Lipid binding in rice nonspecific lipid transfer protein-1 complexes from Oryza sativa

Nonspecific lipid transfer proteins (nsLTPs) facilitate the transfer of phospholipids, glycolipids, fatty acids and steroids between membranes, with wide-ranging binding affinities. Three crystal structures of rice nsLTP1 from Oryza sativa, complexed with myristic (MYR), palmitic (PAL) or stearic acid (STE) were determined. The overall structures of the rice nsLTP1 complexes belong to the four-helix bundle folding with a long C-terminal loop. The nsLTP1-MYR and the nsLTP1-STE complexes bind a single fatty acid while the nsLTP1-PAL complex binds two molecules of fatty acids. The C-terminal loop region is elastic in order to accommodate a diverse range of lipid molecules. The lipid molecules interact with the nsLTP1-binding cavity mainly with hydrophobic interactions. Significant conformational changes were observed in the binding cavity and the C-terminal loop of the rice nsLTP1 upon lipid binding.     

Magnetization transfer from laser-polarized xenon to protons located in the hydrophobic cavity of the wheat nonspecific lipid transfer protein

Nonspecific lipid transfer protein from wheat is studied by liquid-state NMR in the presence of xenon. The gas-protein interaction is indicated by the dependence of the protein proton chemical shifts on the xenon pressure and formally confirmed by the first observation of magnetization transfer from laser-polarized xenon to the protein protons. Twenty-six heteronuclear nOes have allowed the characterization of four interaction sites inside the wheat ns-LTP cavity. Their locations are in agreement with the variations of the chemical shifts under xenon pressure and with solvation simulations. The richness of the information obtained by the noble gas with a nuclear polarization multiplied by approximately 12,000 makes this approach based on dipolar cross-relaxation with laser-polarized xenon promising for probing protein hydrophobic pockets at ambient pressure.     

Effects of ligand binding on the dynamics of rice nonspecific lipid transfer protein 1: a model from molecular simulations

Plant nonspecific lipid transfer proteins (nsLTPs) are small, basic proteins constituted mainly of alpha-helices and stabilized by four conserved disulfide bridges. They are characterized by the presence of a tunnel-like hydrophobic cavity, capable of transferring various lipid molecules between lipid bilayers in vitro. In this study, molecular dynamics (MD) simulations were performed at room temperature to investigate the effects of lipid binding on the dynamic properties of rice nsLTP1. Rice nsLTP1, either in the free form or complexed with one or two lipids was subjected to MD simulations. The C-terminal loop was very flexible both before and after lipid binding, as revealed by calculating the root-mean-square fluctuation. After lipid binding, the flexibility of some residues that were not in direct contact with lipid molecules increased significantly, indicating an increase of entropy in the region distal from the binding site. Essential dynamics analysis revealed clear differences in motion between unliganded and liganded rice nsLTP1s. In the free form of rice nsLTP1, loop1 exhibited the largest directional motion. This specific essential motion mode diminished after binding one or two lipid molecules. To verify the origin of the essential motion observed in the free form of rice nsLTP1, we performed multiple sequence alignments to probe the intrinsic motion encoded in the primary sequence. We found that the amino acid sequence of loop1 is highly conserved among plant nsLTP1s, thus revealing its functional importance during evolution. Furthermore, the sequence of loop1 is composed mainly of amino acids with short side chains. In this study, we show that MD simulations, together with essential dynamics analysis, can be used to determine structural and dynamic differences of rice nsLTP1 upon lipid binding.     

