Rubisco activase requires residues in the large subunit N terminus to remodel inhibited plant Rubisco
2020; Elsevier BV; Volume: 295; Issue: 48 Linguagem: Inglês
10.1074/jbc.ra120.015759
ISSN1083-351X
AutoresJediael Ng, Zhijun Guo, Oliver Mueller‐Cajar,
Tópico(s)Plant biochemistry and biosynthesis
ResumoThe photosynthetic CO2 fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) forms dead-end inhibited complexes while binding multiple sugar phosphates, including its substrate ribulose 1,5-bisphosphate. Rubisco can be rescued from this inhibited form by molecular chaperones belonging to the ATPases associated with diverse cellular activities (AAA+ proteins) termed Rubisco activases (Rcas). The mechanism of green-type Rca found in higher plants has proved elusive, in part because until recently higher-plant Rubiscos could not be expressed recombinantly. Identifying the interaction sites between Rubisco and Rca is critical to formulate mechanistic hypotheses. Toward that end here we purify and characterize a suite of 33 Arabidopsis Rubisco mutants for their ability to be activated by Rca. Mutation of 17 surface-exposed large subunit residues did not yield variants that were perturbed in their interaction with Rca. In contrast, we find that Rca activity is highly sensitive to truncations and mutations in the conserved N terminus of the Rubisco large subunit. Large subunits lacking residues 1–4 are functional Rubiscos but cannot be activated. Both T5A and T7A substitutions result in functional carboxylases that are poorly activated by Rca, indicating the side chains of these residues form a critical interaction with the chaperone. Many other AAA+ proteins function by threading macromolecules through a central pore of a disc-shaped hexamer. Our results are consistent with a model in which Rca transiently threads the Rubisco large subunit N terminus through the axial pore of the AAA+ hexamer. The photosynthetic CO2 fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) forms dead-end inhibited complexes while binding multiple sugar phosphates, including its substrate ribulose 1,5-bisphosphate. Rubisco can be rescued from this inhibited form by molecular chaperones belonging to the ATPases associated with diverse cellular activities (AAA+ proteins) termed Rubisco activases (Rcas). The mechanism of green-type Rca found in higher plants has proved elusive, in part because until recently higher-plant Rubiscos could not be expressed recombinantly. Identifying the interaction sites between Rubisco and Rca is critical to formulate mechanistic hypotheses. Toward that end here we purify and characterize a suite of 33 Arabidopsis Rubisco mutants for their ability to be activated by Rca. Mutation of 17 surface-exposed large subunit residues did not yield variants that were perturbed in their interaction with Rca. In contrast, we find that Rca activity is highly sensitive to truncations and mutations in the conserved N terminus of the Rubisco large subunit. Large subunits lacking residues 1–4 are functional Rubiscos but cannot be activated. Both T5A and T7A substitutions result in functional carboxylases that are poorly activated by Rca, indicating the side chains of these residues form a critical interaction with the chaperone. Many other AAA+ proteins function by threading macromolecules through a central pore of a disc-shaped hexamer. Our results are consistent with a model in which Rca transiently threads the Rubisco large subunit N terminus through the axial pore of the AAA+ hexamer. Virtually all carbon dioxide that enters the biosphere does so via the Calvin Benson Bassham cycle (1Spreitzer R.J. Salvucci M.E. Rubisco: structure, regulatory interactions, and possibilities for a better