Structure-based protein engineering for alpha-amylase inhibitory activity of plant defensin

The structure of a novel plant defensin isolated from the seeds of the mung bean, Vigna radiate, has been determined by (1)H nuclear magnetic resonance spectroscopy. The three-dimensional structure of VrD2, the V. radiate plant defensin 2 protein, comprises an alpha-helix and one triple-stranded anti-parallel beta-sheet stabilized by four disulfide bonds. This protein exhibits neither insecticidal activity nor alpha-amylase inhibitory activity in spite of showing a similar global fold to that of VrD1, an insecticidal plant defensin that has been suggested to function by inhibiting insect alpha-amylase. Our previous study proposed that loop L3 of plant defensins is important for this inhibition. Structural analyses and surface charge comparisons of VrD1 and VrD2 revealed that the charged residues of L3 correlate with the observed difference in inhibitory activities of these proteins. A VrD2 chimera that was produced by transferring the proposed functional loop of VrD1 onto the structurally equivalent loop of VrD2 supported this hypothesis. The VrD2 chimera, which differs by only five residues compared with VrD2, showed obvious activity against Tenebrio molitor alpha-amylase. These results clarify the mode of alpha-amylase inhibition of plant defensins and also represent a possible approach for engineering novel alpha-amylase inhibitors. Plant defensins are important constituents of the innate immune system of plants, and thus the application of protein engineering to this protein family may provide an efficient method for protecting against crop losses.     

Structure of Petunia hybrida defensin 1, a novel plant defensin with five disulfide bonds

The structure of a novel plant defensin isolated from the flowers of Petunia hybrida has been determined by (1)H NMR spectroscopy. P. hybrida defensin 1 (PhD1) is a basic, cysteine-rich, antifungal protein of 47 residues and is the first example of a new subclass of plant defensins with five disulfide bonds whose structure has been determined. PhD1 has the fold of the cysteine-stabilized alphabeta motif, consisting of an alpha-helix and a triple-stranded antiparallel beta-sheet, except that it contains a fifth disulfide bond from the first loop to the alpha-helix. The additional disulfide bond is accommodated in PhD1 without any alteration of its tertiary structure with respect to other plant defensins. Comparison of its structure with those of classic, four-disulfide defensins has allowed us to identify a previously unrecognized hydrogen bond network that is integral to structure stabilization in the family.     

Solution structure of drosomycin, the first inducible antifungal protein from insects.

Drosomycin is the first antifungal protein characterized recently among the broad family of inducible peptides and proteins produced by insects to respond to bacterial or septic injuries. It is a small protein of 44 amino acid residues extracted from Drosophila melanogaster that exhibits a potent activity against filamentous fungi. Its three-dimensional structure in aqueous solution was determined using 1H 2D NMR. This structure, involving an alpha-helix and a twisted three-stranded beta-sheet, is stabilized by three disulfide bridges. The corresponding Cysteine Stabilized alpha beta (CS alpha beta) motif, which was found in other defense proteins such as the antibacterial insect defensin A, short- and long-chain scorpion toxins, as well as in plant thionins and potent antifungal plant defensins, appears as remarkably persistent along evolution.     

Solution structure of the plant defensin VrD1 from mung bean and its possible role in insecticidal activity against bruchids

