A coil–helix instead of a helix–coil motif can be induced in a chloroplast transit peptide from Chlamydomonas reinhardtii

Abstract

A synthetic peptide MQVTMKSSAVSGQRVGGARVATRSVRRAQLQV corresponding to the 32 amino acid chloroplast transit sequence of the ribulose bisphosphatase carboxylase/oxygenase activase preprotein from Chlamydomonas reinhardtii, required for translocation through the envelope of the chloroplast, has been characterized structurally using CD and NMR under the same experimental conditions as used previously for the 32 amino acid presequence of preferredoxin from the same organism [Lancelin, J.-M., Bally, I., Arlaud, G. J., Blackledge, M., Gans, P., Stein, M. & Jacquot, J.-P. (1994) FEBS Lett. 343, 261±266]. The peptide is found to undergo a conformational transition in aqueous 2,2,2-trifluoroethanol, characterized by three turns of amphiphilic a-helix in the C-terminal region preceded by a disordered coil in the N-terminal region. Compared with the preferredoxin transit peptide, the helical and coiled domains are arranged in the reverse order along the peptide sequence, but the positively charged groups are distributed analogously as well as the hydrophobic residues within the amphiphilic a-helix. It is proposed that such coil±helix or helix±coil motifs, occasionally repeated, could be an intrinsic structural feature of chloroplastic transit peptides, adapted to the proper translocase and possibly to each nuclear-encoded chloroplast preproteins. This feature may distinguish chloroplastic transit sequences from the other organelle-targeting peptides in the eukaryotic green alga C. reinhardtii, particularly the mitochondrial transit sequences.Keywords: Chlamydomonas reinhardtii; chloroplast preproteins; NMR; peptide conformation; transit peptide.An extraordinary diversity is the characteristic of transit sequences attached to the N-terminus of nuclear-encoded mitochondrial preproteins in eukaryotes, particularly the nuclear-encoded chloroplast preproteins in plants. The preproteins are expressed in the cytosol from a nuclear gene and have to be addressed and translocated to the proper organelles where they are matured in their native forms. The variability of the transit presequences, both in terms of peptide length and amino acid sequence, precluded recognition of any consensus sequence and yielded only loose secondary-structure predictions from the peptide sequences [1]. We have previously reported NMR structural studies of a synthetic chloroplast transit peptide (cTP) [2] and a mitochondrial transit peptide (mTP) [3] originating from the same photosynthetic organism, the green alga Chlamydomonas reinhardtii. In agreement with several other studies [4±12], both types of transit peptides exhibit a significant propensity to form amphiphilic helices when they are mixed with either lipids or amphipathic solvents such as 2,2,2-trifluoroethanol (TFE). These coil-to-helix transitions can be observed using far-UV CD and solution NMR.It was found that, in C. reinhardtii, the mTP can be characterized in 65% aqueous TFE as fully helical with two short amphiphilic a-helices with their hydrophobic sectors in discontinuity along the he...

Differential Transit Peptide Recognition during Preprotein Binding and Translocation into Flowering Plant Plastids

“…These physicochemical properties may be environmentally sensitive and/or context specific, behaving differently as a function of pH, in a membrane-like environment, or upon receptor binding. One example of this is the tendency of TPs to convert from a random coil in an aqueous inherent environment (von Heijne and Nishikawa, 1991) to an a-helix in the presence of membranes or membrane-mimetic environments (Bruce, 1998;Krimm et al, 1999;Wienk et al, 2000). Finally, it is possible that TP interaction with different components of the TOC and TIC as well as the stromal-localized components, such as stromal processing peptidase (SPP) and molecular chaperones, uses multiple mechanisms of recognition ranging from general physicochemical properties to specific sequence recognition.…”

Identification of a Hsp70 Recognition Domain within the Rubisco Small Subunit Transit Peptide

“…Despite Ͼ250 transit peptides sequences in the CHLPEP database and hundreds of more recently identified transit peptides, few in-depth structural or functional analyses of these sequences are reported (Lancelin et al, 1994;Krimm et al, 1999;Wienk et al, 1999). However, arguments have recently been made to suggest that transit peptides are modular, with discrete domains providing different functional roles.…”

Molecular Mechanisms and Physiological Significance of Organelle Interactions and Cooperation

“…The structure of the transit peptide of ribulose bisphosphate carboxylase (Rubico) activase from the green algae Chlamydomonas reinhardii has been resolved confirming the α-helical domain (Krimm et al, 1999). However, transit peptides are predominantly unstructured in aqueous environment (Bruce, 1998;Krimm et al, 1999), which fits to their amino acid distribution (von Heijne and Nishikawa, 1991), but in hydrophobic environment the fraction of α-helical elements increases (Endo et al, 1992;Bruce, 1998;Krimm et al, 1999). However, these common properties of all transit peptides are complemented by more specific ones, which allow the discrimination of transit peptides by different members of the Toc159 receptor family Demarsy et al, 2014;Dutta et al, 2014).…”

“…On a helical wheel prediction, typical transit peptides encode a domain, which shows amphipathic properties due to a hydrophobic and a positively charged hydrophilic patch on opposite sides of the α-helix, but between these elements polar wedges of hydroxylated residues and occasionally negativly charged residues seem to be present (Bruce, 2000) ( Figure 2G). The structure of the transit peptide of ribulose bisphosphate carboxylase (Rubico) activase from the green algae Chlamydomonas reinhardii has been resolved confirming the α-helical domain (Krimm et al, 1999). However, transit peptides are predominantly unstructured in aqueous environment (Bruce, 1998;Krimm et al, 1999), which fits to their amino acid distribution (von Heijne and Nishikawa, 1991), but in hydrophobic environment the fraction of α-helical elements increases (Endo et al, 1992;Bruce, 1998;Krimm et al, 1999).…”