enzyme.Annu. Rev. Plant Biol. 2002; 53 (12221984): 449-47510.1146/annurev.arplant.53.100301.135233Crossref PubMed Scopus (651) Google Scholar). Aerobic autotrophic organisms such as plants, algae, and cyanobacteria all utilize this somewhat suboptimal CO2-fixation process, which depends on catalysis by the slow and promiscuous enzyme Rubisco. The enzyme binds the five carbon sugar ribulose 1,5-bisphosphate (RuBP), adds a carbon dioxide molecule, and hydrolyzes the six carbon intermediate to form two molecules of 3-phosphoglycerate (3PGA). In plants each active site only processes ∼1–3 reactions/s, and frequently oxygen gas is incorporated instead of CO2, which leads to production of the toxic metabolite 2-phosphoglycolate. To overcome these flux limitations, Rubisco is overexpressed to constitute up to 50% of the leaf soluble protein and is believed to be the most abundant protein on earth (2Raven J.A. Rubisco: still the most abundant protein of Earth?.New Phytol. 2013; 198 (23432200): 1-310.1111/nph.12197Crossref PubMed Scopus (98) Google Scholar, 3Bar-On Y.M. Milo R. The global mass and average rate of Rubisco.Proc. Natl. Acad. Sci. U.S.A. 2019; 116 (30782794): 4738-474310.1073/pnas.1816654116Crossref PubMed Scopus (67) Google Scholar). Recognition that the enzyme catalyzes the rate-limiting step has made its performance the center of multiple ongoing crop improvement strategies (4Long S.P. Marshall-Colon A. Zhu X.G. Meeting the global food demand of the future by engineering crop photosynthesis and yield potential.Cell. 2015; 161 (25815985): 56-6610.1016/j.cell.2015.03.019Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, 5Zhu X.G. Long S.P. Ort D.R. Improving photosynthetic efficiency for greater yield.Annu. Rev. Plant Biol. 2010; 61 (20192734): 235-26110.1146/annurev-arplant-042809-112206Crossref PubMed Scopus (1066) Google Scholar). In addition to its slow speed and inaccuracy, the enzyme is also susceptible to form dead-end inhibited complexes with several sugar phosphates that are present in its environment (6Parry M.A. Keys A.J. Madgwick P.J. Carmo-Silva A.E. Andralojc P.J. Rubisco regulation: a role for inhibitors.J. Exp. Botanz. 2008; 59 (18436543): 1569-158010.1093/jxb/ern084Crossref PubMed Scopus (193) Google Scholar, 7Pearce F.G. Andrews T.J. The relationship between side reactions and slow inhibition of ribulose-bisphosphate carboxylase revealed by a loop 6 mutant of the tobacco enzyme.J. Biol. Chem. 2003; 278 (12783874): 32526-3253610.1074/jbc.M305493200Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). CO2 fixation ceases, unless the inhibitors are constantly removed. This action is performed by a group of dedicated molecular chaperones that have been termed the Rubisco activases (Rcas) (8Salvucci M.E. Portis Jr., A.R. Ogren W.L. A soluble chloroplast protein catalyzes ribulosebisphosphate carboxylase/oxygenase activation in vivo.Photosynth. Res. 1985; 7 (24443088): 193-20110.1007/BF00037012Crossref PubMed Scopus (156) Google Scholar, 9Tsai Y.C. Lapina M.C. Bhushan S. Mueller-Cajar O. Identification and characterization of multiple Rubisco activases in chemoautotrophic bacteria.Nat. Commun. 2015; 6 (26567524): 888310.1038/ncomms9883Crossref PubMed Scopus (55) Google Scholar, 10Mueller-Cajar O. Stotz M. Wendler P. Hartl F.U. Bracher A. Hayer-Hartl M. Structure and function of the AAA+ protein CbbX, a red-type Rubisco activase.Nature. 2011; 479 (22048315): 194-19910.1038/nature10568Crossref PubMed Scopus (111) Google Scholar). Three classes of Rca exist, and although all belong to the superfamily of AAA+ proteins, their primary sequences and mechanisms are highly distinct, indicating convergent evolution (11Mueller-Cajar O. The diverse AAA+ machines that repair inhibited Rubisco active sites.Front. Mol. Biosci. 