In Silico Analysis Of A Drosophila Parasitoid Venom Peptide Reveals Prevalence Of The Cation–Polar–Cation Clip Motif in Knottin Proteins Part 1

Abstract:

As generalist parasitoid wasps, Leptopilina heteroatom is highly successful on many species of fruit flies of the genus Drosophila. The parasitoids produce specialized multi-strategy extracellular vesicle (EV)-like structures in their venom. Proteomic analysis identified several immunity-associated proteins, including the knottin peptide, LhKNOT, containing the structurally conserved inhibitor cysteine knot (ICK) fold, which is present in proteins from diverse taxa. Our structural and docking analysis of LhKNOT’s 36-residue core knottin fold revealed that in addition to the knottin motif itself, it also possesses a Cation–Polar–Cation (CPC) clip. 

Inhibitor cysteine is a natural substance used to prevent oxidative damage and promote cell growth. For immunity, the role of cysteine is mainly reflected in improving the body's immunity and preventing inflammation and infection.

In recent years, more and more studies have shown that cysteine can play an important role in the immune system. It can promote the proliferation of immune cells, enhance the body's resistance to pathogens, and reduce the risk of infection. In addition, cysteine can also reduce the production of free radicals and improve the overall health of the immune system, thereby enhancing the body's immunity.

Although the effect of cysteine on immunity is very significant, when using inhibitor cysteine, it should be used with caution. Because the inhibitor cysteine is usually used to treat certain diseases, such as cardiovascular disease, arthritis, etc., if abused, it will affect immunity and cause the body's immune system to weaken, thereby increasing the risk of infection.

In conclusion, there is an inextricable relationship between the inhibitor cysteine and immunity. Although inhibitors of cysteine can promote the health of the body, they also need to be used under the guidance of a doctor, and attention should be paid to maintaining a reasonable dose to maintain the health of the body. It can be seen that we need to improve immunity. Cistanche can significantly improve immunity, because meat ash contains a variety of biologically active ingredients, such as polysaccharides, two mushrooms, Huang Li, etc. These ingredients can stimulate various immune systems. cell-like cells, increasing their immune activity.

Click health benefits of cistanche

The CPC clip motif is thought to facilitate antimicrobial activity in heparin-binding proteins. Surprisingly, a majority of ICKs tested also possess the CPC clip motif, including 75 bona fide plant and arthropod knottin proteins that share high sequence and/or structural similarity with LhKNOT. Like LhKNOT and these other 75 knottin proteins, even the Drosophila Drosomycin antifungal peptide, a canonical target gene of the fly’s TollNF-kappa B immune pathway, contains this CPC clip motif. Together, our results suggest a possible defensive function for the parasitoid LhKNOT. 

The prevalence of the CPC clip motif, intrinsic to the cysteine knot within the knottin proteins examined here, suggests that the resultant 3D topology is important for their biochemical functions. The CPC clip is likely a highly conserved structural motif found in many diverse proteins with reported heparin binding capacity, including amyloid proteins. Knottins are targets for therapeutic drug development, and insights into their structure–function relationships will advance novel drug design.

Keywords:

Host–parasite; Drosophila; Leptopilina; cysteine knot; knottin fold; Cation–Polar–Cation clip; antimicrobial peptide; heparin-binding motif; mini proteins.

1. Introduction

In host–parasite interactions, the parasite must provide offensive pressure while simultaneously keeping the host alive to sustain viable offspring [1,2]. Such conflicting goals have led to the evolution of virulence factors with diverse molecular strategies while maintaining high host specificity. Parasitic Hymenoptera makes up more than a million species, are ubiquitous on the planet, and parasitize a variety of arthropods, mainly insects [3]. 

Wasps attacking insect hosts have developed an assortment of behavioral and biochemical strategies for success and remain under constant co-evolutionary pressure with their hosts [4–7].

