In Silico Analysis Of A Drosophila Parasitoid Venom Peptide Reveals Prevalence Of The Cation–Polar–Cation Clip Motif in Knottin Proteins Part 2

2.6. A CPC Clip Is Also Present in the Antimicrobial Peptide Drosomycin

Unlike LhKNOT, the structure of Drosomycin contains a different fold with an alpha-helix and a twisted three-stranded beta-sheet, which is stabilized by three disulfide bridges [43]. This structural motif, termed a “cysteine stabilized αβ-motif”, is also found in other host-defense proteins such as the antibacterial insect defensin A, scorpion toxins, plant thionins, and antifungal plant defensins [43,44]. 

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Given the known antimicrobial function of Drosomycin and its related “cysteine stabilized αβ-motif” [43,44], we examined the possibility of a common basis for antimicrobial activity and shared structure–function relationships with LhKNOT to better understand the potential significance of this motif in LhKNOT in the context of host defense. 

Despite a slightly different structural fold, Drosomycin overlays within an RMSD of 2.2Å of both LhKNOT and pB1 (Figure 5A). When the previous CPC clip analysis was extended to Drosomycin, the results revealed the presence of the CPC clip motif in Drosomycin as well. The CPC clip motif in Drosomycin (R6-S4-K38) falls within a 2Å deviation of the motif parameters (Figure 5B, Supplementary Table S5). 

Additionally, surface electrostatics of Drosomycin confirmed the putative CPC clip’s formation of a cationic binding pocket as also observed for LhKNOT and other knottins (Figure 5C, Supplementary Table S5). 

These results suggest that there may be a similarity in the mode of action of Drosomycin and LhKNOT and that the biochemistry embedded in this structural motif may drive antimicrobial function in both peptides.

Figure 5. (A) Superposition of LhKNOT (blue), wound-healing peptide of Pereskia bleo (tan; pB1; cactus; PDB: 5XBD) and Drosomycin (green; Drosomycin; fly; PDB: 1MYN). (B) Distances measured in PyMOL between the centers of mass of participating residues (grey pseudo-atoms) in the Drosomycin–heparin interaction. Distances fall within 2Å of the value ranges of the CPC clip motif. (C) Surface electrostatics of Drosomycin (fly; PDB: 1MYN), showing the positively charged binding surface formed by the CPC clip where heparin is docked. The surface electrostatic potentials are color-graded from −4 kT/e (red) to +4 kT/e (blue).

3. Discussion and Speculation

The biochemical functions of proteins are facilitated by their overall structures, which are reportedly 3–10 times more well-conserved than their primary structures [45]. Consequently, sequence alignments often fall short in investigating the functional potential of proteins such as knottins, where the varied primary structures may obscure shared structure–function relationships. 

In silico methods facilitate broad analysis of potential structure–function correlations for such interesting folds [46–50]. Our detailed analysis of the LhKNOT structure and its structural homologs supports the idea that this parasite protein has the hallmarks of a knottin peptide with functions in host defense: (a) the closest matches to LhKNOT are antimicrobial peptides; (b) LhKNOT’s Pfam signature describes it as cysteine knotted “antifungal peptide”; and (c) a CPC clip is present.

Analysis of a knottin wound-healing peptide login pB1 from the leafy cactus Pereskia bleo initially revealed the presence of a CPC clip, a conserved structural signature of heparin-binding proteins [42]. It has immunity- and signaling-related functions in wound healing and cancer and is used as an anticoagulant [51]. 

GAGs are present on animal cell surfaces and have become an increasingly popular target of studies ranging from clinical to cosmetic [52]. The CPC clip motif of heparin-binding proteins is considered to be the minimum required motif for heparin-binding affinity and is defined by specific spatial relationships between three residues, two cationic (Arg/Lys) and one polar (Asn, Gln, Ser, Thr, Tyr). 

This CPC clip motif is defined by distances between both the α carbons of the involved residues as well as the distances between the centers of mass of the involved residues. Heparin-binding proteins are known to interact promiscuously with other negatively charged substrates, most notably the surface membranes of microbes [41]. 

As a result, heparin-binding proteins can exhibit antimicrobial activity to varying degrees [53]. While the precise mechanisms of this antimicrobial potential are not well defined, it has been hypothesized that the CPC clip may facilitate these protein–microbe interactions [41]. New potential roles of heparin-binding are emerging, such as in the potential therapeutic interventions for amyloidogenesis [54], so understanding the structural determinants of the CPC clip in diverse proteins may be key to delineating the mechanism of the interaction and its disruption.

A clue to a possible antimicrobial mechanism for LhKNOT comes from our finding that its inhibitory cysteine knot and those of other knottins examined here have a CPC clip intrinsic to their folds (Supplementary Table S5). 

It has been noted that the structural and biophysical requirements for heparin-binding proteins’ ability to interact with heparin are strikingly similar to the structural features of many known AMPs [53]. Thus, the CPC clip motif may fit the criteria for both these requirements [41]. 

Our discovery of this CPC clip motif not only in LhKNOT but also in 75 other related or unrelated knottin proteins (Supplementary Table S5) suggests that this CPC clip motif may be a highly conserved feature of the knottin proteins. 