2017; 4 (28580359): 3110.3389/fmolb.2017.00031Crossref PubMed Scopus (36) Google Scholar, 12Hayer-Hartl M. Hartl F.U. Chaperone machineries of Rubisco: the most abundant enzyme.Trends Biochem. Sci. 2020; 45 (32471779): 748-76310.1016/j.tibs.2020.05.001Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Red-type Rca found in red-lineage phytoplankton and proteobacteria transiently threads the C terminus of the Rubisco large subunit through the axial pore of the AAA+ hexamer (10Mueller-Cajar O. Stotz M. Wendler P. Hartl F.U. Bracher A. Hayer-Hartl M. Structure and function of the AAA+ protein CbbX, a red-type Rubisco activase.Nature. 2011; 479 (22048315): 194-19910.1038/nature10568Crossref PubMed Scopus (111) Google Scholar, 13Bhat J.Y. Milicic G. Thieulin-Pardo G. Bracher A. Maxwell A. Ciniawsky S. Mueller-Cajar O. Engen J.R. Hartl F.U. Wendler P. Hayer-Hartl M. Mechanism of enzyme repair by the AAA+ chaperone Rubisco activase.Mol. Cell. 2017; 67 (28803776): 744-75610.1016/j.molcel.2017.07.004Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 14Loganathan N. Tsai Y.-C.C. Mueller-Cajar O. Characterization of the heterooligomeric red-type Rubisco activase from red algae.Proc. Natl. Acad. Sci. U.S.A. 2016; 113 (27872295): 14019-1402410.1073/pnas.1610758113Crossref PubMed Scopus (30) Google Scholar). In contrast the CbbQO-type Rca found in chemoautotrophic proteobacteria consists of a cup-shaped AAA+ hexamer (CbbQ6) bound to a single adaptor protein CbbO, which is essential for Rubisco activation (9Tsai Y.C. Lapina M.C. Bhushan S. Mueller-Cajar O. Identification and characterization of multiple Rubisco activases in chemoautotrophic bacteria.Nat. Commun. 2015; 6 (26567524): 888310.1038/ncomms9883Crossref PubMed Scopus (55) Google Scholar). During Rca function the hexamer remodels CbbO, which is bound to inhibited Rubisco via a von Willebrand Factor A domain (15Tsai Y.C. Ye F. Liew L. Liu D. Bhushan S. Gao Y.G. Mueller-Cajar O. Insights into the mechanism and regulation of the CbbQO-type Rubisco activase, a MoxR AAA+ ATPase.Proc. Natl. Acad. Sci. U.S.A. 2020; 117 (31848241): 381-38710.1073/pnas.1911123117Crossref PubMed Scopus (16) Google Scholar). The detailed molecular mechanism by which inhibitory compounds are removed by higher-plant Rca-mediated modeling of Rubisco's active site has long remained elusive (11Mueller-Cajar O. The diverse AAA+ machines that repair inhibited Rubisco active sites.Front. Mol. Biosci. 2017; 4 (28580359): 3110.3389/fmolb.2017.00031Crossref PubMed Scopus (36) Google Scholar, 16Bracher A. Whitney S.M. Hartl F.U. Hayer-Hartl M. Biogenesis and metabolic maintenance of Rubisco.Annu. Rev. Plant Biol. 2017; 68 (28125284): 29-6010.1146/annurev-arplant-043015-111633Crossref PubMed Scopus (122) Google Scholar). Because functional Rca could be produced recombinantly, a large volume of biochemical information has accumulated on Rca variants (17Portis Jr., A.R. Rubisco activase: Rubisco's catalytic chaperone.Photosynth. Res. 2003; 75 (16245090): 11-2710.1023/A:1022458108678Crossref PubMed Scopus (427) Google Scholar, 18Carmo-Silva E. Scales J.C. Madgwick P.J. Parry M.A. Optimizing Rubisco and its regulation for greater resource use efficiency.Plant Cell Environ. 