Os03g0111300

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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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Function

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.

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]

Reference

[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

Gene Name	Os03g0111300
Description	Nonspecific lipid-transfer protein 2 (nsLTP2) (7 kDa lipid transfer protein)
Version	NM_001055258.1 GI:115450244 GeneID:4331362
Length	476 bp
Definition	Oryza sativa Japonica Group Os03g0111300, complete gene.
Source	Oryza sativa Japonica Group ORGANISM Oryza sativa Japonica Group Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP clade; Ehrhartoideae; Oryzeae; Oryza.
Chromosome	Chromosome 3
Location	Chromosome 3:630670..631145
Sequence Coding Region	630809..631099
Expression	GEO Profiles:Os03g0111300
Genome Context	<gbrowseImage1> name=NC_008396:630670..631145 source=RiceChromosome03 preset=GeneLocation </gbrowseImage1>
Gene Structure	<gbrowseImage2> name=NC_008396:630670..631145 source=RiceChromosome03 preset=GeneLocation </gbrowseImage2>
Coding Sequence	<cdnaseq>atgatgaggaagttggcggtgttggtgttggcggtggcgatggtggcggcgtgcggcggcggcgtcgtgggtgtagcgggggccggttgcaacgctgggcagctgacggtgtgcacgggggcgatcgcgggcggggcgcggccgacggcggcgtgctgctccagcctgcgggcgcagcagggctgcttctgccagttcgccaaggacccgcgctacgggcgctacgtcaacagccccaacgcccgcaaggccgtctcctcctgcggcatcgccctccccacctgccactga</cdnaseq>
Protein Sequence	<aaseq>MMRKLAVLVLAVAMVAACGGGVVGVAGAGCNAGQLTVCTGAIAG GARPTAACCSSLRAQQGCFCQFAKDPRYGRYVNSPNARKAVSSCGIALPTCH</aaseq>
Gene Sequence	<dnaseqindica>47..337#gtacctcgcagcaccaagctagctagcttcgatcagtagctggaggatgatgaggaagttggcggtgttggtgttggcggtggcgatggtggcggcgtgcggcggcggcgtcgtgggtgtagcgggggccggttgcaacgctgggcagctgacggtgtgcacgggggcgatcgcgggcggggcgcggccgacggcggcgtgctgctccagcctgcgggcgcagcagggctgcttctgccagttcgccaaggacccgcgctacgggcgctacgtcaacagccccaacgcccgcaaggccgtctcctcctgcggcatcgccctccccacctgccactgatccatccatccatcgtctcctactcttttattttgtgatgtggtgtacgtgttcgtagtattgtcttggcgtcatctcgtcacgacgcgtatatgcatgcgcagtgcggcttttgaataaaaggggatcgaccgatgtt</dnaseqindica>
External Link(s)	NCBI Gene:Os03g0111300, RefSeq:Os03g0111300