonaphthalene-8-sulfonic acid fluorescence [6] to determine if the absence of nucleotide binding was responsible for the loss or reduction in the ATP hydrolysis and Rubisco activation activities. A K116A mutant in the Walker A domain was used as a negative control, because this mutation in spinach activase
Limited proteolysis to detect nucleotide-induced conformational changes The recombinant wild type tobacco activase and arginine mutants (1 mg/mL) were first incubated with either 5 mM ADP or 5 mM ATP-c-S and 4 mM MgCl2 in a buffer containing 50 mM Tricine (pH 8.0) and 20 mM KCl at room temperature for four minutes. Then chymotrypsin (Sigma–Aldrich) was added at a protease:activase ratio of 1:100 (w/w). Samples were taken at the indicated time intervals, boiled with 2· SDS loading buffer, and analyzed by 15% Tris–Tricine SDS–polyacrylamide gel electrophoresis [23]. Molecular mass determination Recombinant wild type and mutant activases (200 lg in 100 ll) were loaded onto a Superose 6 10/300 GL column equilibrated with 20 mM Bis–tris–propane (BTP) (pH 8.0),
Fig. 2. ATP hydrolysis activities of the recombinant wild type tobacco activase (WT) and the R181A, R241A, R244A, R294A, and R296A mutants. The assays were performed with 50 lg/ml activase. The results are an average of three replicates.
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Fig. 3. Rubisco activation by recombinant wild type tobacco activase (WT) and the R181A, R294A, and R296A mutants. The assays were performed with 5 lg/ml inactive Rubisco-RuBP complex and 80 lg/ml activase. ER represents the activation of the Rubisco–RuBP complex without activase.
exhibited greatly diminished ATP binding as measured with this method [14]. All of the arginine mutants exhibited nucleotide binding affinities comparable to the recombinant wild type (Table 1). Conformational changes indicated by intrinsic fluorescence and resistance to limited proteolysis The intrinsic fluorescence of activase increases after the addition of ATP and Mg2+, and declines as ATP is hydrolyzed [9,25]. The increase reflects an increased hydrophobic environment around tryptophan 109 and 250 [26] and has been attributed to an ATP-induced oligomerization of activase [9,27] because the increase is dependent on activase concentration. In previous studies, activase mutants with minimal ATP hydrolysis activity have exhibited no changes in intrinsic fluorescence [9,15]. Examination of intrinsic fluorescence with the arginine mutants revealed that, unlike wild type and R181A, the fluorescence of R241A, R244A, and R296A remained relatively unchanged upon addition
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of ATP (Fig. 4). Surprisingly, R294A showed a larger increase in fluorescence than the wild type, a phenomenon never observed previously, and the fluorescence declined at a slow rate. The slow quenching is probably due to the low ATP hydrolysis activity and thus is similar to the slow quenching observed with the tobacco D174E mutant [15]. To determine whether the low ATP hydrolysis activity and increased intrinsic fluorescence of the R294A mutant were due to a marked change in the concentration dependence of these parameters, we compared the response of this mutant and the recombinant wild type (Supplemental data). The results indicated that the ATP hydrolysis activity and increased intrinsic fluorescence of R294A saturated at activase concentrations similar to those of wild type. Binding of ATP (or its non-hydrolysable analog, ATPc-S) preserves the enzymatic activity of activase [5] and this protection is also considered to be a consequence of increased oligomerization because the ATP hydrolysis activity is dependent on activase concentration [9,27]. The susceptibility of the wild type and arginine mutants to limited proteolysis by chymotrypsin in the presence of either ADP or ATP-c-S (Fig. 5) was examined as a means to detect conformational changes. R294A and the recombinant wild type were more resistant to chymotrypsin when ATP-c-S was present. Fragments slightly smaller than the undigested protein were more abundant after both 5 and 20 min of digestion (Fig. 5). In contrast, when the R241A, R244A, and R296A mutants were exposed to chymotrysin, essentially no differences were observed in the presence of ATP-c-S versus ADP. Thus, the binding of ATP-c-S offered no protection for these mutants. Similar results were observed when limited proteolysis by trypsin was performed (data not shown).
Table 1 Apparent dissociation constants for ATP and ADP of wild type tobacco activase (TA), K116A, and the various arginine replacement mutants
TA K116A R181A R241A R244A R294A R296A
ADP (lM)
ATP (lM)
0.61 (0.01) 260 (15) 0.37 (0.01) 0.82 (0.04) 0.82 (0.03) 1.29 (0.04) 1.07 (0.16)
4.3 (0.2) 540 (ND) 2.9 (0.3) 5.7 (0.4) 5.4 (1.0) 2.6 (0.3) 4.4 (0.3)
Apparent dissociation constants were determined by the 1-anilinonaphthalene-8-sulfonic acid fluorescence method [6]. K116A, a mutant defective in nucleotide binding, was used as a control. Values in the parenthesis are standard errors of three replicates. ND, not determined.