2015; 38 (25123951): 1817-183210.1111/pce.12425Crossref PubMed Scopus (198) Google Scholar). In summary the data support a canonical AAA+ pore-loop threading mechanism in which the flat top surface of the hexameric disc engages Rubisco, followed by axial pore-loop threading of an element of Rubisco (19Stotz M. Mueller-Cajar O. Ciniawsky S. Wendler P. Hartl F.U. Bracher A. Hayer-Hartl M. Structure of green-type Rubisco activase from tobacco.Nat. Struct. Mol. Biol. 2011; 18 (22056769): 1366-137010.1038/nsmb.2171Crossref PubMed Scopus (85) Google Scholar, 20Shivhare D. Ng J. Tsai Y.C. Mueller-Cajar O. Probing the rice Rubisco–Rubisco activase interaction via subunit heterooligomerization.Proc. Natl. Acad. Sci. U.S.A. 2019; 116 (31712424): 24041-2404810.1073/pnas.1914245116Crossref PubMed Scopus (8) Google Scholar). The intrinsically disordered N-terminal domain, especially a conserved tryptophan, is also important in engaging the holoenzyme (20Shivhare D. Ng J. Tsai Y.C. Mueller-Cajar O. Probing the rice Rubisco–Rubisco activase interaction via subunit heterooligomerization.Proc. Natl. Acad. Sci. U.S.A. 2019; 116 (31712424): 24041-2404810.1073/pnas.1914245116Crossref PubMed Scopus (8) Google Scholar, 21van de Loo F.J. Salvucci M.E. Activation of ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) involves Rubisco activase Trp16.Biochemistry. 1996; 35 (8679566): 8143-814810.1021/bi9604901Crossref PubMed Scopus (54) Google Scholar). Regarding Rubisco, early studies using green algal Chlamydomonas Rubisco were able to pinpoint two residues on the Rubisco large subunits' βC–βD loop that contact the specificity helix (H9) of Rca (22Wang Z.Y. Snyder G.W. Esau B.D. Portis A.R. Ogren W.L. Species-dependent variation in the interaction of substrate-bound Ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) and Rubisco activase.Plant Physiol. 1992; 100 (16653209): 1858-186210.1104/pp.100.4.1858Crossref PubMed Scopus (70) Google Scholar, 23Larson E.M. O'Brien C.M. Zhu G. Spreitzer R.J. Portis Jr, A.R. Specificity for activase is changed by a Pro-89 to Arg substitution in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase.J. Biol. Chem. 1997; 272 (9202018): 17033-1703710.1074/jbc.272.27.17033Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). However, a historical inability to produce plant Rubisco in heterologous organisms such as Escherichia coli hampered further progress. This hurdle was recently removed via the concurrent functional expression of specific plant chaperonins and assembly factors within the heterologous host (24Aigner H. Wilson R.H. Bracher A. Calisse L. Bhat J.Y. Hartl F.U. Hayer-Hartl M. Plant RuBisCo assembly in E. coli with five chloroplast chaperones including BSD2.Science. 2017; 358 (29217567): 1272-127810.1126/science.aap9221Crossref PubMed Scopus (100) Google Scholar). Here, we took advantage of the newly established capability to produce and biochemically characterize many plant Rubisco variants for their interaction with Rca. We find that the highly conserved RbcL N terminus is essential for Rca function, with a particular importance of two threonine residues: Thr5 and Thr7. This is consistent with an N-terminal pore-loop threading mechanism for higher-plant Rca. We used the recently established E. coli plant Rubisco expression platform (24Aigner H. Wilson R.H. Bracher A. Calisse L. Bhat J.Y. Hartl F.U. Hayer-Hartl M. Plant RuBisCo assembly in E. coli with five chloroplast chaperones including BSD2.Science. 2017; 358 (29217567): 1272-127810.1126/science.aap9221Crossref PubMed Scopus (100) Google Scholar, 25Wilson R.H. Thieulin-Pardo G. Hartl F.U. Hayer-Hartl M. Improved recombinant expression and purification of functional plant Rubisco.FEBS Lett. 2019; 593 (30815863): 611-62110.1002/1873-3468.13352Crossref PubMed Scopus (16) Google Scholar) to produce a series of Arabidopsis Rubisco large subunits variants mutated in surface-localized residues in an effort to discover additional regions important for protein–protein interactions. We first tested the βC–βD loop mutations E94K and P89A as positive controls (Fig. 1A), because these substitutions had earlier been shown to greatly perturb the ability of spinach Rca to activate