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Annotated Information

NsLTPs bind to a variety of lipid molecules and catalyze their transfer across membranes in vitro [2]. NsLTPs also have additional biological functions, including biosynthesis of cutin, involvement in defense against pathogens, and managing abiotic stress conditions imparted by temperature or drought [2] and [3]. The nsLTP superfamily possesses eight highly conserved cysteine residues forming four disulfide bonds [1] and [4]. NsLTPs are subdivided into two subfamilies that differ in molecular mass, nsLTP1 (9 kDa), and nsLTP2 (7 kDa) [1].

Sequence similarities

Belongs to the plant LTP family. B11E subfamily.

Mass spectrometry

Molecular mass is 7001.8 Da from positions 1 - 69. Determined by ESI.[5]

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Transfer lipids across membranes. May play a role in plant defense or in the biosynthesis of cuticle layers.

Structure information

The structure of nsLTP2 was obtained using 813 distance constraints, 30 hydrogen bond constraints, and 19 dihedral angle constraints. Fifteen of the 50 random simulated annealing structures satisfied all of the constraints and possessed good nonbonded contacts. The novel three-dimensional fold of rice nsLTP2 contains a triangular hydrophobic cavity formed by three prominent helices. The four disulfide bonds required for stabilization of the nsLTP2 structure show a different pattern of cysteine pairing compared with nsLTP1. The C terminus of the protein is very flexible and forms a cap over the hydrophobic cavity. Molecular modeling studies suggested that the hydrophobic cavity could accommodate large molecules with rigid structures, such as sterols. The positively charged residues on the molecular surface of nsLTP2 are structurally similar to other plant defense proteins (As showed follow)[6]

Structure of Rice nsLTP2—The rice nsLTP2 is a predominantly �-helical protein consisting of three prominent helices within the N-terminal 40 amino acids. The well conserved cysteine residues form four disulfide bonds to stabilize the three-dimensional fold of the protein. The C-terminal amino acid residues, Lys41–His69, constitute a less structured region of the molecule with a high density of positively charged residues. The r.m.s.d. values for the backbone and all heavy atoms were 1.09 � 0.20 and 1.54 � 0.25 Å, respectively. The first 40 amino acids (Ala1–Ala40), constituting the rigid portion of the molecule, have r.m.s.d. values of 0.65 � 0.1 Å for the backbone and 0.95 � 0.15 Å for all heavy atoms. Superposition of the 15 NMR structures are shown as a stereo representation in Fig. 3A. Three helices of rice nsLTP2 positioned at Cys3–Ala16, Thr22–Ala31, and Gln33-Ala40 are colored green, red, and purple, respectively. Helices II and III are connected by a 90° turn to form a very rigid and unique structural motif. The curved helix I accommodates two disulfide bonds (Cys3–Cys35 and Cys11–Cys25). The flexible portion of the polypeptide contains two single-turn helices at positions Tyr45–Tyr48 and Ala54– Val58. A series of hydrophobic residues distributed throughout the nsLTP2 sequence combine to form a hydrophobic cavity. A continuous stretch of hydrophobic residues, Cys61–Ile65, near the C terminus forms a flexible cap over the hydrophobic cavity. The C-terminal region also contains two cysteines bridged to the rigid portion of the molecule (Cys26–Cys61 and Cys37– Cys68). These two disulfide bonds help to maintain the correct orientation of the hydrophobic cap. The final energy-minimized average structure of rice nsLTP2 is shown in Fig. 3B. A Pro- Check analysis of the three-dimensional structure revealed that only Ser59 and Ser60 are in the disallowed region, corresponding to 3.6% of the residues in the protein (19). These residues constitute a portion in the flexible C terminus that makes a very sharp turn to cover the hydrophobic cavity. Comparison of nsLTP2 with nsLTP1—The biophysical properties of the two subfamilies of nsLTP are very different. A higher concentration of GdnHCl is required to denature NsLTP2 (Cm �4.2 M) than nsLTP1 (Cm �3.0 M). NsLTP1 has unusual thermal stability (Tm �95 °C), but nsLTP2 could not be thermally denatured even at temperatures approaching 100 °C (data not shown). A primary sequence analysis using CLUSTAL W revealed a close relationship between these two subfamilies (7). The locations of cysteines, hydrophobic amino acids, and important positively charged residues are well conserved. There are, however, notable differences. In the -CXCmotif, an asparagine between the two cysteines in nsLTP1 is replaced by a hydrophobic amino acid, phenylalanine, in nsLTP2 . The disulfide bond pattern in nsLTP2 differs from nsLTP1 at the -CXC- motif (Fig.2). The hydrophobic residue in the -CXC- motif of nsLTP2 is buried inside the molecule, whereas the hydrophilic residue of nsLTP1 is at the surface . These observations suggest that the central residue of the -CXC- motif may govern the cysteine pairing and influence the overall fold of the protein.