Fig. 4. Time courses of the intrinsic fluorescence changes in recombinant wild type tobacco activase (WT), a K116A mutant and the R181A, R241A, R244A, R294A, and R296A mutants after addition of ATP. The assays were conducted with 40 lg/ml protein and after a short equilibration in the assay buffer, 0.5 mM ATP was added at 0 s.
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activase. The recombinant 43 kDa isoform of spinach activase was examined to eliminate the possible contribution of the larger 45 kDa isoform, which was also present in the native protein that was previously examined [9]. ATP did increase the apparent mass of the 43 kDa spinach isoform to 400 kDa from 200 kDa in the presence of ADP (Fig. 6C and D), consistent with the previous observations. However to clarify whether the increase in the apparent mass of activase is coupled to the increase in its intrinsic fluorescence and its ATP hydrolysis activity as was previously suggested [9], we examined a spinach 43 kDa R239A mutant which is homologous to the R244A replacement in tobacco activase. Similar to the tobacco R244A mutant, this mutant had minimal ATP hydrolysis activity (data not shown) and no increase in its intrinsic fluorescence after addition of ATP (Fig. 7). However, when its molecular mass was determined, the spinach R239A mutant exhibited an increased size in the presence of ATP, similar to the wild type 43 kDa spinach activase (Fig. 6C and D). Thus the ATP-induced size increase observed with gel filtration chromatography can be species and mutant specific and clearly does not necessarily correlate with ATP hydrolysis activity or the conformational changes monitored by intrinsic fluorescence changes. Discussion
Fig. 5. Limited proteolysis of recombinant wild type tobacco activase and arginine mutants with chymotrypsin. (A) Wild type, R241A and R244A; (B) Wild type, R294A and R296A. Samples (10 lg protein) were analyzed by gel electrophoresis after incubation with chymotrypsin for 5 and 20 min in the presence of 5 mM ATP-c-S or ADP as indicated. For other experimental details, see Materials and methods. Standard lane contains the size standards and control is untreated activase.
Molecular mass determination Like most AAA+ proteins, Rubisco activase usually exists in oligomeric forms. With spinach activase, the binding of ATP promoted aggregation into higher-order oligomers as detected by FPLC gel filtration chromatography [9]. Therefore, this method appeared to be ideal to examine the effects of ATP versus ADP on the size of the recombinant tobacco wild type and mutant activases. However, surprisingly and unlike previously reported for spinach activase [9], the apparent molecular masses of recombinant wild type tobacco activase, R244A and R296A were similar in the presence of ATP versus ADP (Fig. 6A and B). The apparent molecular masses of R241A and R294A were significantly smaller than the wild type in the presence of ADP and they increased slightly in the presence of ATP. The inability of ATP to dramatically increase the apparent molecular mass of the recombinant wild type tobacco activase prompted a reexamination of the behavior of spinach
The roles of several conserved arginines in Rubisco activase have been investigated in this research. Comparisons of the roles of these arginines with that in other AAA+ proteins yield some interesting insights. In the AAA subfamily, a conserved arginine in Walker B, near R181 in activase, has been suggested to form a salt bridge with an aspartate in Box VII which stabilizes oligomeric formation and facilitates ATP hydrolysis [18]. The absence of this residue in activase and the minor effects of replacing the nearby R181 clearly distinguish activase from proteins in this subfamily. R244 (Box VII) in activase is a highly conserved residue in the AAA+ superfamily. In almost all cases examined, it has been found to be involved in ATP hydrolysis and in HslU contributes to the stabilization of hexamerization [11,28]. Structural analyses of several AAA+ proteins indicate that this arginine is close to the c-phosphate of ATP bound to an adjacent subunit, which has prompted the idea that this arginine might function as an ‘‘arginine finger,’’ similar to the ‘‘arginine finger’’ in GTPase [12,16]. In activase, replacement of R244 with alanine abolished ATP hydrolysis activity as well as the ATP-induced conformational changes detected by intrinsic fluorescence and limited proteolysis. The second arginine in this region, corresponding to R241 in activase, is often present in the AAA subfamily and a fraction of Clp/Hsp100 subfamily and it has been suggested to be another ‘‘arginine finger’’ [11]. Mutation of this residue in FtsH causes a loss of ATP hydrolysis activity while mutation in NSF has no effect on ATP hydrolysis activity, but affects its SNARE
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Fig. 6. Size determination of wild type (WT) and mutant (R241A, R244A, R294A, and R296A) tobacco activases (A and B) and spinach wild type (SA) and mutant (R239A) activases (C and D) by gel filtration in the presence of 0.1 mM ADP (A and C) or 0.2 mM ATP (B and D) at 4 C. For other experimental details, see Materials and methods. The elution volumes of the protein standards are indicated by the filled squares. Protein standards were cytochrome C (12,400), bovine serum albumin (67,000), b-amylase (200,000), apoferritin (443,000) and thyroglobulin (667,000).