Chlamydomonas Rubisco (26Ott C.M. Smith B.D. Portis Jr., A.R. Spreitzer R.J. Activase region on chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase: nonconservative substitution in the large subunit alters species specificity of protein interaction.J. Biol. Chem. 2000; 275 (10858441): 26241-2624410.1074/jbc.M004580200Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). We then assayed the fully activated holoenzyme (ECM) and the inhibited apo-enzyme bound to the substrate ribulose 1,5-bisphosphate (ER) in the presence and absence of the short (Rcaβ) isoform of Arabidopsis Rca (Fig. 1B). Consistent with the Chlamydomonas–spinach result, the inhibited E94K variant of Arabidopsis Rubisco remained nonfunctional in the presence of its cognate Rca, reconfirming the importance of the N-terminal βC–βD loop in the interaction. The P89A variant, however, was activated well in this system (Fig. 1C), suggesting that the βC–βD loop–Rca interaction is less sensitive to mutation when using Arabidopsis proteins. We next targeted a range of surface-localized Rubisco large subunit residues for mutagenesis (Fig. 2A and Fig. S2). As we found earlier, multiple positively charged residues on the face of the Rca disc are important for its ability to activate Rubisco (20Shivhare D. Ng J. Tsai Y.C. Mueller-Cajar O. Probing the rice Rubisco–Rubisco activase interaction via subunit heterooligomerization.Proc. Natl. Acad. Sci. U.S.A. 2019; 116 (31712424): 24041-2404810.1073/pnas.1914245116Crossref PubMed Scopus (8) Google Scholar, 27Shivhare D. Mueller-Cajar O. Characterization of thermostable CAM Rubisco activase reveals a Rubisco interacting surface loop.Plant Physiol. 2017; 174 (28546437): 1505-151610.1104/pp.17.00554Crossref PubMed Scopus (27) Google Scholar), and therefore the chosen mutations were biased toward probing negatively charged surface residues. This included those located in a negatively charged pocket at the dimer–dimer interface that has recently been implicated in the binding of carboxysomal Rubisco linker proteins in prokaryotic green-type Rubiscos (Fig. 2B) (28Wang H. Yan X. Aigner H. Bracher A. Nguyen N.D. Hee W.Y. Long B.M. Price G.D. Hartl F.U. Hayer-Hartl M. Rubisco condensate formation by CcmM in β-carboxysome biogenesis.Nature. 2019; 566 (30675061): 131-13510.1038/s41586-019-0880-5Crossref PubMed Scopus (93) Google Scholar, 29Oltrogge L.M. Chaijarasphong T. Chen A.W. Bolin E.R. Marqusee S. Savage D.F. Multivalent interactions between CsoS2 and Rubisco mediate α-carboxysome formation.Nat. Struct. Mol. Biol. 2020; 27 (32123388): 281-28710.1038/s41594-020-0387-7Crossref PubMed Scopus (46) Google Scholar). We successfully purified 17 variants (Fig. S1), which were all able to carboxylate RuBP similarly to WT (Fig. 2C, Table S2, and Fig. S3). Rca assays indicated that the different variants could still be activated, effectively indicating that the chosen residues were not of critical importance to the Rubisco–Rca interaction (Fig. 2C and Fig. S3). Only K14A showed a statistically significant 52% increase in its Rca-mediated activation rate, possibly reflecting a reduced stability of the inhibited complex. In several species of plants, Lys14 is trimethylated, a modification that is catalyzed by Rubisco large subunit methyltransferase (Rubisco LSMT) (30Houtz R.L. Poneleit L. Jones S.B. Royer M. Stults J.T. Posttranslational modifications in the amino- terminal region of the large subunit of ribulose- 1,5-bisphosphate carboxylase/oxygenase from several plant species.Plant Physiol. 1992; 98 (16668742): 1170-117410.1104/pp.98.3.1170Crossref PubMed Scopus (54) Google Scholar, 31Houtz R.L. Stults J.T. Mulligan R.M. Tolbert N.E. Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase.Proc. Natl. Acad. Sci. U.S.A. 1989; 86 (2928307): 1855-185910.1073/pnas.86.6.1855Crossref PubMed Scopus (81) Google Scholar, 32Houtz R.L. Magnani R. Nayak N.R. Dirk L.M. Co- and post-translational modifications in Rubisco: unanswered questions.J. Exp. Botany. 2008; 59 (18353761): 1635-164510.1093/jxb/erm360Crossref PubMed Scopus (50) Google Scholar). In the context of activase-mediated remodeling, however, the lack of conservation across all plants diminishes the likelihood of Lys14 trimethylation as essential for reactivation but could possibly represent a mechanism for species-specific regulatory control. Clearly the chosen single amino acid substitutions were insufficient to disrupt the extensive protein–protein interaction interface involved in Rubisco activation. However, attempts to produce combinations of mutations were unsuccessful because of either insolubility or nonfunctionality for all tested cases. The red-type Rubisco activase CbbX transiently threads the RbcL C terminus (10Mueller-Cajar O. Stotz M. Wendler P. Hartl F.U. Bracher A. Hayer-Hartl M. Structure and function of the AAA+ protein CbbX, a red-type Rubisco activase.Nature. 2011; 479 (22048315): 194-19910.1038/nature10568Crossref PubMed Scopus (111) Google Scholar, 13Bhat J.Y. Milicic G. Thieulin-Pardo G. Bracher A. Maxwell A. Ciniawsky S. Mueller-Cajar O. Engen J.R. Hartl F.U. Wendler P. Hayer-Hartl M. Mechanism of enzyme repair by the AAA+ chaperone Rubisco activase.Mol. Cell. 2017; 67 (28803776): 744-75610.1016/j.molcel.2017.07.004Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 14Loganathan N. Tsai Y.-C.C. Mueller-Cajar O. Characterization of the heterooligomeric red-type Rubisco activase from red algae.Proc. Natl. Acad. Sci. U.S.A. 2016; 113 (27872295): 14019-1402410.1073/pnas.1610758113Crossref PubMed Scopus (30) Google Scholar). However, the C terminus of green-type Rubisco large subunits is poorly conserved and is of variable length (33Satagopan S. Spreitzer R.J. Substitutions at the Asp-473 latch residue of Chlamydomonas ribulosebisphosphate carboxylase/oxygenase cause decreases in carboxylation efficiency and CO2/O2 specificity.J. Biol. Chem. 2004; 279 (14734540): 14240-1424410.1074/jbc.M313215200Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), indicating a distinct mechanism for green-type Rca function. In contrast, although sequences at the N terminus of red-type Rubisco large subunits differ between species, both the length and the sequence of the N terminus of higher-plant RbcL is essentially completely conserved (Fig. 3A). In available crystal structures, residues 8–20 of the N terminus are ordered only when the active site is in the closed (ligand-bound) form (Fig. 3B). In the closed conformation, the N terminus is positioned directly above the 60s loop that coordinates P1 of the substrate, with Phe13, Lys14, Gly16, and Lys18 forming interactions with multiple residues of the 60s loop (34Schreuder H.A. Knight S. Curmi P.M.G. Andersson I. Cascio D. Branden C.I. Eisenberg D. Formation of the active-site of ribulose-1,5-bisphosphate carboxylase oxygenase by a disorder order transition from the unactivated to the activated form.Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 9968-997210.1073/pnas.90.21.9968Crossref PubMed Scopus (55) Google Scholar). Coupled with evidence that residues 9–15 of Rubisco from both spinach and wheat are essential for functional carboxylation activity (31Houtz R.L. Stults J.T. Mulligan R.M. Tolbert N.E. Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase.Proc. Natl. Acad. Sci. U.S.A. 1989; 86 (2928307): 1855-185910.1073/pnas.86.6.1855Crossref PubMed Scopus (81) Google Scholar, 35Gutteridge S. Millard B.N. Parry M.A.J. Inactivation of ribulose-bisphosphate carboxylase by limited proteolysis.FEBS Lett. 1986; 196: 263-26810.1016/0014-5793(86)80260-5Crossref Scopus (34) Google Scholar, 36Mulligan R.M. Houtz R.L. Tolbert N.E. Reaction-intermediate analogue binding by ribulose bisphosphate carboxylase/oxygenase causes specific changes in proteolytic sensitivity: the amino-terminal residue of the large subunit is acetylated proline.Proc. Natl. Acad. Sci. U.S.A. 1988; 85 (3422748): 1513-151710.1073/pnas.85.5.1513Crossref PubMed Scopus (35) Google Scholar), the stringent conservation of the first eight residues thus suggested a tantalizing target for mutational analysis. A Rubisco variant with the first seven amino acids replaced by methionine (ΔN7) displayed 83% of WT carboxylation velocity (Fig. 3C). However, when the ER complex was formed, Rca was unable to reactivate ΔN7 (Fig. 3C). This result was consistent with the notion that a higher-plant Rca hexamer engages the disordered N terminus via its axial pore-loops during reactivation and is likely followed by limited threading that leads to active site disruption and inhibitor release. We then performed a detailed mutational analysis of the RbcL N terminus, generating a series of variants that, in the ECM form, were all able to carboxylate at least as well as WT (Fig. 4). Mutant variants were designed either with sequential truncations (ΔN1, ΔN2, and ΔN3), specific deletions or substitutions (ΔTET and TET-AAA), or targeted insertions (M1insAA and T7insAAA) (Fig. 4A). In plants, post-translational processing of the Rubisco large subunit results in the removal of two residues, leaving an acetylated Pro3 as the innate N terminus (30Houtz R.L. Poneleit L. Jones S.B. Royer M. Stults J.T. Posttranslational modifications in the amino- terminal region of the large subunit of ribulose- 1,5-bisphosphate carboxylase/oxygenase from several plant species.Plant Physiol. 1992; 98 (16668742): 1170-117410.1104/pp.98.3.1170Crossref PubMed Scopus (54) Google Scholar, 31Houtz R.L. Stults J.T. Mulligan R.M. Tolbert N.E. Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase.Proc. Natl. Acad. Sci. U.S.A. 1989; 86 (2928307): 1855-185910.1073/pnas.86.6.1855Crossref PubMed Scopus (81) Google Scholar, 32Houtz R.L. Magnani R. Nayak N.R. Dirk L.M. Co- and post-translational modifications in Rubisco: unanswered questions.J. Exp. Botany. 2008; 59 (18353761): 1635-164510.1093/jxb/erm360Crossref PubMed Scopus (50) Google Scholar, 36Mulligan R.M. Houtz R.L. Tolbert N.E. Reaction-intermediate analogue binding by ribulose bisphosphate carboxylase/oxygenase causes specific changes in proteolytic sensitivity: the amino-terminal residue of the large subunit is acetylated proline.Proc. Natl. Acad. Sci. U.S.A. 1988; 85 (3422748): 1513-151710.1073/pnas.85.5.1513Crossref PubMed Scopus (35) Google Scholar). The Rubisco purified using the present E. coli system by Aigner et al. (24Aigner H. Wilson R.H. Bracher A. Calisse L. Bhat J.Y. Hartl F.U. Hayer-Hartl M. Plant RuBisCo assembly in E. coli with five chloroplast chaperones including BSD2.Science. 2017; 358 (29217567): 1272-127810.1126/science.aap9221Crossref PubMed Scopus (100) Google Scholar) was reported to be N-terminally processed by the endogenous machinery. We therefore determined the N terminus of selected variants using de novo mass spectrometric sequencing (Fig. 4A, Table 1, and Table S3).Table 1N-terminal identity of recombinantly purified wild-type and mutant RbcLProteinUnprocessed N terminiObserved N terminiRatios of the peak areas%WTMSPQTETKASMSPQTETKAS36SPQTETKAS15PQTETKAS49ΔN1MPQTETKASPQTETKAS100ΔN2MQTETKASQTETKAS42TETKAS58ΔN3MTETKASTETKAS100ΔN7MKASMKAS100ΔTETMSPQKASMSPQKAS100TET-AAAMSPQAAAKASMSPQAAAKAS72SPQAAAKAS8PQAAAKAS20M1insAAMAASPQTETKASAASPQTETKAS99ASPQTETKAS<1SPQTETKAS<1T7insAAAMSPQTETAAAKASMSPQTETAAAKAS34SPQTETAAAKAS2PQTETAAAKAS64 