Crystal structure of nonspecific lipid transfer protein from Solanum melongena

Lipids are considered to protect protein allergens from proteolysis and are generally seen to exist in a bound form. One of the well‐known plant protein families with bound lipids is non‐specific lipid transfer proteins (nsLTPs). Structure‐function relationships in the case of the members of non‐specific lipid transfer protein family are not clearly understood. As part of exploring the seed proteome, we have analyzed the proteome of a member of Solanaceae family, Solanum melongena (eggplant) and a non‐specific lipid transfer protein from S. melongena, SM80.2 was purified, crystallized and the structure was determined at 1.87 Å resolution. Overall, the tertiary structure is a cluster of α‐helices forming an internal hydrophobic cavity. Absence of conserved Tyr79, known to govern the plasticity of hydrophobic cavity, and formation of hydrogen bond between Asn79 and Asn36 further reduced the pocket size. Structural analysis of SM80.2 thus gives insight about a new hydrogen bond mediated mechanism followed in closure of the binding pocket. Extra electron densities observed at two different places on the protein surface and not in the cavity could provide interesting physiological relevance. In light of allergenic properties, probably overlapping of epitopic region and ligand binding on surface could be a main reason. This work shows first crystal structure of A‐like nsLTP with a close binding pocket and extra density on the surface suggesting a plausible intermediate state during transfer.     

Tolerance to the substitution of buried apolar residues by charged residues in the homologous protein structures

Occurrence and accommodation of charged amino acid residues in proteins that are structurally equivalent to buried non-polar residues in homologues have been investigated. Using a dataset of 1,852 homologous pairs of crystal structures of proteins available at 2A or better resolution, 14,024 examples of apolar residues in the structurally conserved regions replaced by charged residues in homologues have been identified. Out of 2,530 cases of buried apolar residues, 1,677 of the equivalent charged residues in homologues are exposed and the rest of the charged residues are buried. These drastic substitutions are most often observed in homologous protein pairs with low sequence identity (<30%) and in large protein domains (>300 residues). Such buried charged residues in the large proteins are often located in the interface of sub-domains or in the interface of structural repeats, Beyond 7A of residue depth of buried apolar residues, or less than 4% of solvent accessibility, almost all the substituting charged residues are buried. It is also observed that acidic sidechains have higher preference to get buried than the positively charged residues. There is a preference for buried charged residues to get accommodated in the interior by forming hydrogen bonds with another sidechain than the main chain. The sidechains interacting with a buried charged residue are most often located in the structurally conserved regions of the alignment. About 50% of the observations involving hydrogen bond between buried charged sidechain and another sidechain correspond to salt bridges. Among the buried charged residues interacting with the main chain, positively charged sidechains form hydrogen bonds commonly with main chain carbonyls while the negatively charged residues are accommodated by hydrogen bonding with the main chain amides. These carbonyls and amides are usually located in the loops that are structurally variable among homologous proteins.     

Comparison of solution and crystal structures of maize nonspecific lipid transfer protein: A model for a potential in vivo lipid carrier protein

The three-dimensional solution structure of maize nonspecific lipid transfer protein (nsLTP) obtained by nuclear magnetic resonance (NMR) is compared to the X-ray structure. Although both structures are very similar, some local structural differences are observed in the first and the fourth helices and in several side-chain conformations. These discrepancies arise partly from intermolecular contacts in the crystal lattice. The main characteristic of nsLTP structures is the presence of an internal hydrophobic cavity whose volume was found to vary from 237 to 513 ų without major variations in the 15 solution structures. Comparison of crystal and NMR structures shows the existence of another small hollow at the periphery of the protein containing a water molecule in the X-ray structure, which could play an important structural role. A model of the complexed form of maize nsLTP by α-lysopalmitoylphosphatidylcholine was built by docking the lipid inside the protein cavity of the NMR structure. The main structural feature is a hydrogen bond found also in the X-ray structure of the complex maize nsLTP/palmitate between the hydroxyl of Tyr81 and the carbonyl of the lipid. Comparison of 12 primary sequences of nsLTPs emphasizes that all residues delineating the cavities calculated on solution and X-ray structures are conserved, which suggests that this large cavity is a common feature of all compared plant nsLTPs. Furthermore several conserved basic residues seem to be involved in the stabilization of the protein architecture. Proteins 31:160–171, 1998. © 1998 Wiley-Liss, Inc.     