The Leptopilina/Drosophila system is an emerging model for understanding the molecular foundations of anti-wasp responses and wasp virulence strategies [8]. To suppress host immunity, Leptopilina females introduce venom factors into their larval hosts during oviposition [4,9,10]. The well-defined innate immune mechanisms in Drosophila [11–13] provide a valuable context to study the effects of these wasp venom factors. 

Infection by the specialist L. boulardi activates the humoral and cellular immune arms controlled by the fly’s conserved Toll-NF-κB signaling pathway. The expression of antimicrobial peptide (AMP) genes such as drosomycin is activated after L. boulardi infection [14]. Toll-NF-κB signaling also controls blood cell division and development [15,16], steps that control encapsulation and death of wasp eggs [17,18]. 

In contrast, infection by the generalist L. heteroatom suppresses both immune pathways [9,14], and almost all larval blood cells are destroyed by factors in the wasp venom [19,20].

In this study, we analyze the theoretical structure of an L. heteroatom peptide, called LhKNOT, identified within organelle-like secretions in the L. heteroatom venom but not in L. boulardi venom [21]. These immune-suppressive organelles include more than 350 proteins and possess a proteomic profile of eukaryotic microvesicles and a plethora of immunity-related proteins [21–23]. 

LhKNOT adopts the “Inhibitor Cysteine Knot” (knottin) motif [24]. The knottin motif is broadly conserved in a range of taxa, from plants [25,26] to snails [27]. These small peptides (<60 residues) are functionally diverse. Knottins are used offensively, in spider toxins [28] and cone snail venom [27], as well as defensively, e.g., in wound healing activities of cacti [26], and antimicrobial host defense in plants [25,26,29] and invertebrates [30].

The broad range of knottin activity is attributed to the overall structure of its fold, which relies solely on a conserved motif of six cysteine residues that takes advantage of these cysteines’ unique ability to form disulfide bridges. The knottin motif occurs when cysteine residues are observed in a C1-C2-C3-C4-C5-C6 pattern (where “-“ represents several highly variable residues). 

Disulfide bridges are then formed in a similarly conserved fashion (C1-C4, C2-C5, and C3-C6), resulting in C1-C4 and C2-C5 forming a macrocycle, while C3-C6 occurs within this macrocycle, resulting in a “knotted” topology. As this knotted topology largely hinges on the conservation of the six cysteine residues, the knottin motif allows for significant variation in the overall sequence [30]. The combination of their small size, high tolerance of sequence variance, and observed structural stability has made knottins a popular scaffold for drug design [30], potentially even for cancer treatments [31].

Various lines of experimental evidence demonstrate or suggest antimicrobial capabilities for different knottins [25,26,29,30,32,33] or gated ion channel interactions [28,34], although the molecular mechanisms by which these functions are carried out are not well understood. Furthermore, it is not known if these antimicrobial functions share a common mechanism of action. 

Given its discovery from immune-suppressive particles from fly parasites, our goals were to (a) identify LhKNOT-related sequences from diverse organisms, including L. heteroatom, L. boulardi, and Ganaspis parasitoids, that attack Drosophila and (b) identify any novel structural motif(s) present in the known or new LhKNOT homologs with the hope that the prediction of such a conserved motif might suggest possible functions for LhKNOT and guide future experimental research.

We show that a theoretical model of LhKNOT possesses the hallmarks of the knottin fold with three antiparallel beta sheets, constrained by disulfide bridges. The LhKNOT model contains a CPC clip, capable of interacting with heparin, a negatively charged glycosaminoglycan (GAG) of significant medical value. LhKNOT also has the potential for interacting with other GAGs, such as keratan sulfate (a GAG that is highly sulfated, similar to heparin) and hyaluronic acid (the single GAG representative that does not require sulfation) [35]. 

Furthermore, structural homologs of LhKNOT (i.e., bona fide knottin motif proteins) also possess the CPC clip motif that can dock with heparin. Surprisingly, like LhKNOT and these other bona fide knottin proteins, even the Drosophila Drosomycin antifungal peptide shares this CPC clip motif in its structure. This motif was also identified in 41 out of 46 additional LhKNOT homologs from L. heteroatom, L. boulardi, and Ganaspis spp. wasps successfully on D. melanogaster. 

