Cleavage of a synthetic COOH‐terminal oligopeptide of D1 precursor protein by a purified processing enzyme

Fujita, Shuji; Inagaki, Noritoshi; Ono, T.; Inoue, Yorinao; Satoh, Kimiyuki

doi:10.1016/0014-5793(89)81049-x

Cited by 17 publications

(8 citation statements)

References 12 publications

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“…The function of the D1 C-terminal-processing protease is to remove the C-terminal extension of the D1 polypeptide of photosystem II of oxygenic photosynthesis, which is necessary for assembly of the photosynthesis complex (23,24). The suggested function of the PDZ of D1p is to serve as binding site of the C-terminal extension of the target protein (15).…”

Section: Resultsmentioning

confidence: 99%

Folding and Misfolding in a Naturally Occurring Circularly Permuted PDZ Domain

Ivarsson¹,

Travaglini-Allocatelli

Brunori

et al. 2008

Journal of Biological Chemistry

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One of the most extreme and fascinating examples of naturally occurring mutagenesis is represented by circular permutation. Circular permutations involve the linking of two chain ends and cleavage at another site. Here we report the first description of the folding mechanism of a naturally occurring circularly permuted protein, a PDZ domain from the green alga Scenedesmus obliquus. Data reveal that the folding of the permuted protein is characterized by the presence of a low energy off-pathway kinetic trap. This finding contrasts with what was previously observed for canonical PDZ domains that, although displaying a similar primary structure when structurally re-aligned, fold via an on-pathway productive intermediate. Although circular permutation of PDZ domains may be necessary for a correct orientation of their functional sites in multidomain protein scaffolds, such structural rearrangement may compromise their folding pathway. This study provides a straightforward example of the divergent demands of folding and function.A crucial development of our knowledge on protein folding has been contributed by correlating rate constants of folding of small proteins with their topology as measured by the gross parameter of the contact order (1). The contact order represents the average distance, on the primary structure, between interacting residues in the tertiary structure. A protein with a low contact order will by and large present interacting residues that are close in sequence. On the other hand, high contact order implies a large number of long-range interactions. Baker and coworkers (2) first showed a strong correlation between kinetic and structural parameters, suggesting that protein topology is a key factor in determining the folding pathways and speed. An important corollary of these observations is that folding transition states must reflect a distorted version of the native state. This feature was already captured by the nucleation condensation model (3, 4), which suggested the protein to fold all at once around an extended, weakly formed, folding nucleus.The notion that protein folding pathways are governed by protein topology (1) has recently been challenged by ingenious experiments using topological mutants such as circularly permuted variants (5-9). Despite the dramatic change experienced by the primary structure, circular permutations seem well tolerated by several protein sequences. In nature, circular permutations have been recognized in ϳ5% of proteins of known structures (10,11). Folding studies on artificially permuted proteins have been specifically aimed at monitoring the effect on both folding speed and mechanism. By systematically altering the sequence connectivity of the ribosomal protein S6, Oliveberg and coworkers (12) showed that protein folding rate constants of circularly permuted variants are well predicted by the contact order parameter. On the other hand, analysis of protein folding pathways reveals apparently contradicting results. In particular, while the folding pathway of chymotr...

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Section: Resultsmentioning

confidence: 99%

Folding and Misfolding in a Naturally Occurring Circularly Permuted PDZ Domain

Ivarsson¹,

Travaglini-Allocatelli

Brunori

et al. 2008

Journal of Biological Chemistry

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“…from Asn-335 to Gly-353 in pD1, as the substrate, which has been studied in detail (25,28). When the oligopeptide was incubated with the purified enzyme, two products with the sequences AIEAPSTNG and NAHNFPLDLA, corresponding to the C-terminal extension and the C terminus of mD1, respectively, were eluted from the reverse-phase HPLC column (Fig.…”

Section: Cleavage Of C-terminal Oligopeptides By Ctpa Expressed In Ementioning

confidence: 99%

“…In previous studies, we analyzed the mechanism of the Cterminal processing of pD1 by utilizing partially purified protease from spinach chloroplasts and two types of substrate in solution: 1) synthetic oligopeptides corresponding to the Cterminal sequence of pD1 (25,28) and 2) in vitro transcribed/ translated full-length pD1 (18). An unexpected finding from these studies was that the proteolytic activity exhibited a strong pH dependence, with an optimum in the pH range of 7.5-8.0.…”

mentioning

confidence: 99%

Overexpression and Characterization of Carboxyl-terminal Processing Protease for Precursor D1 Protein