Vigna radiata plant defensin 1 (VrD1) is the first reported plant defensin exhibiting insecticidal activity. We report herein the nuclear magnetic resonance solution structure of VrD1 and the implication on its insecticidal activity. The root-mean-square deviation values are 0.51 +/- 0.35 and 1.23 +/- 0.29 A for backbone and all heavy atoms, respectively. The VrD1 structure comprises a triple-stranded antiparallel beta-sheet, an alpha-helix, and a 3(10) helix stabilized by four disulfide bonds, forming a typical cysteine-stabilized alphabeta motif. Among plant defensins of known structure, VrD1 is the first to contain a 3(10) helix. Glu26 is highly conserved among defensins; VrD1 contains an arginine at this position, which may induce a shift in the orientation of Trp10, thereby promoting the formation of this 3(10) helix. Moreover, VrD1 inhibits Tenebrio molitor alpha-amylase. Alpha-amylase has an essential role in the digestion of plant starch in the insect gut, and expression of the common bean alpha-amylase inhibitor 1 in transgenic pea imparts complete resistance against bruchids. These results imply that VrD1 insecticidal activity has its basis in the inhibition of a polysaccharide hydrolase. Sequence and structural comparisons between two groups of plant defensins having different specificity toward insect alpha-amylase reveal that the loop between beta2 and beta3 is the probable binding site for the alpha-amylase. Computational docking experiments were used to study VrD1-alpha-amylase interactions, and these results provide information that may be used to improve the insecticidal activity of VrD1.     

Human OLA1 defines an ATPase subfamily in the Obg family of GTP-binding proteins

Purine nucleotide-binding proteins build the large family of P-loop GTPases and related ATPases, which perform essential functions in all kingdoms of life. The Obg family comprises a group of ancient GTPases belonging to the TRAFAC (for translation factors) class and can be subdivided into several distinct protein subfamilies. The founding member of one of these subfamilies is the bacterial P-loop NTPase YchF, which had so far been assumed to act as GTPase. We have biochemically characterized the human homologue of YchF and found that it binds and hydrolyzes ATP more efficiently than GTP. For this reason, we have termed the protein hOLA1, for human Obg-like ATPase 1. Further biochemical characterization of YchF proteins from different species revealed that ATPase activity is a general but previously missed feature of the YchF subfamily of Obg-like GTPases. To explain ATP specificity of hOLA1, we have solved the x-ray structure of hOLA1 bound to the nonhydrolyzable ATP analogue AMPPCP. Our structural data help to explain the altered nucleotide specificity of YchF homologues and identify the Ola1/YchF subfamily of the Obg-related NTPases as an exceptional example of a single protein subfamily, which has evolved altered nucleotide specificity within a distinct protein family of GTPases.     

Solution structure of Pisum sativum defensin 1 by high resolution NMR: plant defensins, identical backbone with different mechanisms of action.

Pisum sativum defensin 1 (Psd1) is a 46 amino acid residue plant defensin isolated from seeds of pea. The three-dimensional structure in solution of Psd1 was determined by two-dimensional NMR data recorded at 600 MHz. Experimental restraints were used for structure calculation using CNS and torsion-angle molecular dynamics. The 20 lowest energy structures were selected and further subjected to minimization, giving a root-mean-square deviation of 0.78(+/- 0.22) A in the backbone and 1.91(+/-0.60) A for over all atoms of the molecule. The protein has a globular fold with a triple-stranded antiparalell beta-sheet and an alpha-helix (from residue Asn17 to Leu27). Psd1 presents the so called "cysteine stabilized alpha/beta motif" and presents identical three-dimensional topology in the backbone with other defensins and neurotoxins. Comparison of the electrostatic surface potential among proteins with high three-dimensional (selected using the softwares TOP and DALI) topology gave insights into the mode of action of Psd1. The surface topologies between proteins that present antifungal activity or sodium channel inhibiting activity are different. On the other hand the surface topology presents several common features with potassium channel inhibitors, suggesting that Psd1 presents this activity. Other common features with potassium channel inhibitors were found including the presence of a lysine residue essential for inhibitory activity. The identity of Psd1 in primary sequence is not enough to infer a mechanism of action, in contrast with the strategy proposed here.     