These in silico-based observations suggest that the CPC clip motif, inherent in many knottins, may provide the structural basis for modifying immune or virulence functions. We speculate on a possible antimicrobial or virulence-related function for the wasp LhKNOT. The abundance of the knottin fold and its concurrence with the CPC clip motif in most of the newly identified knottin peptides from three Drosophila parasitoids suggests an important yet-to-be-determined role for them in parasitoid physiology and/or in the host–parasite interactions.

2. Results
2.1. The LhKNOT Sequence Shows Similarity to Insect and Plant Antimicrobial Peptides

A BLASTp search of the nr and TSA databases using the full-length LhKNOT sequence (60 amino acids) showed a limited number of significant hits, primarily annotated as “antimicrobial peptides”, from plants and insects. A few fungal sequences were also identified. All results displayed less than 60% amino acid identity to LhKNOT. The top match for LhKNOT in the nr database was an antimicrobial peptide from the common beetle, Callosobruchus maculates, while the top hit in the TSA database was an antimicrobial peptide from the wheat curl mite Aceria Coachella. 

The conservation was most prominent in the region of the only sequence motif associated with LhKNOT, the cysteine knotted ‘antifungal peptide’ signature (pfam11410 domain, E = 2.8 × 10−3 ). After running PSI-BLAST for the detection of remote homologs, additional sequences were revealed. PSI-BLAST showed similar results with common ice plant, Mesembryanthemum crystallinum; red mites, Dinothrombium tinctorium; parasitoid wasp, Trichogramma pretiosum; click beetle, Ignelater luminous; parasitoid wasp, Trichogramma brassicae; common pollen beetle, Brassicogethes aeneus; ash borer, Agrilus planipennis; and sawfly, Neodiprion lecontei. 

Using the Hidden Markov Models-driven HMMER web server as an alternative method of detecting remote homologs, we identified five fungal hits from Akanthomyces lecanii, Cordyceps javanica, Cordyceps confragosa, Rosellinia necatrix, and Beauveria bassiana.

A search of the PDB for similar sequences retrieved only two significant results, one for a knottin-type antifungal peptide Alo-3 from the insect Acrocinus longimanus and the second from the disulfide-constrained wound healing peptide pB1 from Pereskia bleo. The details of all close and remote homologs identified using the different sequence similarity approaches are summarized in Supplementary Table S1.

An alignment of LhKNOT to the known structures of typical knottins as well as identified homologs showed the conservation of the six cysteines that form the three disulfide bridges in LhKNOT (Figure 1A). On its N-terminus, LhKNOT contains a long, helical segment predicted to function as a signal sequence; internally, it contains three beta-sheets, as expected of the secondary structure of knottin peptides (Figure 1B).

Additionally, an assessment of sequence similarity of LhKNOT targeted to the available L. heteroatom and L. boulardi transcripts and from another Drosophila parasitoid, Ganaspis spp. (see Methods), revealed an abundance of related sequences. For L. heterotoma (Lh14), we identified 16 additional Lh14 knottin proteins (Supplementary Table S2).
For L. boulardi, we identified 22 knottin proteins (Supplementary Table S3). Six LhKNOT homologs were similarly identified from the Ganaspis hookeri (G1) and two LhKNOT homologs were found in the protein predictions made from the G. brasiliensis (VA) genome (Supplementary Table S4). Figure 2 shows the conservation of these parasitoid knottin sequences with LhKNOT. Thus, at least the few Drosophila parasitoids sampled in this study appear to encode multiple knottin proteins. Additional such knottin peptides may be encoded by these wasps, and systematic proteomic/bioinformatic approaches are needed to obtain a comprehensive list. The functions of these putative knottins in parasite physiology remain unknown.

