New insights into ligand binding by plant lipid transfer proteins: A case study of the lentil Lc-LTP2

https://doi.org/10.1016/j.bbrc.2020.04.139Get rights and content

Highlights

  • Arg45 and Tyr80 play an important role in stabilization of lentil Lc-LTP2 complexes with both fatty acids and lysolipids.

  • Mutant analogs R45A, Y80A, R45A/Y80A worse than wild-type Lc-LTP2 bind lipid ligands.

  • Replacement of Arg45 or/and Tyr80 leads to a change of the internal hydrophobic cavity dimensions.

  • Replacement of one or both residues changes ligand orientation in the protein cavity.

  • Possible orientation C for a ligand into the hydrophobic cavity of plant LTPs has been described for the first time.

Abstract

Lipid transfer proteins (LTPs) are an important class of plant proteins containing an internal cavity and binding hydrophobic ligands. Although LTP structures and functions are well studied, mechanisms of ligand binding remain unclear. Earlier, we discovered the lentil lipid transfer protein Lc-LTP2 capable of binding and transfer various ligands. We have shown that the “bottom” entrance of the Lc-LTP2 cavity takes part in attachment to the micelle surface and in lipids uptake. Here, we studied the role of Arg45 and Tyr80, located at the “bottom” entrance, in Lc-LTP2 ligand binding. We obtained recombinant mutant analogs of Lc-LTP2 (R45A, Y80A, R45A/Y80A), investigated their ability to bind fatty acids and lysolipids, as well as performed molecular modeling of the protein-ligand complexes. We showed that replacement of one or both residues led to a change of the internal hydrophobic cavity dimensions. As a result, lipids may change their orientation into the protein cavity, and thereby binding ability of mutant analogs may be affected as well. In the present work, we revealed an important role of Arg45 and Tyr80 in stabilization of the Lc-LTP2 complexes with both fatty acids and lysolipids with different ligand orientation.

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)

There are more references available in the full text version of this article.

Cited by (7)

View all citing articles on Scopus
View full text