Structural insights into the lipid transfer mechanism of a non‐specific lipid transfer protein

The non‐specific lipid transfer proteins (nsLTPs) are multifunctional seed proteins engaged in several different physiological processes. The nsLTPs are stabilized by four disulfide bonds and exhibit a characteristic hydrophobic cavity, which is the primary lipid binding site. While these proteins are known to transfer lipids between membranes, the mechanism of lipid transfer has remained elusive. Four crystal structures of nsLTP from Solanum melongena, one in the apo‐state and three myristic acid bound states were determined. Among the three lipid bound states, two lipid molecules were bound on the nsLTP surface at different positions and one was inside the cavity. The lipid‐dependent conformational changes leading to opening of the cavity were revealed based on structural and spectroscopic data. The surface‐bound lipid represented a transient intermediate state and the lipid ultimately moved inside the cavity through the cavity gate as revealed by molecular dynamics simulations. Two critical residues in the loop regions played possible ‘gating’ role in the opening and closing of the cavity. Antifungal activity and membrane permeabilization effect of nsLTP against Fusarium oxysporum suggested that it could possibly involve in bleaching out the lipids. Collectively, these studies support a model of lipid transfer mechanism by nsLTP via intermediate states.     

Solution structure of a tobacco lipid transfer protein exhibiting new biophysical and biological features

Plant lipid transfer proteins are small soluble extracellular proteins that are able to bind and transfer a variety of lipids in vitro. Recently, it has been proposed that lipid transfer proteins may play a key role in plant defence mechanisms, especially during the induction of systemic acquired resistance. However, very little is known about the proteins expressed in developing plants and tissues, since almost all the biophysical and structural data available to date on lipid transfer proteins originate from proteins present in storage tissues of monocot cereal seeds. In this paper, we report the structural and functional characteristics of a lipid transfer protein (named LTP1_1) constitutively expressed in young aerial organs of Nicotiana tabacum (common tobacco). The unlabelled and uniformly labelled proteins were produced in the yeast Pichia pastoris, and we determined the three-dimensional (3D) structure of LTP1_1 using nuclear magnetic resonance (NMR) spectroscopy and molecular modeling techniques. The global fold of LTP1_1 is very close to the previously published structures of LTP1 extracted from cereal seeds, including an internal cavity. However, the chemical shift variations of several NMR signals upon lipid binding show that tobacco LTP1_1 is able to bind only one LysoMyristoylPhosphatidylCholine (LMPC), while wheat and maize LTPs can bind either one or two. Titration experiments using intrinsic tyrosine fluorescence confirm this result not only with LMPC but also with two fatty acids. These differences can be explained by the presence in tobacco LTP1_1 of a hydrophobic cluster closing the second possible access to the protein cavity. This result suggests that LTP1 lipid binding properties could be modulated by subtle changes in a conserved global structure. The biological significance of this finding is discussed in the light of the signalling properties of the tobacco LTP1_1-jasmonate complex described elsewhere.     

Multiple binding modes for palmitate to barley lipid transfer protein facilitated by the presence of proline 12

Molecular dynamics simulations have been used to characterise the binding of the fatty acid ligand palmitate in the barley lipid transfer protein 1 (LTP) internal cavity. Two different palmitate binding modes (1 and 2), with similar protein–ligand interaction energies, have been identified using a variety of simulation strategies. These strategies include applying experimental protein–ligand atom–atom distance restraints during the simulation, or protonating the palmitate ligand, or using the vacuum GROMOS 54B7 force‐field parameter set for the ligand during the initial stages of the simulations. In both the binding modes identified the palmitate carboxylate head group hydrogen bonds with main chain amide groups in helix A, residues 4 to 19, of the protein. In binding mode 1 the hydrogen bonds are to Lys 11, Cys 13, and Leu 14 and in binding mode 2 to Thr 15, Tyr 16, Val 17, Ser 24 and also to the OH of Thr 15. In both cases palmitate binding exploits irregularity of the intrahelical hydrogen‐bonding pattern in helix A of barley LTP due to the presence of Pro 12. Simulations of two variants of barley LTP, namely the single mutant Pro12Val and the double mutant Pro12Val Pro70Val, show that Pro 12 is required for persistent palmitate binding in the LTP cavity. Overall, the work identifies key MD simulation approaches for characterizing the details of protein–ligand interactions in complexes where NMR data provide insufficient restraints.     