File:Figure 2

Potential application in drug delivery

Potential application in drug delivery Plant non-specific lipid-transfer proteins (nsLTPs) have received an increasing interest as potential drug carriers in drug delivery systems. NsLTPs are subdivided into nsLTP1 (9 kDa) and nsLTP2 (7 kDa) according to the molecular weight. All of nsLTPs are highly stable proteins because they possess eight highly conserved cysteine residues forming four disulfide bonds. These highly stable proteins can protect drugs against oxidation or degradation. In this paper, the application of nsLTPs in a drug carrier systemwas comprehended through scanning chemical compounds to obtain the potential nsLTPs-binding drugs from the comprehensive medicinal chemistry (CMC) database. These results helped us to realize the binding differences for preferred drugs between maize nsLTP1 and rice nsLTP2. We have successfully constructed a rice nsLTP2 mutant (Y45W) to improve fluorescence sensitivity. The fluorescence binding assay showed that nsLTP2 can associate with sterol-like or triphenylmethane-like molecules but the binding affinities of nsLTP2 with both of nsLTP2-binding drug candidates are quite different. Dissociation constants (Kd) for sterol/nsLTP2 complexes is below one micromolar and it is sufficient for these molecules to slowly release in a controlled-release drug delivery process. In addition, titration curve shows that binding model for nsLTP2 with the triphenylmethyl moiety of the molecule is more complicated. The basic triphenyl ring system may be critical for the nsLTP2 association. These results imply that rice nsLTP2 have highly potential applications in pharmaceuticals. The procedure combined a unique computer-based high throughput screening (HTS) method with an experimental binding assay, can effectively determine potential nsLTPs-binding drugs from the compound library, thus increasing the added value of nsLTPs in a drug carrier system. [7]

Fatty acids binds affinity

Fatty acids binds affinity A computational study was carried out to identify the structural determinant controlling the affinity, specificity and binding strength of several saturated and unsaturated fatty acids with Oryza sativa(Indica group) nonspecific lipid transfer protein(nsLTP2).Association between the number, position and conformation of hydrophobic patches and lipid binding properties of the protein was evidenced by docking analysis .Binding affinity is influenced by the number of carbon atoms, location of double bonds and hydroxyl group in the acyl chain. The results may direct at developing application sinLTP-mediated transport and control led release o flow molecular weight drugs [8] The results would pave the routes for application of current methodology in nsLTP structural properties and may provide a convenient plat form for the development of protein-based drug carriers. The results would pave the routes for application of current methodology in nsLTP structural properties and may provide a convenient plat form for the development of protein-based drug carriers. Plant nonspecific lipid transfer protein 2 (nsLTP2) is a small (7 kDa) protein that binds lipid-like ligands. An inner hydrophobic cavity surrounded by a-helices is the defining structural feature of nsLTP2. Although nsLTP2 structures have been reported earlier, the detailed mechanisms of ligand binding and lipid transfer remain unclear. In this study, we used site-directed mutagenesis to determine the role of various hydrophobic residues (L8, I15, F36, F39, Y45, Y48, and V49) in the structure, stability, ligand binding, and lipid transfer activity of rice nsLTP2. Three single mutations (L8A, F36A, and V49A) drastically alter the native tertiary structure and perturb ligand binding and lipid transfer activity. Therefore, these three residues are structurally important. The Y45A mutant, however, retains a native-like structure but has decreased lipid binding affinity and lipid transfer activity, implying that this aromatic residue is critical for these biological functions. The mutants, I15A and Y48A, exhibit quite different ligand binding affinities. Y48 is involved in planar sterol binding but not linear lysophospholipid association. As for I15A, it had the highest dehydroergosterol binding affinity in spite of the lower lipid binding and transfer abilities. Our results suggest that the long alkyl side chain of I15 would restrict the flexibility of loop I (G13-A19) for sterol entry. Finally, F39A can markedly increase the exposed hydrophobic surface to maintain its transfer efficiency despite reduced ligand binding affinity. These findings suggest that the residues forming the hydrophobic cavity play various important roles in the structure and function of rice nsLTP2.


[1]J.P Douliez, T Michon, K Elmorjani, D Marion Structure, biological and technological functions of lipid transfer proteins and indolines, the major lipid binding proteins from cereal kernels J. Cereal Sci., 32 (2000), pp. 1–20

[2]J.C Kader Lipid transfer proteins in plants Annu. Rev. Plant Physiol. Plant Mol. Biol., 47 (1996), pp. 627–654

[3]K.L Larsen, J.R Winther Surprisingly high stability of barley lipid transfer protein, LTP1, towards denaturant, heat, and proteases FEBS Lett., 488 (2001), pp. 145–148

[4]J.P Douliez, C Pato, H Rabesona, D Molle, D Marion Disulfide bond assignment, lipid transfer activity and secondary structure of a 7-kDa plant lipid transfer protein, LTP2 Eur. J. Biochem., 268 (2001), pp. 1400–1403

[5]"Purification and characterization of a novel 7-kDa non-specific lipid transfer protein-2 from rice (Oryza sativa)." Liu Y.-J., Samuel D., Lin C.H., Lyu P.-C.

[6]Solution Structure of Plant Nonspecific Lipid Transfer Protein-2 from Rice (Oryza sativa)*(2012) Ping-Chiang Lyu

[7] Evaluation of plant non-specific lipid-transfer proteins for potential application in drug delivery Ping-Chiang Lyu

[8] Computational evaluation on the binding affinity of non-specific lipid-transfer protein-2 with fatty acids. Adam Matkowski

Structured Information