Open table in a new tab Our recombinantly purified WT RbcL was found to contain a mixture of unprocessed, partially processed, and fully processed (∼50%) N termini (Table 1 and Fig. 4A). Shortening the N terminus of RbcL by a single amino acid after methionine (ΔN1) resulted in a homogenous pool of post processed native-like N termini that did not negatively affect Rca function (Fig. 4A). Sequential removal of the next amino acid (ΔN2) resulted in a heterogenous mix of post processed RbcL states (with the first amino acid being either Gln4 or Thr5), which resulted in a 67% increase in Rca functionality. In contrast, removal of the first three amino acids after methionine (ΔN3) or deleting residues 5–7 (ΔTET) almost completely eliminated the ability of Rca to activate Rubisco (Fig. 4A). The dramatic difference between ΔN2 and ΔN3 suggests that Gln4 is critical for Rca function, but the N-terminal sequencing result suggests it does not have to be present on every large subunit. Lengthening the N terminus by inserting two alanine residues upstream of residue 2 (M1insAA), which resulted in a postprocessed population of mostly lengthened N termini states, was found to greatly reduce Rca function by ∼64%. Changing the register of the N-terminal sequence by inserting an AAA sequence upstream of Lys8 (T7insAAA) in the WT or ΔTET variant (TET-AAA) also eliminated Rca function. These results indicated that Rca function was highly sensitive to both length and identity of the RbcL N terminus. Next, we evaluated the effect of single amino acid substitutions in the N-terminal motif. Whereas E6A and K8A substitutions were well-tolerated, both T5A and T7A resulted in ∼70% reductions in Rca functionality. This finding indicates that the two threonine residues are likely to play an important role in the threading process, possibly via specific interactions with residues in Rca's pore-loops 1 and 2 (19Stotz M. Mueller-Cajar O. Ciniawsky S. Wendler P. Hartl F.U. Bracher A. Hayer-Hartl M. Structure of green-type Rubisco activase from tobacco.Nat. Struct. Mol. Biol. 2011; 18 (22056769): 1366-137010.1038/nsmb.2171Crossref PubMed Scopus (85) Google Scholar, 20Shivhare D. Ng J. Tsai Y.C. Mueller-Cajar O. Probing the rice Rubisco–Rubisco activase interaction via subunit heterooligomerization.Proc. Natl. Acad. Sci. U.S.A. 2019; 116 (31712424): 24041-2404810.1073/pnas.1914245116Crossref PubMed Scopus (8) Google Scholar). We also further note that the observed 2-amino acid step interval would be consistent with successive zipper-like interactions that have been described to be utilized for substrate engagement and threading by the central pore in multiple other AAA+ proteins (37Puchades C. Rampello A.J. Shin M. Giuliano C.J. Wiseman R.L. Glynn S.E. Lander G.C. Structure of the mitochondrial inner membrane AAA+ protease YME1 gives insight into substrate processing.Science. 2017; 358 (29097521): eaao046410.1126/science.aao0464Crossref PubMed Scopus (123) Google Scholar, 38Shin M. Puchades C. Asmita A. Puri N. Adjei E. Wiseman R.L. Karzai A.W. Lander G.C. Structural basis for distinct operational modes and protease activation in AAA+ protease Lon.Sci. Adv. 2020; 6 (32490208): eaba840410.1126/sciadv.aba8404Crossref PubMed Scopus (21) Google Scholar, 39Gates S.N. Yokom A.L. Lin J. Jackrel M.E. Rizo A.N. Kendsersky N.M. Buell C.E. Sweeny E.A. Mack K.L. Chuang E. Torrente M.P. Su M. Shorter J. Southworth D.R. Ratchet-like polypeptide translocation mechanism of the AAA+ disaggregase Hsp104.Science. 2017; 357 (28619716): 273-27910.1126/science.aan1052Crossref PubMed Scopus (166) Google Scholar
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