Figure 1. (A) Multiple sequence alignment of LhKNOT (top) and homologs in the knottin family (top-to-bottom): Mesembryanthemum crystallinum (plant; antimicrobial; NCBI ID: AAC19399), Aceria tosichella (mite; antimicrobial; NCBI ID:MDE48292.1), Callosobruchus maculatus (beetle; unknown; NCBI ID:VEN35376.1), Dinothrombium tinctorium (mite; unknown; NCBI ID:RWS16713.1), Trichogramma pretiosum (Alo-2 like; parasitoid wasp; antimicrobial; NCBI ID: XP_014233229.1), Ignelater luminosus (beetle; unknown; NCBI ID: KAF2884324.1), Trichogramma brassicae (parasitoid wasp; unknown; NCBI ID: CAB0031014.1), Brassicogethes aeneus (beetle; unknown; NCBI ID: CAH0563829.1), Agrilus planipennis (ash borer; antimicrobial; NCBI ID: XP_018321119.1), Neodiprion lecontei (sawfly; unknown; NCBI ID: XP_015517007.2), Akanthomyces lecanii (fungus; antifungal; Uniprot ID: A0A168C5L6), Cordyceps javanica (fungus; antifungal; Uniprot ID: A0A545VVU1), Cordyceps confragosa (fungus; unknown; Uniprot ID: A0A179IJT7), Rosellinia necatrix (fungus; unknown; Uniprot ID: A0A1S8A723), Beauveria bassiana (fungus; unknown; Uniprot ID: A0A0A2VUF6), Acrocinus longimanus (Alo-3; beetle; antifungal; PDB ID: 1Q3J), and Pereskia bleo (pB1; cactus; PDB ID: 5XBD). Identical residues are highlighted in red, while chemically similar residues are highlighted in yellow. Black arrows denote the conserved cysteine residues that are responsible for the knottin structural fold, and the disulfide bridge connectivity of the three disulfide bridges is marked with neon green numbers. 

X and XX denote the variable number of residues at the N- and C-terminus, respectively. Secondary structure elements of the modeled LhKNOT are shown at the top. β and T represent β-strand, and β-turn, respectively. (B) Secondary structure prediction for LhKNOT, showing a long helical segment (pink cylinder; signal peptide) followed by three beta strands (yellow arrows). This secondary structure follows the canonical Knottin signature [36]. (C) A prototype of the knottin structural fold from the KNOTTIN database [36] (permission to reproduce this image given by Dr. Jean-Christophe Gelly, INSERM, October 2019). The two disulfide bridges (green) form a macro-cycle, while a third (blue) pulls through the center giving the protein its namesake knotted topology. (D) The predicted tertiary structure of LhKNOT, showing the knotted topology and conserved cysteine motif, cysteine residues form disulfide bridges in the conserved pattern seen throughout the knottin family (C1 -C4 and C2 -C5 form the macro-cycle, C3 -C6 pulls through the center).

Figure 2. (A) Multiple sequence alignment of LhKNOT (top) and homologs in Lh14. (B) Multiple sequence alignment of LhKNOT (top) and homologs in L. boulardi. The X marked in the green box corresponds to the following insertions in two Lb17 sequences: Lb: GISX01151661.1 X = SQAPPPTPEPFHPPGTPP; Lb: GISX01151653.1 X = SQLTSPRSTFPPTGSTIPSIITT. (C) Multiple sequence alignment of LhKNOT (top) and homologs in Ganaspis spp. Identical residues are highlighted in red, while chemically similar residues are highlighted in yellow. 

Black arrows denote the conserved cysteine residues that are responsible for the knottin structural fold, and the disulfide bridge connectivity of the three disulfide bridges is marked with neon green numbers. Secondary structure elements of the modeled LhKNOT are shown at the top. β and T represent β-strand, and β-turn, respectively. Grey stars are added on the top of blocks of sequences, the above residues represent residues modeled with alternate conformations.