Solution structures of the antifungal heliomicin and a selected variant with both antibacterial and antifungal activities

In response to an experimental infection, the lepidopteran Heliothis virescens produces an antifungal protein named heliomicin. Heliomicin displays sequence similarities with antifungal plant defensins and antibacterial or antifungal insect defensins. To gain information about the structural elements required for either antifungal or antibacterial activity, heliomicin and selected point-mutated variants were expressed in yeast as fusion proteins. The effects of mutations, defined by comparing the primary structure of heliomicin with the sequences of members of the insect defensin family, were analyzed using antibacterial and antifungal assays. One of the variants shows significant activity against Gram-positive bacteria while remaining efficient against fungi. The three-dimensional structures of this variant and of the wild-type protein were determined by two-dimensional (1)H NMR to establish a correlation between structure and antibacterial or antifungal activity. Wild-type and mutated heliomicins adopt a similar scaffold, including the so-called cysteine-stabilized alphabeta motif. A comparison of their structures with other defensin-type molecules indicates that common hydrophobic characteristics can be assigned to all the antifungal proteins. A comparative analysis of various structural features of heliomicin mutant and of antibacterial defensins enables common properties to be assessed, which will help to design new mutants with increased antibacterial activity.     

The three-dimensional solution structure of NaD1, a new floral defensin from Nicotiana alata and its application to a homology model of the crop defense protein alfAFP

NMR spectroscopy and simulated annealing calculations have been used to determine the three-dimensional structure of NaD1, a novel antifungal and insecticidal protein isolated from the flowers of Nicotiana alata. NaD1 is a basic, cysteine-rich protein of 47 residues and is the first example of a plant defensin from flowers to be characterized structurally. Its three-dimensional structure consists of an alpha-helix and a triple-stranded antiparallel beta-sheet that are stabilized by four intramolecular disulfide bonds. NaD1 features all the characteristics of the cysteine-stabilized alphabeta motif that has been described for a variety of proteins of differing functions ranging from antibacterial insect defensins and ion channel-perturbing scorpion toxins to an elicitor of the sweet taste response. The protein is biologically active against insect pests, which makes it a potential candidate for use in crop protection. NaD1 shares 31% sequence identity with alfAFP, an antifungal protein from alfalfa that confers resistance to a fungal pathogen in transgenic potatoes. The structure of NaD1 was used to obtain a homology model of alfAFP, since NaD1 has the highest level of sequence identity with alfAFP of any structurally characterized antifungal defensin. The structures of NaD1 and alfAFP were used in conjunction with structure-activity data for the radish defensin Rs-AFP2 to provide an insight into structure-function relationships. In particular, a putative effector site was identified in the structure of NaD1 and in the corresponding homology model of alfAFP.     

Specific binding sites for an antifungal plant defensin from Dahlia (Dahlia merckii) on fungal cells are required for antifungal activity

Dm-AMP1, an antifungal plant defensin from seeds of dahlia (Dahlia merckii), was radioactively labeled with t-butoxycarbonyl-[35S]-L-methionine N-hydroxy-succinimi-dylester. This procedure yielded a 35S-labeled peptide with unaltered antifungal activity. [35S]Dm-AMP1 was used to assess binding on living cells of the filamentous fungus Neurospora crassa and the unicellular fungus Saccharomyces cerevisiae. Binding of [35S]Dm-AMP1 to fungal cells was saturable and could be competed for by preincubation with excess, unlabeled Dm-AMP1 as well as with Ah-AMP1 and Ct-AMP1, two plant defensins that are highly homologous to Dm-AMP1. In contrast, binding could not be competed for by more distantly related plant defensins or structurally unrelated antimicrobial peptides. Binding of [35S]Dm-AMP1 to either N. crassa or S. cerevisiae cells was apparently irreversible. In addition, whole cells and microsomal membrane fractions from two independently obtained S. cerevisiae mutants selected for resistance to Dm-AMP1 exhibited severely reduced binding affinity for [35S]Dm-AMP1, compared with wild-type yeast. This finding suggests that binding of Dm-AMP1 to S. cerevisiae plasma membranes is required for antifungal activity of this protein.     