Solution structure of barley lipid transfer protein complexed with palmitate. Two different binding modes of palmitate in the homologous maize and barley nonspecific lipid transfer proteins.

The structure of a nonspecific lipid transfer protein from barley (ns-LTPbarley) in complex with palmitate has been determined by NMR spectroscopy. The structure has been compared to the structure of ns-LTPbarley in the absence of palmitate, to the structure of ns-LTPbarley in complex with palmitoyl coenzyme A, to the structure of ns-LTPmaize in its free form, and to the maize protein complexed with palmitate. Binding of palmitate only affects the structure of ns-LTPbarley moderately in contrast to the binding of palmitoyl coenzyme A, which leads to a considerable expansion of the protein. The modes of binding palmitate to the maize and barley protein are different. Although in neither case there are major conformational changes in the protein, the orientation of the palmitate in the two proteins is exactly opposite.     

Insights into nonspecific binding of homeodomains from a structure of MATalpha2 bound to DNA

The 2.1-A resolution crystal structure of the MATalpha2 homeodomain bound to DNA reveals the unexpected presence of two nonspecifically bound alpha2 homeodomains, in addition to the two alpha2 homeodomains bound to canonical alpha2 binding sites. One of the extra homeodomains makes few base-specific contacts, while the other extra homeodomain binds to DNA in a previously unobserved manner. This unusually bound homeodomain is rotated on the DNA, making possible major groove contacts by side-chains that normally do not contact the DNA. This alternate docking may represent one way in which homeodomains sample nonspecific DNA sequences.     

The importance of valine 114 in ligand binding in β2‐adrenergic receptor

G‐protein coupled receptors (GPCRs) are transmembrane signaling molecules, with a majority of them performing important physiological roles. β₂‐Adrenergic receptor (β₂‐AR) is a well‐studied GPCRs that mediates natural responses to the hormones adrenaline and noradrenaline. Analysis of the ligand‐binding region of β₂‐AR using the recently solved high‐resolution crystal structures revealed a number of highly conserved amino acids that might be involved in ligand binding. However, detailed structure‐function studies on some of these residues have not been performed, and their role in ligand binding remains to be elucidated. In this study, we have investigated the structural and functional role of a highly conserved residue valine 114, in hamster β₂‐AR by site‐directed mutagenesis. We replaced V114 in hamster β₂‐AR with a number of amino acid residues carrying different functional groups. In addition to the complementary substitutions V114I and V114L, the V114C and V114E mutants also showed significant ligand binding and agonist dependent G‐protein activation. However, the V114G, V114T, V114S, and V114W mutants failed to bind ligand in a specific manner. Molecular modeling studies were conducted to interpret these results in structural terms. We propose that the replacement of V114 influences not only the interaction of the ethanolamine side‐chains but also the aryl‐ring of the ligands tested. Results from this study show that the size and orientation of the hydrophobic residue at position V114 in β₂‐AR affect binding of both agonists and antagonists, but it does not influence the receptor expression or folding.     

Computational design and biochemical characterization of maize nonspecific lipid transfer protein variants for biosensor applications

Lipid transfer proteins (LTPs) are a family of proteins that bind and transfer lipids. Utilizing the maize LTP, we have successfully engineered fluorescent reagentless biosensors for the natural ligand of LTPs; this was achieved by using computational protein design to remove a disulfide bridge and attaching a thio-reactive fluorophore. Conformational change induced by ligand titration is thought to affect the fluorescence of the fluorophore, allowing detection of ligand binding. Fluorescence measurements show that our LTP variants have affinity to palmitate that is consistent with wild-type LTP. These molecules have the potential to be utilized as scaffolds to design hydrophobic ligand biosensors or to serve as drug carriers.     