Fig. 7. Time course of the intrinsic fluorescence change with wild type spinach activase (SA) and R239A spinach activase (R239A SA) after addition of ATP-c-S. The assays were conducted with 40 lg/ml protein and after 60 s incubation at room temperature, 0.5 mM ATP-c-S was added as indicated.
assembly [16,29]. Except for gel filtration, replacement of R241 in activase resulted in similar effects as replacement of R244. In a predicted 3D structure of the activase AAA+ domains (Fig. 1B), both of these arginines are located distant from the ATP binding site, which makes it impossible for them to interact with the ATP bound by their own subunit. Thus our data are consistent with the
hypothesis that these two arginines might also interact with ATP from an adjacent subunit as ‘‘arginine fingers.’’ It has been suggested [11] that the arginine corresponding to R241 in tobacco activase is mutually exclusive with the conserved arginine in Sensor 2 (R296 in tobacco activase), but both arginines are present in activase and important for ATP hydrolysis and ATP-induced conformational changes. R296 in the Sensor 2 domain of activase also may project into the nucleotide binding site and thereby mediate conformational changes associated with nucleotide binding or hydrolysis. In the predicted structure (Fig. 1B) R296 is not in a position to project into the nucleotide binding domain. However, the predicted structure may be inaccurate in this area because in contrast to the conserved Rossman fold domain, the arrangement of the helices within the C-terminal, a-helical domain varies in the known structures of AAA+ proteins. Our results indicate that R296 is critical for ATP hydrolysis, Rubisco activation and conformational changes associated with proteolytic protection and intrinsic fluorescence changes. Thus R296 in activase may function in a manner similar to the suggested role of this arginine in mediating conformation changes in the Sensor 2 domain in response to ATP [12]. Replacement of the adjacent R294 residue resulted in retention of the nucleotide-dependent proteolytic protection and some ATP hydrolysis activity, enhanced the ATP-induced intrinsic fluorescence change, but almost eliminated Rubisco activation activity. Thus this residue appears to be playing a more subtle role in maintaining the proper interactions necessary to activate Rubisco.
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An ATP-induced aggregation in Rubisco activase, observed in gel filtration experiments with spinach activase, has been suggested to closely correlate with the ATP-induced increase in intrinsic fluorescence and ATP hydrolysis [9,27]. However, we found that recombinant wild type tobacco activase (and two of the arginine mutants) did not exhibit an ATP-induced increase in apparent molecular mass monitored by gel filtration. Furthermore, spinach R239A, a mutant without ATP hydrolysis activity as well as the ATP-induced intrinsic fluorescence increase, exhibited an ATP-induced increase in molecular mass which was similar to the wild type 43 kDa spinach activase. The contrasting results with tobacco, spinach and the R239A spinach mutant suggest that the intrinsic fluorescence increase induced by ATP may not require increased aggregation, but perhaps only reflects reorientations between subunits in an oligomeric complex. However, the possibility remains that higher-order oligomers transiently form during ATP binding/hydrolysis which may not be detected by current methods. Recently, Salvucci [30] has demonstrated that a carboxyl-terminal cross-linked activase dimer retained its enzymatic function, which indicates that complete dissociation to monomers might not be required for activase to fulfill its function. In any case, the increased aggregation of spinach activase observed by gel filtration analysis appears to be species specific and its significance for activase function with Rubisco remains unclear. Acknowledgment This work was supported in part by a grant from the US Department of Energy (97ER20268). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.abb.2006. 04.002. References [1] V.J. Streusand, A.R. Portis Jr., Plant Physiol. 85 (1987) 152–154.