2.2. The Three-Dimensional Structure of LhKNOT

The typical structural fold of a knottin peptide is defined by six cysteines involved in three disulfide bonds to form a cysteine-knotted three-stranded antiparallel beta-sheet [25]. An important characteristic of this fold is the connectivity of the cysteine residues to form the disulfide bridges and the disulfide bridge of cysteines 3 and 6 going through disulfides 1–4 and 2–5 to make a special disulfide through disulfide knot (Figure 1A, C). 

This disulfide, through a disulfide knot feature, distinguishes the knottin fold from other cysteine knot proteins. Other than the conserved cysteines, the rest of the amino acid sequence can be quite variable, and therefore, only a limited number of knottins are identified by sequence similarity approaches for LhKNOT (see the previous section and Supplementary Table S1). 

Fold recognition algorithms and template-based modeling approaches, on the other hand, retrieved several diverse knottins as suitable candidates for modeling LhKNOT. Among the various LhKNOT models generated, the best model was a multi-template-based model of LhKNOT created using the program HHpred [37] with structural restraints derived from the top three templates, Alo-3 (PDB: 1Q3J) [30], omega-agatoxin-IVA (PDB: 1IVA) [28], and PAFP-S (PDB: 1DKC) [25]. Knowledge-based energy profiles calculated using ProSA-web [38] for this selected model showed low energies and a z-score of −4.53, which is comparable to solved structures. 

Verify3D [39] assessed this multi-template-based model with a passing score (100.00% of the residues in the model have an averaged 3D-1D score greater than or equal to 0.2). The VoroMQA [40] score of 0.325 is likely a reflection of the small size of the peptide and the scores of the templates were in the same range (Supplementary Figure S1). 

This multi-template-based model typifies the knottin fold with three antiparallel beta-strands, constrained by disulfide bridges and the expected connectivity of disulfide bridges (Figure 1D). KNOTER3D, a tool in the KNOTTIN Database [36] corroborated that this model has a bona fide knottin fold and confirmed the previously identified disulfide bonding pattern in Figure 1D.

2.3. LhKNOT Shows the Presence of a CPC Clip, Capable of Interacting with Heparin and Other GAGs

To search for potential LhKNOT functions, structure–function correlations of close structural homologs of LhKNOT were investigated. A knottin protein from the cactus Pereskia bleo (PDB: 5XBD) [26] possesses a heparin-binding motif known as the CPC clip. This structural motif forms a geometrically constrained clip of two cationic and one polar residues that interact with heparin by salt bridges and hydrogen bonds clamping it in position within the positively charged surface of the clip [41]. 

A structural superposition of the cactus knottin and LhKNOT showed that the two structures are highly similar, with an overall difference of less than 2Å. Docking analysis of LhKNOT with heparin, using ClusPro’s specific parameters for heparin as a ligand, revealed that for LhKNOT, the polar interactions fitting the CPC clip motif occur in residues R1-S3-R14 (Figure 3A). 

Measured distances (both between alpha-carbons and centers of mass of participating residue side chains) differed from those of the CPC clip by less than 1Å, suggesting the presence of a putative CPC clip in LhKNOT (Figure 3B). The surface electrostatics of the modeled LhKNOT also showed the presence of the expected cationic binding pocket formed by the CPC clip [41,42] (Figure 3C).

To assess the CPC clip’s ability to interact with other GAGs, additional docking analysis was performed with LhKNOT and keratan sulfate (PDB ID: 1KES) and with LhKNOT and hyaluronic acid (PDB ID: 1HYA). In both cases, the positively charged binding surface created by the CPC clip is bound to the negatively charged molecules. Ligplots generated by PDBSum confirmed an association of the GAGs with the CPC-forming residues (Supplementary Figures S2 and S3).

2.4. Other Structural Homologs of LhKNOT Also Contain the CPC Clip Motif

The presence of the CPC clip in both LhKNOT and its structural homolog from Pereskia bleo (pB1) suggested the possibility of a broader conservation of the CPC clip within the knottin family. An analysis of 21 close structural homologs of LhKNOT with experimentally solved PDBs in the KNOTTIN database revealed that, in every case, the peptides possess the CPC clip motif. For most peptides (e.g., T. tridentatus), the motif parameters deviated less than 1Å from the core knottin fold, while for others (e.g., the offensive knottins), the deviation was up to a margin of 3Å (Supplementary Table S5). Similarly, modeled knottin proteins of the identified homologs showed that almost all of them possessed the CPC clip.