Plant defensins

Plant defensins are small, basic peptides that have a characteristic three-dimensional folding pattern that is stabilized by eight disulfide-linked cysteines. They are termed plant defensins because they are structurally related to defensins found in other types of organism, including humans. To date, sequences of more than 80 different plant defensin genes from different plant species are available. In Arabidopsis thaliana, at least 13 putative plant defensin genes (PDF) are present, encoding 11 different plant defensins. Two additional genes appear to encode plant defensin fusions. Plant defensins inhibit the growth of a broad range of fungi but seem nontoxic to either mammalian or plant cells. Antifungal activity of defensins appears to require specific binding to membrane targets. This review focuses on the classification of plant defensins in general and in Arabidopsis specifically, and on the mode of action of plant defensins against fungal pathogens.     

Expression of BrD1, a plant defensin from Brassica rapa, confers resistance against brown planthopper (Nilaparvata lugens) in transgenic rices

Plant defensins are small (5-10 kDa) basic peptides thought to be an important component of the defense pathway against fungal and/or bacterial pathogens. To understand the role of plant defensins in protecting plants against the brown planthopper, a type of insect herbivore, we isolated the Brassica rapa Defensin 1 (BrD1) gene and introduced it into rice (Oryza sativa L.) to produce stable transgenic plants. The BrD1 protein is homologous to other plant defensins and contains both an N-terminal endoplasmic reticulum signal sequence and a defensin domain, which are highly conserved in all plant defensins. Based on a phylogenetic analysis of the defensin domain of various plant defensins, we established that BrD1 belongs to a distinct subgroup of plant defensins. Relative to the wild type, transgenic rices expressing BrD1 exhibit strong resistance to brown planthopper nymphs and female adults. These results suggest that BrD1 exhibits insecticidal activity, and might be useful for developing cereal crop plants resistant to sap-sucking insects, such as the brown planthopper.     

<Emphasis Type="Italic">Germin-like protein</Emphasis> gene family of a moss, <Emphasis Type="Italic">Physcomitrella patens</Emphasis>, phylogenetically falls into two characteristic new clades

We identified 77 EST clones encoding germin-like proteins (GLPs) from a moss, Physcomitrella patens in a database search. These Physcomitrella GLPs (PpGLPs) were separated into seven groups based on DNA sequence homology. Phylogenetic analysis showed that these groups were divided into two novel clades clearly distinguishable from higher plant germins and GLPs, named bryophyte subfamilies 1 and 2. PpGLPs belonging to bryophyte subfamilies 1 lacked two cysteines at the conserved positions observed in higher plant germins or GLPs. PpGLPs belonging to bryophyte subfamily 2 contained two cysteines as observed in higher plant germins and GLPs. In bryophyte subfamily 1, 12 amino acids, in which one of two cysteines is included, were deleted between boxes A and B. Further, we determined the genomic structure of all of seven PpGLP genes. The sequences of PpGLPs of bryophyte subfamily 1 contained one or two introns, whereas those of bryophyte subfamily 2 contained no introns. Other GLPs from bryophytes, a liverwort GLP from Marchantia polymorpha, and two moss GLPs from Barbula unguiculata and Ceratodon purpureus also fell into bryophyte subfamily 1 and bryophyte subfamily 2, respectively. No higher plant germins and GLPs were grouped into the bryophyte subfamilies 1 and 2 by our analysis. Moreover, we revealed that PpGLP6 had manganese-containing extracellular superoxide dismutase activity. These results indicated that bryophyte possess characteristic GLPs, which phylogenetically are clearly distinguishable from higher plant GLPs.     

Cyclic and Acyclic Defensins Inhibit Human Immunodeficiency Virus Type-1 Replication by Different Mechanisms