[2] M.E. Salvucci, A.R. Portis Jr., W.L. Ogren, Photosyn. Res. 7 (1985) 193–201. [3] Z.Y. Wang, A.R. Portis Jr., Plant Physiol. 99 (1992) 1348–1353. [4] S.P. Robinson, V.J. Streusand, J.M. Chatfield, A.R. Portis Jr., Plant Physiol. 88 (1988) 1008–1014. [5] S.P. Robinson, A.R. Portis Jr., Arch. Biochem. Biophys. 268 (1989) 93–99. [6] Z.Y.Wang,A.R.PortisJr.,Biochim.Biophys.Acta1079(1991)263–267. [7] M.E. Salvucci, A.J. Chavan, R.R. Klein, K. Rajagopolan, B.E. Haley, Biochemistry 33 (1994) 14879–14886. [8] M.E. Salvucci, K. Rajagopalan, G. Sievert, B.E. Haley, D.S. Watt, J. Biol. Chem. 268 (1993) 14239–14244. [9] Z.Y. Wang, R.T. Ramage, A.R. Portis Jr., Biochim. Biophys. Acta 1202 (1993) 47–55. [10] A.F. Neuwald, L. Aravind, J.L. Spouge, E.V. Koonin, Genome Res. 9 (1999) 27–43. [11] T. Ogura, S.W. Whiteheart, A.J. Wilkinson, J. Struct. Biol. 146 (2004) 106–112. [12] T. Ogura, A.J. Wilkinson, Genes Cells 6 (2001) 575–597. [13] J.B. Shen, W.L. Ogren, Plant Physiol. 99 (1992) 1201–1207. [14] J.B. Shen, E.M. Orozco, W.L. Ogren, J. Biol. Chem. 266 (1991) 8963– 8968. [15] F.J. van de Loo, M.E. Salvucci, Biochemistry 37 (1998) 4621– 4625. [16] K. Karata, T. Inagawa, A.J. Wilkinson, T. Tatsuta, T. Ogura, J. Biol. Chem. 274 (1999) 26225–26232. [17] C. Li, M.E. Salvucci, A.R. Portis Jr., J. Biol. Chem. 280 (2005) 24864–24869. [18] K. Karata, C.S. Verma, A.J. Wilkinson, T. Ogura, Mol. Microbiol. 39 (2001) 890–903. [19] Z.Y. Wang, G.W. Synder, B.D. Esau, A.R. Portis Jr., W.L. Ogren, Plant Physiol. 100 (1992) 1858–1862. [20] N. Zhang, A.R. Portis Jr., Proc. Natl. Acad. Sci. USA 96 (1999) 9438–9443. [21] B. Esau, G.W. Snyder, A.R. Portis Jr., Arch. Biochem. Biophys. 326 (1996) 100–105. [22] F.J. van de Loo, M.E. Salvucci, Biochemistry 35 (1996) 8143–8148. [23] H. Schagger, G. von Jagow, Anal. Biochem. 166 (1987) 368–379. [24] M.E. Salvucci, R.R. Klein, Arch. Biochem. Biophys. 314 (1994) 178–185. [25] A.R. Portis Jr., Photosyn. Res. 75 (2003) 11–27. [26] D. Wang, A.R. Portis, Jr., Photosyn. Res. (in press), doi:10.1007/ s1120-006-9051-2. [27] R.M. Lilley, A.R. Portis Jr., Plant Physiol. 114 (1997) 605–613. [28] H.K. Song, C. Hartmann, R. Ramachandran, M. Bochtler, R. Behrendt, L. Moroder, R. Huber, Proc. Natl. Acad. Sci. USA 97 (2000) 14103–14108. [29] E.A. Matveeva, A.P. May, P. He, S.W. Whiteheart, Biochemistry 41 (2002) 530–536. [30] M.E. Salvucci, FEBS Lett. 560 (2004) 205–209.
Identification of critical arginine residues in the functioning of Rubisco activase Cishan Li a, Dafu Wang a, Archie R. Portis Jr. b
a,b,*
a Department of Plant Biology, University of Illinois, 190 ERML, 1201 W Gregory Ave., Urbana IL 61801, USA United States Department of Agriculture, Agricultural Research Service, Photosynthesis Research Unit, 1201 W Gregory Ave., Urbana IL 61801, USA
Received 9 February 2006, and in revised form 30 March 2006 Available online 2 May 2006
Abstract Rubisco activase is a member of the AAA+ family in which arginines located in the Box VII and Sensor 2 domains are a recurrent feature and typically contribute to ATP-binding/hydrolysis or an inter-subunit interface. Replacement of R241 or R244 in Box VII or R294 or R296 in Sensor 2 with alanine in tobacco activase did not greatly alter the binding of ATP or ADP. However, ATP hydrolysis was minimal (R241A and R244A) or greatly diminished (R296A) and none of these mutants were able to activate Rubisco. R241, R244 and R296 were also required for nucleotide-dependent conformational changes detected by intrinsic fluorescence and limited proteolysis. ATP-induced oligomerization, monitored by gel filtration, was not observed with the wild type and mutant tobacco activases in contrast to spinach activase and a R239A mutant (corresponding to R244A in tobacco). Thus, there is not a strict correlation of oligomerization with ATP hydrolysis and intrinsic fluorescence. Published by Elsevier Inc. Keywords: ATP binding/hydrolysis; Arginine finger; AAA+ protein; Conformational change; Oligomerization
Rubisco activase facilitates the activation of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco;1 EC 4.1.1.39) by the removal of sugar phosphate inhibitors from various inactive forms of Rubisco [1–3]. This process requires the hydrolysis of ATP by activase [1,4–6], which is inhibited by the presence of ADP [4,5]. The hydrolysis of ATP by activase is considered to proceed in three steps: first, ATP and Mg2+ bind to activase, which can be detected by fluorescence quenching of 1-anilinonaphthalene-8-sulfonic acid [6] or photoaffinity labeling [7,8]. Then activase undergoes a conformational change which can be detected by an increase in its intrinsic fluorescence [9] and which has been associated with aggregation into high-ordered oligomers, as detected by gel filtration [9]. Finally, ATP hydrolysis occurs [1,9]. *
Corresponding author. Fax: +1 217 244 4419. E-mail address: [email protected] (A.R. Portis Jr.). 1 Abbreviations used: Rubisco, ribulose 1,5-bisphosphate carboxylase/ oxygenase; RuBP, ribulose 1,5-bisphosphate. 0003-9861/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.abb.2006.04.002
Rubisco activase belongs to an AAA+ superfamily whose members share a common two-domain nucleotidebinding fold consisting of a Rossmann fold (a b-sheet flanked by a-helices) and a separate C-terminal, a-helical domain. A nucleotide-binding pocket is formed from residues in the Walker A, Walker B, Sensor 1, Box VII and Sensor 2 domains [10–12]. The involvement of residues in the Walker A, Walker B and Sensor 1 domains has been demonstrated by either site-directed mutagenesis [13–15] or ATP analog photoaffinity labeling [7,8]. Critical roles for residues in the other two domains, Box VII and Sensor 2, have not yet been demonstrated with activase although highly conserved arginines in these domains in other AAA+ proteins were reported to play important roles in ATP binding/hydrolysis [11]. Structural studies of several AAA+ proteins indicate that conserved arginines in Box VII, upon oligomerization, come close to the c-phosphate of ATP from an adjacent subunit and facilitate the hydrolysis of ATP in a manner resembling the ‘‘arginine finger’’ in GTPase [16], while an arginine in Sensor 2 may mediate
