The K-Segment of Maize DHN1 Mediates Binding to Anionic Phospholipid Vesicles and Concomitant Structural Changes

Dehydrins (DHNs; late embryogenesis abundant D11 family) are a family of intrinsically unstructured plant proteins that accumulate in the late stages of seed development and in vegetative tissues subjected to water deficit, salinity, low temperature, or abscisic acid treatment. We demonstrated previously that maize (Zea mays) DHNs bind preferentially to anionic phospholipid vesicles; this binding is accompanied by an increase in α-helicity of the protein, and adoption of α-helicity can be induced by sodium dodecyl sulfate. All DHNs contain at least one “K-segment,” a lysine-rich 15-amino acid consensus sequence. The K-segment is predicted to form a class A2 amphipathic α-helix, a structural element known to interact with membranes and proteins. Here, three K-segment deletion proteins of maize DHN1 were produced. Lipid vesicle-binding assays revealed that the K-segment is required for binding to anionic phospholipid vesicles, and adoption of α-helicity of the K-segment accounts for most of the conformational change of DHNs upon binding to anionic phospholipid vesicles or sodium dodecyl sulfate. The adoption of structure may help stabilize cellular components, including membranes, under stress conditions.When plants encounter environmental stresses such as drought or low temperature, various responses take place to adapt to these conditions. Typical responses include increased expression of chaperones, signal transduction pathway and late embryogenesis abundant (LEA) proteins, osmotic adjustment, and induction of degradation and repair systems (Ingram and Bartels, 1996).Dehydrins (DHNs; LEA D11 family) are a subfamily of group 2 LEA proteins that accumulate to high levels during late stages of seed development and in vegetative tissues subjected to water deficit, salinity, low temperature, or abscisic acid (ABA) treatment (Svensson et al., 2002). Some DHNs are expressed constitutively during normal growth (Nylander et al., 2001; Rorat et al., 2004, 2006; Rodriguez et al., 2005). DHNs exist in a wide range of photosynthetic organisms, including angiosperms, gymnosperms, algae, and mosses (Svensson et al., 2002). DHNs are encoded by a dispersed multigene family and are differentially regulated, at least in higher plants. For example, 13 Dhn genes have been identified in barley (Hordeum vulgare), dispersed over seven genetic map locations (Choi et al., 1999; Svensson et al., 2002) and regulated variably by drought, low temperature, and embryo development (Tommasini et al., 2008). DHNs are localized in various subcellular compartments, including cytosol (Roberts et al., 1993), nucleus (Houde et al., 1995), chloroplast (Artus et al., 1996), vacuole (Heyen et al., 2002), and proximal to the plasma membrane and protein bodies (Asghar et al., 1994; Egerton-Warburton et al., 1997; Puhakainen et al., 2004). Elevated expression of Dhn genes generally has been correlated with the acquisition of tolerance to abiotic stresses such as drought (Whitsitt et al., 1997), salt (Godoy et al., 1994; Jayaprakash et al., 1998), chilling (Ismail et al., 1999a), or freezing (Houde et al., 1995; Danyluk et al., 1998; Fowler et al., 2001). The differences in expression and tissue location suggest that individual members of the Dhn multigene family have somewhat distinct biological functions (Close, 1997; Zhu et al., 2000; Nylander et al., 2001). Many studies have observed a positive correlation between the accumulation of DHNs and tolerance to abiotic stresses (Svensson et al., 2002). However, overexpression of a single DHN protein has not, in general, been sufficient to confer stress tolerance (Puhakainen et al., 2004).DHNs are subclassified by sequence motifs referred to as the K-segment (Lys-rich consensus sequence), the Y-segment (N-terminal conserved sequence), the S-segment (a tract of Ser residues), and the φ-segment (Close, 1996). Because of high hydrophilicity, high content of Gly (>20%), and the lack of a defined three-dimensional structure in the pure form (Lisse et al., 1996), DHNs have been categorized as “intrinsically disordered/unstructured proteins” or “hydrophilins” (Wright and Dyson, 1999; Garay-Arroyo et al., 2000; Tompa, 2005; Kovacs et al., 2008). On the basis of compositional and biophysical properties and their link to abiotic stresses, several functions of DHNs have been proposed, including ion sequestration (Roberts et al., 1993), water retention (McCubbin et al., 1985), and stabilization of membranes or proteins (Close, 1996, 1997). Observations from in vitro experiments include DHN binding to lipid vesicles (Koag et al., 2003; Kovacs et al., 2008) or metals (Svensson et al., 2000; Heyen et al., 2002; Kruger et al., 2002; Alsheikh et al., 2003; Hara et al., 2005), protection of membrane lipid against peroxidation (Hara et al., 2003), retention of hydration or ion sequestration (Bokor et al., 2005; Tompa et al., 2006), and chaperone activity against the heat-induced inactivation and aggregation of various proteins (Kovacs et al., 2008).Intrinsically disordered/unstructured proteins that lack a well-defined three-dimensional structure have recently been recognized to be prevalent in prokaryotes and eukaryotes (Oldfield et al., 2005). They fulfill important functions in signal transduction, gene expression, and binding to targets such as protein, RNA, ions, and membranes (Wright and Dyson, 1999; Tompa, 2002; Dyson and Wright, 2005). The disorder confers structural flexibility and malleability to adapt to changes in the protein environment, including water potential, pH, ionic strength, and temperature, and to undergo structural transition when complexed with ligands such as other proteins, DNA, RNA, or membranes (Prestrelski et al., 1993; Uversky, 2002). Structural changes from disorder to ordered functional structure also can be induced by the folding of a partner protein (Wright and Dyson, 1999; Tompa, 2002; Mouillon et al., 2008).The idea that DHNs interact with membranes is consistent with many immunolocalization studies, which have shown that DHNs accumulate near the plasma membrane or membrane-rich areas surrounding lipid and protein bodies (Asghar et al., 1994; Egerton-Warburton et al., 1997; Danyluk et al., 1998; Puhakainen et al., 2004). The K-segment is predicted to form a class A2 amphipathic α-helix, in which hydrophilic and hydrophobic residues are arranged on opposite faces (Close, 1996). The amphipathic α-helix is a structural element known to interact with membranes and proteins (Epand et al., 1995). Also, in the presence of helical inducers such as SDS and trifluoroethanol (Dalal and Pio, 2006), DHNs take on α-helicity (Lisse et al., 1996; Ismail et al., 1999b). We previously examined the binding of DHN1 to liposomes and found that DHNs bind preferentially to anionic phospholipids and that this binding is accompanied by an increase in α-helicity of the protein (Koag et al., 2003). Similarly, a mitochondrial LEA protein, one of the group III LEA proteins, recently has been shown to interact with and protect membranes subjected to desiccation, coupled with the adoption of amphipathic α-helices (Tolleter et al., 2007).Here, we explore the basis of DHN-vesicle interaction using K-segment deletion proteins. This study reveals that the K-segment is necessary and sufficient for binding to anionic phospholipid vesicles and that the adoption of α-helicity of DHN proteins can be attributed mainly to the K-segment.