Structural Mechanism and Photoprotective Function of Water-soluble Chlorophyll-binding Protein

Abstract

A water-soluble chlorophyll-binding protein (WSCP) is the single known instance of a putative chlorophyll (Chl) carrier in green plants. Recently the photoprotective function of WSCP has been demonstrated by EPR measurements; the light-induced singlet-oxygen formation of Chl in the WSCP tetramer is about four times lower than that of unbound Chl. This paper describes the crystal structure of the WSCP-Chl complex purified from leaves of Lepidium virginicum (Virginia pepperweed) to clarify the mechanism of its photoprotective function. The WSCP-Chl complex is a homotetramer comprising four protein chains of 180 amino acids and four Chl molecules. At the center of the complex one hydrophobic cavity is formed in which all of the four Chl molecules are tightly packed and isolated from bulk solvent. With reference to the novel Chl-binding mode, we propose that the photoprotection mechanism may be based on the inhibition of physical contact between the Chl molecules and molecular oxygen.Water-soluble chlorophyll-binding proteins (WSCPs), 4 which form a complex with chlorophyll (Chl), have been isolated from Amaranthaceae, Chenopodiaceae, and Polygonaceae (class-I) and from Brassicaceae (class-II). The two WSCP classes differ in that the protein-Chl complexes of the former change their absorption spectra upon illumination, whereas those of the latter show no photoconversion of their absorption behavior (2). The amino acid sequence of class-I WSCP shows no similarity to that of class-II WSCP.The class-II WSCPs (hereafter referred to as "WSCPs") are water-soluble proteins of ϳ20 kDa, which form a tetrameric assembly upon binding Chl molecules with a Chl-to-protein ratio of one or less, and exhibit high thermal and photostability (1, 3). The physiological function of WSCPs is still not known. Although WSCPs have a sequence similarity of ϳ30% with Künitz-type proteinase inhibitors, no significant protease inhibitor activity of WSCPs has yet been identified (4 -6). The low Chl content per protein makes it unlikely that WSCPs are involved in the light reaction of photosynthesis. Meanwhile, it has been speculated (7, 8) that WSCP acts as a scavenger of free Chl, by transporting it from the thylakoid membrane to the chloroplast envelope, where Chlase, the enzyme that initiates the Chl catabolism, is thought to reside (9).An in vitro binding assay with the recombinant apo-WSCP cloned from cauliflower (Brassica oleralcea var. Botrys) and Chl or its derivatives showed that the central Mg 2ϩ ion and the phytyl tail of Chl were essential for protein-pigment binding and tetrameric assembly, respectively (1). Furthermore, the recombinant apo-WSCP was able to remove Chl from the thylakoid membrane in vitro and organize the tetrameric WSCPChl complex (3). The absorption spectrum of the reconstituted complex is very similar to that of the native WSCP-Chl complex purified from fresh leaves (1, 3).The WSCP-Chl complex retains its fresh green color under dim light for months, enough for a long term crystallization experiment. Free Chl, on t...

MP-4 Contributes to Snake Venom Neutralization by Mucuna pruriens Seeds through an Indirect Antibody-mediated Mechanism

“…2B). It was therefore unlikely that MP-4 belongs to the WSCP class because these proteins are normally tetrameric (37). Using mass spectrometry, the molecular mass of MP-4 was found to be 20.9 kDa (Fig.…”

A Proteomic Profiling Approach to Reveal a Novel Role of Brassica napus Drought 22 kD/Water-Soluble Chlorophyll-Binding Protein in Young Leaves during Nitrogen Remobilization Induced by Stressful Conditions

“…Interestingly, BnD22 is a class II WSCP that is able to bind chlorophyll (Chl a/b) and chlorophyll precursors such as chlorophyllides a and b in tetrameric forms (Nishio and Satoh, 1997;Schmidt et al, 2003;Horigome et al, 2007). It has been proposed that WSCP as a chlorophyll carrier implied either chlorophyll degradation (Satoh et al, 1998) or chlorophyll biosynthesis (Schmidt et al, 2003;Reinbothe et al, 2004).…”

“…This WSCP is able to bind chlorophyll molecules, suggesting a role as a chlorophyll carrier (Satoh et al, 2001;Reinbothe et al, 2004) and an involvement in the regulation of both the development and senescence of chloroplasts. Recently, Horigome et al (2007) have suggested this WSCP may contribute to the photoprotection of chlorophyll molecules.…”

Protein and solvent dynamics of the water-soluble chlorophyll-binding protein (WSCP)

“…Class-II WSCPs are watersoluble and possess a molecular weight of about 20 kDa. Up to now, a crystal structure obtained by X-ray diffraction is available only for WSCP from Lepidium virginicum [9]. However, all class-II WSCPs occur in tetrameric form binding 1-4 chlorophyll molecules.…”