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Fig. 1. (A) Alignment of tobacco activase with E. coli RuvB and E. coli FtsH to highlight some conserved subdomains in the AAA domains related to nucleotide binding and hydrolysis. Critical regions in the domains are colored pink and conserved arginine residues are colored blue. (B) Predicted 3D structure of the activase AAA+ domains [25] using Modbase to highlight the ATP binding pocket (http://alto.compbio.ucsf.edu/modbase-cgi/index.cgi). ADP is colored red and Walker A, Walker B, Sensor 1, Box VII and Sensor 2 are colored orange, yellow, light green, dark green, and blue respectively. The numbered residues in (A) are labeled. For clarity, some regions surrounding the core b-sheets are omitted.
a conformational change of the C-terminal a-helix bundle in response to the presence of ATP [11]. Similar to its role in several AAA+ proteins, Sensor 2 has been shown to be a substrate recognition domain in activase and a conformational change of this domain has been suggested to be important in Rubisco activation [17]. Comparisons of the domains that contribute residues to the ATP-binding pocket in tobacco activase and two representatives of AAA+ proteins, E. coli RuvB and E. coli FtsH, which have 21% sequence identity and 53% to 55% similarity within the AAA module to tobacco activase, indicate that activase has corresponding arginine residues at several positions (Fig. 1). In this report, we examined the effects of mutating the three matching residues (R241, R244, and R296) to alanine, along with two more arginines (R181 and R294), on several properties of activase. R181 is near an arginine residue in FtsH that is critical for ATP hydrolysis [18] and R294 was of interest because of its close proximity to R296.
Materials and methods Materials Wild type Rubiscos from spinach/tobacco were isolated as previously reported [19]. Site-directed mutagenesis primers were obtained from Integrated DNA Technologies.2 A QuikChange site-directed mutagenesis kit was purchased from Stratagene. Chymotrypsin was purchased from Sigma–Aldrich. Trypsin was purchased from Promega. ATP-c-S was purchased from Boehringer–Mannheim. All other reagents were of the highest purity readily available.
2 Mention of a trademark, proprietary product or vendor does not constitute a guarantee or warranty of the product by the US Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.
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Site-directed mutagenesis, protein expression, and purification The desired activase mutations were created according to protocols provided in the QuikChange kit. Six single mutants were generated from the wild type tobacco activase (WT) construct: K116A, R181A, R241A, R244A, R294A, and R296A. A mutant in spinach activase, R239A corresponding to R244A in tobacco, was generated from the spinach activase small isoform (SA). All mutations were confirmed by DNA sequencing. All verified constructions/mutations were introduced into BL21 (DE3) cells (Novagen). The procedures for expression and purification were the same as previously reported [19,20]. ATP hydrolysis and Rubisco activation assays ATP hydrolysis activity of activase (50 lg/ml) was assayed spectrophotometrically at 25 C by coupling ADP production to NADH oxidation as reported previously [19]. Rubisco activation by activase was also assayed spectrophotometrically as previously reported [21] using 5 lg/ml inactive Rubisco-RuBP (ER) complex and 80 lg/ ml activase. Fluorescence nucleotide binding assays and intrinsic fluorescence
20 mM KCl, 0.2 mM EDTA, and 0.2 mM ATP (or 0.1 mM ADP). The column was run with an FPLC system at 4 C with a flow rate of 0.25 ml/min. Results Enzymatic activities All mutants were successfully expressed and purified, but only R181A retained an ATP hydrolysis activity comparable to wild type (Fig. 2). R241A and R244A (in Box VII domain) exhibited essentially no ATP hydrolysis activity which indicated that they have a direct role in ATP hydrolysis. Both R294A and R296A (in Sensor 2 domain) exhibited low ATP hydrolysis activity, similar to a K247R mutant previously reported [24]. Thus the Sensor 2 arginines play important but not critical roles in ATP hydrolysis. Rubisco activation by the arginine mutants was also examined (Fig. 3). R241A and R244A were not assayed, because it is has been repeatedly found that significant ATP hydrolysis activity is required for measurable rates of Rubisco activation [1,14,21,24]. Rubisco activation by R181A was similar to the recombinant wild type, whereas both R294A and R296A were unable to effectively activate Rubisco. Nucleotide binding
Changes in the fluorescence of 1-anilinonaphthalene-8sulfonic acid after the addition of ADP or ATP were determined to estimate nucleotide affinity as previously described [6,15]. Apparent dissociation constants (Kd) and goodness of fit were calculated as previously described [6]. Intrinsic fluorescence measurements with activase (40 lg/ml) were performed as described previously [9,22] with the exception that all measurements were conducted at room temperature.