Defensins are antimicrobial peptides expressed by plants and animals. In mammals there are three subfamilies of defensins, distinguished by structural features: α, β and θ. Alpha and β-defensins are linear peptides with broad anti-microbial activity that are expressed by many mammals including humans. In contrast, θ-defensins are cyclic anti-microbial peptides made by several non-human primates but not humans. All three defensin types have anti-HIV-1 activity, but their mechanisms of action differ. We studied the anti-HIV-1 activity of one defensin from each group, HNP-1 (α), HBD-2 (β) and RTD-1 (θ). We examined how each defensin affected HIV-1 infection and demonstrated that the cyclic defensin RTD-1 inhibited HIV-1 entry, while acyclic HNP-1 and HBD-2 inhibited HIV-1 replication even when added 12 hours post-infection and blocked viral replication after HIV-1 cDNA formation. We further found that all three defensins downmodulated CXCR4. Moreover, RTD-1 inactivated X4 HIV-1, while HNP-1 and HBD-2 inactivated both X4 and R5 HIV-1. The data presented here show that acyclic and cyclic defensins block HIV-1 replication by shared and diverse mechanisms. Moreover, we found that HNP-1 and RTD-1 directly inhibited firefly luciferase enzymatic activity, which may affect the interpretation of previously published data.     

NMR studies of defensin antimicrobial peptides. 2. Three-dimensional structures of rabbit NP-2 and human HNP-1.

The solution structure of two homologous naturally occurring antimicrobial peptides, rabbit defensin NP-2 and human defensin HNP-1, have been determined by two-dimensional nuclear magnetic resonance spectroscopy, distance geometry, and restrained molecular dynamics calculations. The structure of these defensins consists of an antiparallel beta-sheet in a hairpin conformation, a short region of triple-stranded beta-sheet, several tight turns, and a loop region that has a well-defined local structure but with a global orientation that is not well-defined with respect to the rest of the molecule. The solution structures of these two peptides are compared with the solution and crystal structures of two other homologous defensins. The structures for the defensins are also compared with known structures of other naturally occurring antimicrobial peptides.     

The Tomato Defensin TPP3 Binds Phosphatidylinositol (4,5)-Bisphosphate via a Conserved Dimeric Cationic Grip Conformation To Mediate Cell Lysis

Defensins are a class of ubiquitously expressed cationic antimicrobial peptides (CAPs) that play an important role in innate defense. Plant defensins are active against a broad range of microbial pathogens and act via multiple mechanisms, including cell membrane permeabilization. The cytolytic activity of defensins has been proposed to involve interaction with specific lipid components in the target cell wall or membrane and defensin oligomerization. Indeed, the defensin Nicotiana alata defensin 1 (NaD1) binds to a broad range of membrane phosphatidylinositol phosphates and forms an oligomeric complex with phosphatidylinositol (4,5)-bisphosphate (PIP2) that facilitates membrane lysis of both mammalian tumor and fungal cells. Here, we report that the tomato defensin TPP3 has a unique lipid binding profile that is specific for PIP2 with which it forms an oligomeric complex that is critical for cytolytic activity. Structural characterization of TPP3 by X-ray crystallography and site-directed mutagenesis demonstrated that it forms a dimer in a “cationic grip” conformation that specifically accommodates the head group of PIP2 to mediate cooperative higher-order oligomerization and subsequent membrane permeabilization. These findings suggest that certain plant defensins are innate immune receptors for phospholipids and adopt conserved dimeric configurations to mediate PIP2 binding and membrane permeabilization. This mechanism of innate defense may be conserved across defensins from different species.     

Molecular cloning, characterization and expression of a novel jasmonate-dependent defensin gene from Ginkgo biloba

A novel defensin gene was isolated from Ginkgo biloba. The full-length cDNA of G. biloba defensin (designated as Gbd) was 534bp. The cDNA contained a 240-bp open reading frame encoding an 80-amino acid protein of 5.68 kDa with a potential 30 aa signal peptide. The putative GbD mature protein showed striking similarity to other plant defensins, representing low molecular size antimicrobial polypeptides. Eight cysteine sites conserved in plant defensins were also found in GbD at similar positions. Three-dimensional structure modeling showed that GbD strongly resembled defensin from tobacco (NaD1) and consisted of an alpha-helix and a triple-strand antiparallel beta-sheet that were stabilized by four intramolecular disulfide bonds, implying GbD may have functions similar to NaD1. The genomic DNA gel blot indicated that Gbd belonged to a multigene family. Expression analysis revealed that Gbd was up-regulated by wounding and methyl jasmonate treatments, suggesting that Gbd is potentially involved in plant resistance or tolerance to pathogens during wounding.     

