New insights into ligand binding by plant lipid transfer proteins: A case study of the lentil Lc-LTP2
Introduction
Plant lipid transfer proteins (LTPs) comprise an important class of basic proteins that are capable of binding and transfer lipid molecules. They are characterized by presence a tunnel-like hydrophobic cavity in their fold. LTP structure is stabilized by four disulfide bridges and includes four helices connected to the C-terminal part formed by a series of turns [1].
Biophysical studies have demonstrated that LTPs can bind a broad range of hydrophobic molecules such as phospho- and glycolipids, acyl-coenzyme A, fatty acids, and prostaglandins [2].Their ability to form complexes with ligands depends on the size of the protein hydrophobic cavity and the chemical nature of amino acid residues located there. Functions of plant LTPs in vivo are not completely understood, but most likely, their ability to intermediate a systemic, intracellular, and extracellular transport of hydrophobic ligands plays a key role [3,4]. Presumably, this ability predetermines their multifunctionality: they are involved in growth and development of plants, their protection under stress conditions, and cuticular wax formation [4]. In addition, these proteins are clinically significant plant allergens involved in IgE-mediated allergic reactions of various severity [5]. Recent data revealed that the presence of lipid ligands affected the stability and sensitization potential of plant LTPs [6]. All the above creates the need to deepen our knowledge of lipid-binding properties of plant LTPs.
LTPs have been shown to interact with a ligand according to the cooperative binding model. Hydrophobic cavity of LTPs has two entrances differing by size. The cavity can accommodate one or two lipid molecules placed in two opposite orientations. Despite conserved sequences and fold of the proteins, not all LTPs bind identical ligands in the same orientation [7,8]. Amino acid residues inside the hydrophobic cavity may be considered as the determinants of the specificity of the protein-ligand molecular association.
Earlier, we showed that Lc-LTP2 from the lentil Lens culinaris seeds bound and transferred fatty acids and lysolipids with different effectiveness [9]. High selectivity of Lc-LTP2 towards anionic lipids was also demonstrated. We showed that the “bottom” entrance of Lc-LTP2 played an important role in the protein attachment to the membrane or micelle surface and in the lipid uptake [10]. In this study, we investigated the role of two residues belonging to the “bottom” entrance of Lc-LTP2 - aromatic polar Y80 and basic R45, their effects on the microarchitecture of the protein hydrophobic cavity, ligand specificity and effectiveness of lipid binding (see Table 1).
Section snippets
Production of Lc-LTP2 and its mutant analogs
The expression plasmids pET-His8-TrxL-Lc-LTP2(R45A), pET-His8-TrxL-Lc-LTP2(Y80A) and pET-His8-TrxL-Lc-LTP2(R45A/Y80A) were obtained by site-directed mutagenesis of the original plasmid pET-His8-TrxL-Lc-LTP2 using full-length inverse PCR amplification with mutagenizing primers (Table S1; Supplementary material). The resulting amplicons were recircularized in vivo after E. coli DH10B transformation through a mechanism of RecA-independent homology recombination [11]. The length of the homology
Secondary structure and relative stability of Lc-LTP2 and its mutant analogs
Protein secondary structures were determined by CD spectroscopy in the far-UV region (Fig. 1A). The CD spectra of Lc-LTP2 and the mutant analogs (R45A, Y80A and R45A/Y80A) had a maximum at 192 nm and double minima at 208 and 222 nm, typical of a predominantly α-helical structure. The presence of LPPG, unlike STE, slightly increased α-helical content for all proteins except the Y80A analog (Fig. 1, Table S2; Supplementary material). The results of thermal denaturation indicated that the
Discussion
For the last 20 years, ligands binding by plant LTPs were studied. However, it still remains obscure what is the mechanism of this process. Ligand binding includes its uptake and retention inside the protein cavity. An initial protein-ligand interaction plays a key role in the lipid uptake. It was shown that myristic acid binds to the N-terminal part of LTP from Solanum melongena leading to partial unlocking of the protein hydrophobic cavity and ligand internalization into it [19]. On the other
Conclusions
In the present work, we showed that Arg45 and Tyr80 play an important role in the Lc-LTP2 binding of both fatty acids and lysolipids. Moreover, functional significances of these residues are similar at some points. We assume that Arg45 and Tyr80 located at the “bottom” entrance of the Lc-LTP2 cavity take part in stabilization of the protein-ligand complexes with different ligand orientation. The replacement of Arg45 and Tyr80 leads to a significant change in space dimensions of the hydrophobic
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by the Russian Science Foundation (project no.19-74-00150).
References (20)
- et al.
Plant lipid transfer proteins: are we finally closing in on the roles of these enigmatic proteins?
J. Lipid Res.
(2018) - et al.
In vivo DNA assembly using common laboratory bacteria: a re-emerging tool to simplify molecular cloning
J. Biol. Chem.
(2019) - et al.
Recombinant production and solution structure of lipid transfer protein from lentil Lens culinaris
Biochem. Biophys. Res. Commun.
(2013) - et al.
Lipid transfer proteins as components of the plant innate immune system: structure, functions, and applications
Acta Naturae
(2016) - et al.
Plant pathogenesis-related proteins binding lipids and other hydrophobic ligands
Russ. J. Bioorg. Chem.
(2018) - et al.
New aspects of Phloem-mediated long-distance lipid signaling in plants
Front. Plant Sci.
(2012) - et al.
The clinical relevance of lipid transfer protein
Clin. Exp. Allergy
(2018) - et al.
Interaction of non-specific lipid-transfer proteins with plant-derived lipids and its impact on allergic sensitization
Front. Immunol.
(2018) - et al.
Computational study of ligand binding in lipid transfer proteins: structures, interfaces, and free energies of protein-lipid complexes
J. Comput. Chem.
(2012) - et al.
Molecular dynamics simulations of barley and maize lipid transfer proteins show different ligand binding preferences in agreement with experimental data
Biochemistry
(2013)
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Plant non-specific lipid transfer proteins: An overview
2022, Plant Physiology and BiochemistryCitation Excerpt :The fact that lipid ligands prefer to access the inner cavity of nsLTPs in a specific orientation is supported by nuclear magnetic resonance and high-resolution X-ray studies performed in maize (Han et al., 2001), lentil (Shenkarev et al., 2017), rice (Cheng et al., 2004), and wheat nsLTPs. Nevertheless, the specific location in the protein to initiate this interaction seems to be dependent on the nsLTP (Melnikova et al., 2020b). In some cases, even the same ligand can be allocated in different positions inside the cavity; e.g., nsLTP1 from wheat is able to interact simultaneously with two molecules of the same ligand displaying opposite orientations (Cheng et al., 2004).
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