The ability of the mutants to bind ADP or ATP was assayed by changes in 1-anilinonaphthalene-8-sulfonic acid fluorescence [6] to determine if the absence of nucleotide binding was responsible for the loss or reduction in the ATP hydrolysis and Rubisco activation activities. A K116A mutant in the Walker A domain was used as a negative control, because this mutation in spinach activase
Limited proteolysis to detect nucleotide-induced conformational changes The recombinant wild type tobacco activase and arginine mutants (1 mg/mL) were first incubated with either 5 mM ADP or 5 mM ATP-c-S and 4 mM MgCl2 in a buffer containing 50 mM Tricine (pH 8.0) and 20 mM KCl at room temperature for four minutes. Then chymotrypsin (Sigma–Aldrich) was added at a protease:activase ratio of 1:100 (w/w). Samples were taken at the indicated time intervals, boiled with 2· SDS loading buffer, and analyzed by 15% Tris–Tricine SDS–polyacrylamide gel electrophoresis [23]. Molecular mass determination Recombinant wild type and mutant activases (200 lg in 100 ll) were loaded onto a Superose 6 10/300 GL column equilibrated with 20 mM Bis–tris–propane (BTP) (pH 8.0),
Fig. 2. ATP hydrolysis activities of the recombinant wild type tobacco activase (WT) and the R181A, R241A, R244A, R294A, and R296A mutants. The assays were performed with 50 lg/ml activase. The results are an average of three replicates.
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Fig. 3. Rubisco activation by recombinant wild type tobacco activase (WT) and the R181A, R294A, and R296A mutants. The assays were performed with 5 lg/ml inactive Rubisco-RuBP complex and 80 lg/ml activase. ER represents the activation of the Rubisco–RuBP complex without activase.
exhibited greatly diminished ATP binding as measured with this method [14]. All of the arginine mutants exhibited nucleotide binding affinities comparable to the recombinant wild type (Table 1). Conformational changes indicated by intrinsic fluorescence and resistance to limited proteolysis The intrinsic fluorescence of activase increases after the addition of ATP and Mg2+, and declines as ATP is hydrolyzed [9,25]. The increase reflects an increased hydrophobic environment around tryptophan 109 and 250 [26] and has been attributed to an ATP-induced oligomerization of activase [9,27] because the increase is dependent on activase concentration. In previous studies, activase mutants with minimal ATP hydrolysis activity have exhibited no changes in intrinsic fluorescence [9,15]. Examination of intrinsic fluorescence with the arginine mutants revealed that, unlike wild type and R181A, the fluorescence of R241A, R244A, and R296A remained relatively unchanged upon addition
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of ATP (Fig. 4). Surprisingly, R294A showed a larger increase in fluorescence than the wild type, a phenomenon never observed previously, and the fluorescence declined at a slow rate. The slow quenching is probably due to the low ATP hydrolysis activity and thus is similar to the slow quenching observed with the tobacco D174E mutant [15]. To determine whether the low ATP hydrolysis activity and increased intrinsic fluorescence of the R294A mutant were due to a marked change in the concentration dependence of these parameters, we compared the response of this mutant and the recombinant wild type (Supplemental data). The results indicated that the ATP hydrolysis activity and increased intrinsic fluorescence of R294A saturated at activase concentrations similar to those of wild type. Binding of ATP (or its non-hydrolysable analog, ATPc-S) preserves the enzymatic activity of activase [5] and this protection is also considered to be a consequence of increased oligomerization because the ATP hydrolysis activity is dependent on activase concentration [9,27]. The susceptibility of the wild type and arginine mutants to limited proteolysis by chymotrypsin in the presence of either ADP or ATP-c-S (Fig. 5) was examined as a means to detect conformational changes. R294A and the recombinant wild type were more resistant to chymotrypsin when ATP-c-S was present. Fragments slightly smaller than the undigested protein were more abundant after both 5 and 20 min of digestion (Fig. 5). In contrast, when the R241A, R244A, and R296A mutants were exposed to chymotrysin, essentially no differences were observed in the presence of ATP-c-S versus ADP. Thus, the binding of ATP-c-S offered no protection for these mutants. Similar results were observed when limited proteolysis by trypsin was performed (data not shown).