Rational design of peptides active against the gram positive bacteria Staphylococcus aureus

In an attempt to increase the antimicrobial activity of the insect defensin from Anopheles gambiae, which is active against Staphylococcus aureus at low concentration, hybrid defensins were designed by combining conserved sequence regions and variable regions of insect defensins. Their activity against S. aureus strains sensitive and resistant to conventional antibiotics was evaluated, and the toxicity of the most active molecules was tested. The three-dimensional structure of Anopheles gambiae defensin and five hybrids were determined by NMR and molecular modelling. This strategy led to the design of two chimeric defensins with increased activity compared with the native molecule, but one of them appears to be toxic to mice at a rather low concentration. The structure of the CS alphabeta motif, which is a characteristic of insect defensin, is sensitive to sequence modifications, in particular in the N-terminal loop. The existence of the CS alphabeta is most probably a prerequisite for the stability and the activity of the molecule, but is not sufficient by itself since the hybrid displaying the best defined structure is not active against the tested strains. The analysis of the structure, in relation with the activity and the toxicity data, underlines the importance of turns and of the N-terminal loop. Residues located in the turns contributing to the preservation of positive electrostatic areas at the surface of the molecules seem particularly important for the activity of the molecule, while residues involved in the N-terminal loop are both involved in the modulation of the activity and the toxicity of the molecule.     

Plant defensins and virally encoded fungal toxin KP4 inhibit plant root growth

Plant defensins are small, highly stable, cysteine-rich antimicrobial proteins that are thought to constitute an important component of plant defense against fungal pathogens. There are a number of such defensins expressed in various plant tissues with differing antifungal activity and spectrum. Relatively little is known about the modes of action and biological roles of these proteins. Our previous work on a virally encoded fungal toxin, KP4, from Ustilago maydis and subsequently with the plant defensin, MsDef1, from Medicago sativa demonstrated that some of these proteins specifically blocked calcium channels in both fungi and animals. The results presented here demonstrate that KP4 and three plant defensins, MsDef1, MtDef2, and RsAFP2, all inhibit root growth in germinating Arabidopsis seeds at low micromolar concentrations. We have previously demonstrated that a fusion protein composed of Rab GTPase (RabA4b) and enhanced yellow fluorescent protein (EYFP) is dependent upon calcium gradients for localization to the tips of the growing root hairs in Arabidopsis thaliana. Using this tip-localized fusion protein, we demonstrate that all four proteins rapidly depolarize the growing root hair and block growth in a reversible manner. This inhibitory activity on root and root hair is not directly correlated with the antifungal activity of these proteins and suggests that plants apparently express targets for these antifungal proteins. The data presented here suggest that plant defensins may have roles in regulating plant growth and development. A. Allen and A.K. Snyder contributed equally.     

Structure and dynamics of the neutrophil defensins NP-2, NP-5, and HNP-1: NMR studies of amide hydrogen exchange kinetics

The exchange kinetics for the slowly exchanging amide hydrogens in three defensins, rabbit NP-2, rabbit NP-5, and human HNP-1, have been measured over a range of pH at 25°C using 1D and 2D NMR methods. These NHs have exchange rates 10² to 10⁵ times slower than rates from unstructured model peptides. The observed distribution of exchange rates under these conditions can be rationalized by intramolecular hydrogen bonding of the individual NHs, solvent accessibility of the NHs, and local fluctuations in structure. The temperature dependencies of NH chemical shifts (NH temperature coefficients) were measured for the defensins and these values are consistent with the defensin structure. A comparison is made between NH exchange kinetics, NH solvent accessibility, and NH temperature coefficients of the defensins and other globular proteins. Titration of the histidine side chain in NP-2 was examined and the results are mapped to the three-dimensional structure. © 1994 Wiley-Liss, Inc.