Rubisco in complex with Rubisco large subunit methyltransferase

Abstract

SET domain protein lysine methyltransferases (PKMT) are a structurally unique class of enzymes that catalyze the specific methylation of lysine residues in a number of different substrates. Especially histone-specific SET domain PKMTs have received widespread attention because of their roles in the regulation of epigenetic gene expression and the development of some cancers. Rubisco large subunit methyltransferase (RLSMT) is a chloroplastlocalized SET domain PKMT responsible for the formation of trimethyl-lysine-14 in the large subunit of Rubisco, an essential photosynthetic enzyme. Here, we have used cryoelectron microscopy to produce an 11-Å density map of the Rubisco-RLSMT complex. The atomic model of the complex, obtained by fitting crystal structures of Rubisco and RLSMT into the density map, shows that the extensive contact regions between the 2 proteins are mainly mediated by hydrophobic residues and leucine-rich repeats. It further provides insights into potential conformational changes that may occur during substrate binding and catalysis. This study presents the first structural analysis of a SET domain PKMT in complex with its intact polypeptide substrate.electron microscopy ͉ LSMT ͉ SET domain ͉ single particle S ET [SU(VAR)3-9, E(Z), and TRX] domain protein lysine methyltransferases (PKMTs) are a structurally unique class of enzymes that catalyze the formation of site-specific methylated lysine residues in a number of different polypeptide substrates including cytochrome c (1, 2), histones (3), ribosomal proteins (3, 4), p53 (5), TAF10 (6), ␥-tocopherol methyltransferase (7), and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (8). All SET domain PKMTs have a unique and conserved structural motif that contains separate binding sites for the target protein substrate and the methyl donor, Sadenosylmethionine (AdoMet) (9). The mechanisms by which these enzymes achieve site-specific methylation is of great interest and histone-specific SET domain protein methyltransferases (HKMTs) have received widespread attention because of their roles in the regulation of epigenetic gene expression and the development of some cancers (9, 10). The specificity of several SET domain PKMTs has been examined through structural and biochemical analyses of ternary complexes with bound polypeptide substrates, but all of these studies used short synthetic polypeptide mimetics of the intact polypeptide substrate and therefore do not account for the potential influence of areas outside the immediate residues flanking the target lysine methylation site (11)(12)(13)(14)(15)(16). Rubisco large subunit methyltransferase (RLSMT) is a chloroplast-localized SET domain PKMT responsible for the formation of trimethyl-lysine-14 in the large subunit of Rubisco (8,17), an essential photosynthetic enzyme with a hexadecameric structure and large molecular mass (Ϸ534 kDa). Detailed information is available regarding the structure, catalytic mechanism, active site residues, and the kinetic reaction mechanism for pea RLSMT (7,8,17,18)...

Characterization of Chloroplastic Fructose 1,6-Bisphosphate Aldolases as Lysine-methylated Proteins in Plants

“…Also, the unmethylated status of Rubisco in Arabidopsis and other species like spinach or wheat does not reflect sequence divergence of LS around the Lys-14 methylation site (see supplemental Fig. 4) or the residues mediating the interaction with the methyltransferase (23). The structure of the active site of PsLSMT indicated that the LS Rubisco and AdoMet substrates bind to separate clefts connected by a pore large enough to accommodate the transfer of methyl groups to Lys-14 (16).…”

“…This protein, referred to as AtLSMT-L for Arabidopsis LSMTlike, shares 62% sequence identity (plus 15% conservative changes) with PsLSMT. Hence, both the N-terminal located catalytic SET domain and the C-terminal lobe that forms extensive contacts with the LS Rubisco substrate are highly conserved between both proteins (23). AtLSMT-L is a 482-residue and 54.6-kDa protein harboring a predicted chloroplastic transit peptide.…”

“…Rubisco was purified from spinach stroma by ion exchange chromatography. This source of unmethylated enzyme was shown to be representative of the interaction that occurs between LSMT and Rubisco (23). In addition to FBA2 and FBA3, two candidate protein substrates from Arabidopsis were also produced in E. coli as fusions with His tags at their N termini, i.e.…”

Advancing Our Understanding and Capacity to Engineer Nature’s CO2-Sequestering Enzyme, Rubisco

“…of L-subunit pairs in L 8 S 8 Rubisco but not the S-subunits (Raunser et al, 2009), suggesting that trimethylation of Lys-14 occurs after L 2 assembly (see below) and prior to S-subunit assembly (Fig. 5).…”

Posttranslational Modifications of Chloroplast Proteins: An Emerging Field

“…RLSMT was shown to exist in complex with Rubisco (Raunser et al, 2009) in which the Lys-14 is trimethylated in several species belonging to the Fabaceae, Solanaceae, and Cucurbitaceae families but not in wheat (Triticum aestivum), spinach (Spinacia oleracea), or Arabidopsis (Houtz et al, 1992;Mininno et al, 2012). Despite numerous studies, the effects of Lys-14 trimethylation of RBCL have not been defined, and RLSMT knockdown plants do not show marked defects in growth and CO 2 assimilation (Mininno et al, 2012).…”