Table 1 Apparent dissociation constants for ATP and ADP of wild type tobacco activase (TA), K116A, and the various arginine replacement mutants
TA K116A R181A R241A R244A R294A R296A
ADP (lM)
ATP (lM)
0.61 (0.01) 260 (15) 0.37 (0.01) 0.82 (0.04) 0.82 (0.03) 1.29 (0.04) 1.07 (0.16)
4.3 (0.2) 540 (ND) 2.9 (0.3) 5.7 (0.4) 5.4 (1.0) 2.6 (0.3) 4.4 (0.3)
Apparent dissociation constants were determined by the 1-anilinonaphthalene-8-sulfonic acid fluorescence method [6]. K116A, a mutant defective in nucleotide binding, was used as a control. Values in the parenthesis are standard errors of three replicates. ND, not determined.
Fig. 4. Time courses of the intrinsic fluorescence changes in recombinant wild type tobacco activase (WT), a K116A mutant and the R181A, R241A, R244A, R294A, and R296A mutants after addition of ATP. The assays were conducted with 40 lg/ml protein and after a short equilibration in the assay buffer, 0.5 mM ATP was added at 0 s.
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activase. The recombinant 43 kDa isoform of spinach activase was examined to eliminate the possible contribution of the larger 45 kDa isoform, which was also present in the native protein that was previously examined [9]. ATP did increase the apparent mass of the 43 kDa spinach isoform to 400 kDa from 200 kDa in the presence of ADP (Fig. 6C and D), consistent with the previous observations. However to clarify whether the increase in the apparent mass of activase is coupled to the increase in its intrinsic fluorescence and its ATP hydrolysis activity as was previously suggested [9], we examined a spinach 43 kDa R239A mutant which is homologous to the R244A replacement in tobacco activase. Similar to the tobacco R244A mutant, this mutant had minimal ATP hydrolysis activity (data not shown) and no increase in its intrinsic fluorescence after addition of ATP (Fig. 7). However, when its molecular mass was determined, the spinach R239A mutant exhibited an increased size in the presence of ATP, similar to the wild type 43 kDa spinach activase (Fig. 6C and D). Thus the ATP-induced size increase observed with gel filtration chromatography can be species and mutant specific and clearly does not necessarily correlate with ATP hydrolysis activity or the conformational changes monitored by intrinsic fluorescence changes. Discussion
Fig. 5. Limited proteolysis of recombinant wild type tobacco activase and arginine mutants with chymotrypsin. (A) Wild type, R241A and R244A; (B) Wild type, R294A and R296A. Samples (10 lg protein) were analyzed by gel electrophoresis after incubation with chymotrypsin for 5 and 20 min in the presence of 5 mM ATP-c-S or ADP as indicated. For other experimental details, see Materials and methods. Standard lane contains the size standards and control is untreated activase.
Molecular mass determination Like most AAA+ proteins, Rubisco activase usually exists in oligomeric forms. With spinach activase, the binding of ATP promoted aggregation into higher-order oligomers as detected by FPLC gel filtration chromatography [9]. Therefore, this method appeared to be ideal to examine the effects of ATP versus ADP on the size of the recombinant tobacco wild type and mutant activases. However, surprisingly and unlike previously reported for spinach activase [9], the apparent molecular masses of recombinant wild type tobacco activase, R244A and R296A were similar in the presence of ATP versus ADP (Fig. 6A and B). The apparent molecular masses of R241A and R294A were significantly smaller than the wild type in the presence of ADP and they increased slightly in the presence of ATP. The inability of ATP to dramatically increase the apparent molecular mass of the recombinant wild type tobacco activase prompted a reexamination of the behavior of spinach
The roles of several conserved arginines in Rubisco activase have been investigated in this research. Comparisons of the roles of these arginines with that in other AAA+ proteins yield some interesting insights. In the AAA subfamily, a conserved arginine in Walker B, near R181 in activase, has been suggested to form a salt bridge with an aspartate in Box VII which stabilizes oligomeric formation and facilitates ATP hydrolysis [18]. The absence of this residue in activase and the minor effects of replacing the nearby R181 clearly distinguish activase from proteins in this subfamily. R244 (Box VII) in activase is a highly conserved residue in the AAA+ superfamily. In almost all cases examined, it has been found to be involved in ATP hydrolysis and in HslU contributes to the stabilization of hexamerization [11,28]. Structural analyses of several AAA+ proteins indicate that this arginine is close to the c-phosphate of ATP bound to an adjacent subunit, which has prompted the idea that this arginine might function as an ‘‘arginine finger,’’ similar to the ‘‘arginine finger’’ in GTPase [12,16]. In activase, replacement of R244 with alanine abolished ATP hydrolysis activity as well as the ATP-induced conformational changes detected by intrinsic fluorescence and limited proteolysis. The second arginine in this region, corresponding to R241 in activase, is often present in the AAA subfamily and a fraction of Clp/Hsp100 subfamily and it has been suggested to be another ‘‘arginine finger’’ [11]. Mutation of this residue in FtsH causes a loss of ATP hydrolysis activity while mutation in NSF has no effect on ATP hydrolysis activity, but affects its SNARE
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Fig. 6. Size determination of wild type (WT) and mutant (R241A, R244A, R294A, and R296A) tobacco activases (A and B) and spinach wild type (SA) and mutant (R239A) activases (C and D) by gel filtration in the presence of 0.1 mM ADP (A and C) or 0.2 mM ATP (B and D) at 4 C. For other experimental details, see Materials and methods. The elution volumes of the protein standards are indicated by the filled squares. Protein standards were cytochrome C (12,400), bovine serum albumin (67,000), b-amylase (200,000), apoferritin (443,000) and thyroglobulin (667,000).