A rice lipid transfer protein binds to plasma membrane proteinaceous sites

Nonspecific lipid transfer protein (nsLTP) is usually basic and secreted low-molecular-mass protein in plants. The 3-D structure of nsLTP1 resembles that of elicitin produced by the plant pathogen Phytophthora cryptogea, which can bind to the plant plasma membrane putative receptor and activate the downstream responses. It is inferred that nsLTP1 may have similar binding sites on the plasma membranes. In this work, rice recombinant protein TRX-nsLTP110 labeled with ¹²⁵I was shown to bind to rice plasma membrane preparations in a saturable curve, with an apparent K_d of 13.6 nM and B_max of 150 fmol/mg proteins. Competition experiments revealed that the binding of TRX-nsLTP110 was specific, in contrast to the nonspecific binding of the fusion tag thioredoxin. Protease treatment assay showed that the binding sites were proteinaceous. Our results suggest that the binding sites of nsLTPs on plasma membranes may be ubiquitous in the plant kingdom. They may be competed out from the binding sites under pathogen attack, supporting a role for nsLTP1 in host defense response to pathogens.     

Effects of acylation on the structure,lipid binding,and transfer activity of wheat lipid transfer protein

Study of the effect of protein chemical acylation on their functional properties or activity often brings valuable information regarding structure-function relationships. We performed such work on wheat lipid transfer protein, LTP1, to investigate the role of grafted acyl chains on the lipid binding and transfer properties. LTP1 was acylated by using anhydride derivatives of various chain lengths from C2 to C6. Only the chemical modifications with hexanoic acid yielded a marked effect on the tertiary structure and a slight change in the secondary structure. The affinity of the modified proteins for myristoyl-lysophosphatidylcholine was similar to that of the native protein accompanied by a slight decrease in stoichiometry. Interestingly, the acylation of LTP1 enhanced the lipid transfer activity by at least a factor of 10 for hexanoic chain length. Finally, the grafting of acyl chains was investigated by means of molecular modelling, and an attempt is made to correlate with our experimental data.     

Distal heme pocket regulation of ligand binding and stability in soybean leghemoglobin

Leghemoglobins facilitate diffusion of oxygen through root tissue to a bacterial terminal oxidase in much the same way that myoglobin transports oxygen from blood to muscle cell mitochondria. Leghemoglobin serves an additional role as an oxygen scavenger to prevent inhibition of nitrogen fixation. For this purpose, the oxygen affinity of soybean leghemoglobin is 20-fold greater than myoglobin, resulting from an 8-fold faster association rate constant combined with a 3-fold slower dissociation rate constant. Although the biochemical mechanism used by myoglobin to bind oxygen has been described in elegant detail, an explanation for the difference in affinity between these two structurally similar proteins is not obvious. The present work demonstrates that, despite their similar structures, leghemoglobin uses methods different from myoglobin to regulate ligand affinity. Oxygen and carbon monoxide binding to a comprehensive set of leghemoglobin distal heme pocket mutant proteins in comparison to their myoglobin counterparts has revealed some of these mechanisms. The "distal histidine" provides a crucial hydrogen bond to stabilize oxygen in myoglobin but has little effect on bound oxygen in leghemoglobin and is retained mainly for reasons of protein stability and prevention of heme loss. Furthermore, soybean leghemoglobin uses an unusual combination of HisE7 and TyrB10 to sustain a weak stabilizing interaction with bound oxygen. Thus, the leghemoglobin distal heme pocket provides a much lower barrier to oxygen association than occurs in myoglobin and oxygen dissociation is regulated from the proximal heme pocket.     

Atg2: A novel phospholipid transfer protein that mediates de novo autophagosome biogenesis

The degradation of cytoplasmic components via autophagy is crucial for intracellular homeostasis. In the process of autophagy, a newly synthesized isolation membrane (IM) is developed to sequester degradation targets and eventually the IM seals, forming an autophagosome. One of the most poorly understood autophagy‐related proteins is Atg2, which is known to localize to a contact site between the edge of the expanding IM and the exit site of the endoplasmic reticulum (ERES). Recent advances in structural and biochemical analyses have been applied to Atg2 and have revealed it to be a novel multifunctional protein that tethers membranes and transfers phospholipids between them. Considering that Atg2 is essential for the expansion of the IM that requires phospholipids as building blocks, it is suggested that Atg2 transfers phospholipids from the ERES to the IM during the process of autophagosome formation, suggesting that lipid transfer proteins can mediate de novo organelle biogenesis.     

