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  • Moreover recent structural studies of VEEV nsP


    Moreover, recent structural studies of VEEV nsP2pro in complex with E-64d revealed an important molecular interaction at the interface of two subdomains. In this structure, Asn475 which is in polar contact with carbonyl oxygen of Asp507 of the protease subdomain makes an H-bond with Arg662 of the MTase-like subdomain and aligns itself to assist the nucleophilic attack by the catalytic cysteine (PDB ID: 5EZS) and thus proposed to act like a transition state stabilizing residue [53] (Fig. 7A). In CHIKV nsP2pro, Asn476 is homologous toAsn475 of VEEV and is found to make polar contact with the backbone carbonyl oxygen of Asp509 (Fig. 7B & Fig. S3). Sequence alignment of CHIKV nsP2pro with VEEV revealed that Arg662 of VEEV nsP2pro is substituted by Thr665 in CHIKV nsP2pro. However, an adjacent residue Glu669 seems to be playing a role similar to Arg662 of VEEV nsP2pro, as Glu669 is positioned in such a way that it makes molecular contact with Asn476 in CHIKV nsP2pro structure (Fig. 7C). This structural analysis suggests that Asn475 might potentially play a role of a transition state stabilizing residue in CHIKV nsP2pro. This Asn residue is also found in other viral papain-like cysteine proteases like SARS PLpro, MERS PLpro and corona viral protease, where its function has been established as the transition state stabilizing residue [[58], [59], [60]]. The molecular interactions at the interface of the two subdomains near the active site specify the regulatory role of interface residues in the substrate binding and enzymatic activity of alphavirus nsP2pro. Additionally, Trp549, a highly conserved S2 subsite residue of the alphavirus nsP2pro is located in a shallow depression at the interface between the two subdomains and makes significant contribution in substrate binding. In CHIKV nsP2 crystal structure, the side chain of Trp549 is H-bonded to a glycerol molecule present near the active site at the substrate binding subsites P2/P3, which might mimic the molecular interaction with that of actual substrate (Fig. 4C).
    Availability of supporting data The atomic coordinates and structure factor amplitudes are available in the Protein Data Bank repository ( with accession number 4ZTB.
    Introduction Cereal seeds undergo two phases of growth, development and germination, which are separated by a 2-Deoxyadenosine 5-diphosphate of dormancy (Domínguez and Cejudo, 2014). While both phases require the growth and differentiation of new tissues, proteolysis is also an essential process for the programed cell death of individual seed tissues and the metabolism of cereal proteins (Bozhkov et al., 2004). Cysteine proteases (CPs) have been shown to play essential roles during the growth of cereal seeds. These enzymes participate in the maturation of various proteins, the degradation of storage proteins in germinating seeds and the elimination of endogenous, unnecessary or incorrectly synthesized proteins and peptides (Cejudo et al., 2001). Moreover, it should be emphasized that the lack of cysteine protease-mediated control of these processes causes irreversible and damaging alterations of plant development, such as premature germination of seeds in the ear, which is called pre-harvest sprouting (PHS). The most direct regulators of cysteine protease activities are their natural inhibitors, which are called phytocystatins (PhyCys) (Arai et al., 2002, Bode and Huber, 1992). PhyCys have been shown to control the activities of the endogenous cysteine proteases involved in the growth of cereal seeds and the mobilization of storage proteins (Arai et al., 2002, Margis et al., 1998, Martínez et al., 2005a).
    Development and germination of cereal seeds Caryopses (cereal grains) are the propagation units of many monocot species, such as wheat, triticale, barley and other cereal species. A caryopsis is a type of dry fruit composed of three tissues: the diploid embryo, the triploid endosperm and the maternal pericarp and seed coat (Sreenivasulu et al., 2006) (Fig. 1A). The embryo is a miniature of the new plant which contains: 1. the embryonic axis with the embryonic bud (plumule) and the embryonic root (radicle) surrounded by the coleoptile (leaf sheath) and coleorhiza (root sheath) respectively; 2. the cotyledon (one) called the scutellum. During germination the scutellum participates in the transfer of gibberellin phytohormones (secreted by the embryo), which signals the aleurone layer to begin or to accelerate the synthesis and secretion of hydrolytic enzymes (Waterworth et al., 2005). Subsequently, the scutellum transports hydrolytic products from endosperm to embryo (Subbarao et al., 1998). The endosperm is a storage tissue of cereal seeds, divided into the internal part, called starch endosperm and the external part, called the aleurone layer. The main function of this tissue is to provide nutrients to the embryo during development and germination (Sabelli and Larkins, 2009).