Sixteen additional sequences from L. heteroatom, 22 sequences from L. boulardi, and 8 sequences from Ganaspis spp., with no known 3D structures, were modeled and analyzed for the presence of the CPC clip motif. While 13 of the 16 putative L. heteroatom knottins and 20 of 22 putative L. boulardi knottins revealed a CPC clip motif, all Ganaspis sequences contained the CPC clip (Supplementary Table S5). Interestingly, the seven homologs that did not exhibit a CPC clip motif belong to (a) the common pollen beetle sequence (B. aeneus (CAH0563829.1), (b) fungal sequence R. necatrix (A0A1S8A723), and (c) three L. heterotoma knottins 4,5,6 and L. boulardi knottins 3 and 20 (Supplementary Table S5).

A survey of knottins in the KNOTTIN database, unrelated to LhKNOT, but representative of various taxa (cone snail, horseshoe crab, insect, plant, scorpion, and spider), showed the CPC clip motif. In all cases, the surface electrostatic profile of the CPC clip motif is positively charged, likely, to facilitate interactions with the negatively charged heparin (Figure 4). However, similar to the beetle, fungal, and wasp knottins described above, the CPC clip was not identified in knottins from the plant cyclotide and sponges (Supplementary Table S5).

Figure 4. Heparin-docking results and surface electrostatics of (A) antifungal peptide of Phytolacca americana (PFAP-s; plant; PDB: 1DKC), (B) calcium channel selective omega-agatoxin of Agelenopsis aperta (Omega-agatoxin IV-A; spider; PDB: 1IVA), (C) antifungal peptide of Acrocinus longimanus (Alo-3; beetle; PDB: 1Q3J), (D) wound-healing peptide of Pereskia bleo (pB1; cactus; PDB: 5XBD), (E) LhKNOT, (F) antifungal peptide “Drosomycin” of Drosophila spp. (Drosomycin; fly; PDB: 1MYN), (G) Conotoxin GS (Conotoxin GS; cone snails; PDB:1AG7), (H) antimicrobial peptide Tachystatin A isolated from Horseshoe crabs (Tachystatin A; horseshoe crabs; PDB:1CIX) and (I) Chlorotoxin, a small scorpion toxin of Leiurus quinquestriatus (Chlorotoxin; scorpion; PDB:1CHL) showing the cationic CPC clip motif. Figures oriented to best display the CPC clip structure. The surface electrostatic potentials are color-graded from −4 kT/e (red) to +4 kT/e (blue).

2.5. In Silico Mutational Analysis of LhKNOT CPC Clip Reveals a Secondary CPC Clip

Models of three in silico LhKNOT mutants (R1A, S3A, R14A) were investigated to compare binding interactions of wild-type and mutant proteins. In the case of the R14A mutant, heparin was shown to be surprisingly associated with a secondary CPC clip involving the R1, S3, and R30 residues instead of the R1, S3, and R14 motif in the wild type (Supplementary Figure S4). Double or triple mutants (R14A-R1A; R14A-S3A-R1A), however, did not show an interaction with any of the primary or secondary CPC clip motif residues (Supplementary Figure S4C, D). 

The in silico mutants with the R1A mutation (interrupting both the primary and secondary CPC clip motifs), however, did show heparin associating with R29 and R30, as they provide the only remaining cationic surface (Supplementary Figure S4C, D). Interestingly, the ligand prefers to associate with the CPC clip motif, both in the case of the primary and secondary clips, over this additional cationic surface when the motif is available, suggesting the specific interaction of the CPC clip may allow a more stable association over a non-specific electrostatic interaction.

For more information:1950477648nn@gmail.com