Fig. 7. Time course of the intrinsic fluorescence change with wild type spinach activase (SA) and R239A spinach activase (R239A SA) after addition of ATP-c-S. The assays were conducted with 40 lg/ml protein and after 60 s incubation at room temperature, 0.5 mM ATP-c-S was added as indicated.
assembly [16,29]. Except for gel filtration, replacement of R241 in activase resulted in similar effects as replacement of R244. In a predicted 3D structure of the activase AAA+ domains (Fig. 1B), both of these arginines are located distant from the ATP binding site, which makes it impossible for them to interact with the ATP bound by their own subunit. Thus our data are consistent with the
hypothesis that these two arginines might also interact with ATP from an adjacent subunit as ‘‘arginine fingers.’’ It has been suggested [11] that the arginine corresponding to R241 in tobacco activase is mutually exclusive with the conserved arginine in Sensor 2 (R296 in tobacco activase), but both arginines are present in activase and important for ATP hydrolysis and ATP-induced conformational changes. R296 in the Sensor 2 domain of activase also may project into the nucleotide binding site and thereby mediate conformational changes associated with nucleotide binding or hydrolysis. In the predicted structure (Fig. 1B) R296 is not in a position to project into the nucleotide binding domain. However, the predicted structure may be inaccurate in this area because in contrast to the conserved Rossman fold domain, the arrangement of the helices within the C-terminal, a-helical domain varies in the known structures of AAA+ proteins. Our results indicate that R296 is critical for ATP hydrolysis, Rubisco activation and conformational changes associated with proteolytic protection and intrinsic fluorescence changes. Thus R296 in activase may function in a manner similar to the suggested role of this arginine in mediating conformation changes in the Sensor 2 domain in response to ATP [12]. Replacement of the adjacent R294 residue resulted in retention of the nucleotide-dependent proteolytic protection and some ATP hydrolysis activity, enhanced the ATP-induced intrinsic fluorescence change, but almost eliminated Rubisco activation activity. Thus this residue appears to be playing a more subtle role in maintaining the proper interactions necessary to activate Rubisco.
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An ATP-induced aggregation in Rubisco activase, observed in gel filtration experiments with spinach activase, has been suggested to closely correlate with the ATP-induced increase in intrinsic fluorescence and ATP hydrolysis [9,27]. However, we found that recombinant wild type tobacco activase (and two of the arginine mutants) did not exhibit an ATP-induced increase in apparent molecular mass monitored by gel filtration. Furthermore, spinach R239A, a mutant without ATP hydrolysis activity as well as the ATP-induced intrinsic fluorescence increase, exhibited an ATP-induced increase in molecular mass which was similar to the wild type 43 kDa spinach activase. The contrasting results with tobacco, spinach and the R239A spinach mutant suggest that the intrinsic fluorescence increase induced by ATP may not require increased aggregation, but perhaps only reflects reorientations between subunits in an oligomeric complex. However, the possibility remains that higher-order oligomers transiently form during ATP binding/hydrolysis which may not be detected by current methods. Recently, Salvucci [30] has demonstrated that a carboxyl-terminal cross-linked activase dimer retained its enzymatic function, which indicates that complete dissociation to monomers might not be required for activase to fulfill its function. In any case, the increased aggregation of spinach activase observed by gel filtration analysis appears to be species specific and its significance for activase function with Rubisco remains unclear. Acknowledgment This work was supported in part by a grant from the US Department of Energy (97ER20268). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.abb.2006. 04.002. References [1] V.J. Streusand, A.R. Portis Jr., Plant Physiol. 85 (1987) 152–154.
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