Yamamoto

Inagaki

Satoh

2001

Journal of Biological Chemistry

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CtpA, which is classified as a novel type of serine protease with a Ser/Lys catalytic dyad, is responsible for the C-terminal processing of precursor D1 protein (pD1) of the photosystem II reaction center, a process that is indispensable for the integration of water-splitting machinery in photosynthesis. In this study, overexpression in Escherichia coli and one-step purification of spinach CtpA were carried out to analyze the characteristics of this new type of protease and to elucidate the molecular interactions in the C-terminal processing of pD1 on the thylakoid membrane. The successful accumulation of functional CtpA in E. coli may argue against the possibility, based on homology to E. coli Tsp, that the enzyme is involved in the degradation of incomplete proteins in chloroplasts, e.g. by utilizing the ssrA-tagging system. Analysis using a synthetic pD1 oligopeptide demonstrated that the enzymatic properties (including substrate recognition) of overexpressed CtpA with an extra sequence of GSHMLE at the N terminus were indistinguishable from those of the native enzyme. CtpA was insensitive to penem, which has been shown to inhibit some Ser/Lys-type proteases, suggesting that the catalytic center of CtpA is quite unique. By using the substrate in different molecular environments (i.e. synthetic pD1 oligopeptide in solution and pD1 in photosystem II-enriched thylakoid membrane), we observed a dramatic difference in the pH profile and affinity for the substrate, suggesting the presence of a specific interaction of CtpA with a factor(s) that modulates the pH dependence of proteolytic action in response to physiological conditions. The D1 protein is a membrane-spanning subunit constituting the core part of the photosystem II (PSII) 1 reaction center (1, 2). In the predicted secondary structure, the D1 protein consists of five transmembrane ␣-helices and a C-terminal extension protruding into the luminal space of thylakoids (reviewed in Ref. 2). A curious feature of this protein is the extraordinarily high rate of metabolic turnover in vivo, despite its fundamental importance in the structure and function of PSII (3). In the dynamic process, the D1 protein, which is encoded by the psbA gene in the plastid genome in the case of eukaryotic organisms, is synthesized on membrane-associated ribosomes on the cytosolic or stromal surface of thylakoids in a precursor form with a short C-terminal extension consisting of 8 -16 amino acids (4 -6). This part of the protein is co-translationally translocated into the luminal space and then immediately removed by the action of an endopeptidase called the C-terminal processing protease (CtpA) (7-10). In eukaryotic organisms, the enzyme is nuclear encoded (8 -10) and imported from the cytosol to thylakoidal lumen (11). When present on the D1 protein, this extension's removal is absolutely essential for the integration of the machinery for water oxidation, i.e. the manganese cluster in PSII (12-15). However, the absence of this extension by itself at least under the conditions exam...

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“…In order to analyze the mechanism of this unique enzymatic process and to understand the role of the proteolytic processing in the integration of oxygen‐evolving machinery in the PSII, synthetic oligopeptides corresponding to the C‐terminal sequence of spinach pD1 have been utilized in in vitro analyses [11, 12]. In these studies, substituted C‐terminal oligopeptides were synthesized in order to analyze the recognition signal for the protease on pD1 [12].…”

Section: Introductionmentioning

confidence: 99%

Competitive inhibition analysis of the enzyme‐substrate interaction in the carboxy‐terminal processing of the precursor D1 protein of photosystem II reaction center using substituted oligopeptides

Yamamoto

Satoh

1998

FEBS Letters

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A clear parallelism was demonstrated between the efficiency as substrate of the substituted oligopeptides corresponding to the carboxy-terminal (C-terminal) sequence of the precursor D1 protein (pD1) in the in vitro enzymatic assay and their competitive inhibitory capacity toward the proteolytic Cterminal processing of the full-length pD1 integrated in the intact photosystem II complex embedded in the thylakoid membrane of Scenedesmus obliquus LF-1 mutant, as shown e.g. by the influence of L343A, A345G and A345V substitutions and the effect of C-terminal fragments. This suggests that the basic mechanism for substrate recognition by the processing protease elucidated in the enzymatic analysis using synthetic oligopeptides is also effective in vivo, although it can sometimes be difficult to detect the consequence of amino acid substitution in the integrated systems.z 1998 Federation of European Biochemical Societies.

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Cleavage of a synthetic COOH‐terminal oligopeptide of D1 precursor protein by a purified processing enzyme

Cited by 17 publications

References 12 publications

Folding and Misfolding in a Naturally Occurring Circularly Permuted PDZ Domain

Folding and Misfolding in a Naturally Occurring Circularly Permuted PDZ Domain

Overexpression and Characterization of Carboxyl-terminal Processing Protease for Precursor D1 Protein

Competitive inhibition analysis of the enzyme‐substrate interaction in the carboxy‐terminal processing of the precursor D1 protein of photosystem II reaction center using substituted oligopeptides

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