The role of Trp-82 in the folding of intestinal fatty acid binding protein

Multiple phases have been observed during the folding and unfolding of intestinal fatty acid binding protein (WT-IFABP) by stopped-flow fluorescence. Site-directed mutagenesis has been used to examine the role of each of the two tryptophans of this protein in these processes. The unfolding and refolding kinetics of the mutant protein containing only tryptophan 82 (W6Y-IFABP) showed that the tryptophan at this location was critical to the fluorescence signal changes observed throughout the unfolding reaction and early in the refolding reaction. However, the kinetic patterns of the mutant protein containing only tryptophan 6 (W82Y-IFABP) indicated that the tryptophan at this location participated in the fluorescence signal changes observed early in the unfolding reaction and late in the refolding reaction. Together, these data suggest that native-like structure was formed first in the vicinity of tryptophan 82, near the center of the hydrophobic core of this beta-sheet protein, prior to formation of native-like structure in the periphery of the protein.     

Native cysteine residues are dispensable for the structure and function of all five yeast mitotic septins

Budding yeast septins assemble into hetero‐octamers and filaments required for cytokinesis. Solvent‐exposed cysteine (Cys) residues provide sites for attaching substituents useful in assessing assembly kinetics and protein interactions. To introduce Cys at defined locations, site‐directed mutagenesis was used, first, to replace the native Cys residues in Cdc3 (C124 C253 C279), Cdc10 (C266), Cdc11 (C43 C137 C138), Cdc12 (C40 C278), and Shs1 (C29 C148) with Ala, Ser, Val, or Phe. When plasmid‐expressed, each Cys‐less septin mutant rescued the cytokinesis defects caused by absence of the corresponding chromosomal gene. When integrated and expressed from its endogenous promoter, the same mutants were fully functional, except Cys‐less Cdc12 mutants (which were viable, but exhibited slow growth and aberrant morphology) and Cdc3(C124V C253V C279V) (which was inviable). No adverse phenotypes were observed when certain pairs of Cys‐less septins were co‐expressed as the sole source of these proteins. Cells grew less well when three Cys‐less septins were co‐expressed, suggesting some reduction in fitness. Nonetheless, cells chromosomally expressing Cys‐less Cdc10, Cdc11, and Cdc12, and expressing Cys‐less Cdc3 from a plasmid, grew well at 30°C. Moreover, recombinant Cys‐less septins—or where one of the Cys‐less septins contained a single Cys introduced at a new site—displayed assembly properties in vitro indistinguishable from wild‐type. Proteins 2013; 81:1964–1979. © 2013 Wiley Periodicals, Inc.     

A divide-and-conquer approach to determine the Pareto frontier for optimization of protein engineering experiments

In developing improved protein variants by site-directed mutagenesis or recombination, there are often competing objectives that must be considered in designing an experiment (selecting mutations or breakpoints): stability versus novelty, affinity versus specificity, activity versus immunogenicity, and so forth. Pareto optimal experimental designs make the best trade-offs between competing objectives. Such designs are not "dominated"; that is, no other design is better than a Pareto optimal design for one objective without being worse for another objective. Our goal is to produce all the Pareto optimal designs (the Pareto frontier), to characterize the trade-offs and suggest designs most worth considering, but to avoid explicitly considering the large number of dominated designs. To do so, we develop a divide-and-conquer algorithm, Protein Engineering Pareto FRontier (PEPFR), that hierarchically subdivides the objective space, using appropriate dynamic programming or integer programming methods to optimize designs in different regions. This divide-and-conquer approach is efficient in that the number of divisions (and thus calls to the optimizer) is directly proportional to the number of Pareto optimal designs. We demonstrate PEPFR with three protein engineering case studies: site-directed recombination for stability and diversity via dynamic programming, site-directed mutagenesis of interacting proteins for affinity and specificity via integer programming, and site-directed mutagenesis of a therapeutic protein for activity and immunogenicity via integer programming. We show that PEPFR is able to effectively produce all the Pareto optimal designs, discovering many more designs than previous methods. The characterization of the Pareto frontier provides additional insights into the local stability of design choices as well as global trends leading to trade-offs between competing criteria.