The coincident occurrence of the CPC clip motif within the cysteine knot structure in diverse proteins suggests that the CPC clip motif itself may underlie the antimicrobial function. However, because this structural motif is also identified in offensive knottins (Supplementary Table S5), it may also serve a more general role via this more generalized CPC clip interaction motif.

The discovery of a putative CPC clip motif in Drosomycin is intriguing, as it sheds light on the possible mechanism of action of this antifungal peptide and supports a possible antimicrobial function for the CPC clip motif itself. Previous studies have shown the importance of Drosomycin’s residues R6 and K38, which are part of its CPC clip motif (R6-S4-K38), for its antifungal potential; alterations of these residues reduce antifungal activity [55]. Our structural analysis of the wasp LhKNOT and its similarities with Drosophila Drosomycin offers possible insights into the L. heterotoma’s ability to broadly suppress the TollNF-κB pathway in D. melanogaster and yet succeed on immune-compromised hosts. 

In the absence of a functional Toll immune pathway, and consequently, without Drosomycin production, Drosophila larvae can often succumb to opportunistic pathogens [11,13,56]. LhKNOT’s putative antimicrobial activities might defend the host from opportunistic microbial pathogens in wasp-infected, immune-compromised host larvae and developing pupae. Alternatively, similar to Drosomycin, LhKNOT could induce hemocyte apoptosis [57]. L. heteroatom EVs enter hemocytes after infection [58], and their activities within EVs compromise encapsulation [19,21].

The CPC clip has been shown to associate promiscuously with GAGs [41], which are nevertheless ubiquitous in insects including Drosophila [59]. Heparin sulfate GAGs in flies serve as growth factor receptors and participate in creating and maintaining morphogenic gradients [60]. The CPC clip has also been shown to associate promiscuously with lipopolysaccharides (LPS) [41]. Bacterial LPS, being major surface components of Gram-negative bacteria, are extremely potent stimulators of the innate immune response in various eukaryotic species including insects [61]. GAGs in Drosophila also control the binding of α C protein, a virulence determinant of group B streptococcus [62]. 

Thus, it is possible that a CPC clip containing LhKNOT and Drosomycin (and possibly other fly AMPs, such as Defensin [63]) can modulate immune signaling (and/or binding to the pathogen’s virulence proteins), similarly affecting the bacterial pathogen/metazoan parasite outcome. Previous studies have shown that three homologous knottins (Alo-1, 2, 3) from the harlequin beetle Acrocinus longimanus are active against yeast, Candida glabrata [30]. Additionally, a pore-forming candidacidal peptide, Psacotheasin, attacks Candida albicans [64]. Lastly, a plant pathogenic rust fungus, Melampsora lyrics-popular, produces a knottin peptide as its candidate effector [65].

Our finding that multiple putative knottin peptides may be secreted by adult parasitoid wasps of Drosophila was unexpected and intriguing. While their functions are not known, the possible existence of a knottin gene family suggests some redundancy in their functions that may be linked to the parasitic life history. The exploration of the knottin structural fold in unrelated and yet-to-be-discovered knottins will prove to be valuable. Our work provides a short list of residues significant to the proposed physiological activity for the Knottin family protein investigation. Such information can be incorporated into rational drug discovery and design for infectious diseases and related therapies.

4. Methods

4.1. Sequence Analysis

Initial scans of the NCBI non-redundant protein sequence (nr) database (https:// www.ncbi.nlm.nih.gov/protein; accessed on 4 April 2018), the Transcriptome Shotgun Assembly (TSA) sequences database (https://www.ncbi.nlm.nih.gov/genbank/tsa; accessed on 16 April 2018) [66], and the Protein Data Bank (PDB; https://www.rcsb.org; accessed on 5 July 2018) [67] with the full-length amino acid sequence of the L. heteroatom knottin peptide (GAJC01011813.1) using NCBI BLAST (BLASTp; https://blast.ncbi.nlm.nih.gov/ Blast. cgi; accessed on 16 April 2018) [68] were used to retrieve related protein sequences in other organisms. Remote homologs were searched using Position-Specific Iterative BLAST (PSI-BLAST; https://blast.ncbi.nlm.nih.gov/Blast.cgi; accessed on 4 April 2022) [69], and Hidden Markov Models (HMMER; https://www.ebi.ac.uk/Tools/hmmer; accessed on 6 April 2022) [70,71]. BLAST searches were adjusted to word size 2, and scoring matrix BLOSUM45, while default parameters were used for HMMER.

To identify knottin homologs in select Leptopilina and Ganaspis spp. not accessible in the protein databases, targeted searches using translated BLASTs against genomic and transcriptomic datasets were employed. To identify additional L. heteroatom knottins, the L. heteroatom female abdominal transcriptome GAJC [72] and the whole-body transcriptomes from Ground GHUQ (GenBank Accession: GHUP00000000; TSA: Leptopilina heteroatom strain Lh14, transcriptome shotgun) and Space GHUP (GenBank Accession: GHUQ00000000; TSA: Leptopilina heteroatom strain Lh14, transcriptome shotgun) were searched using tblastn (e-value threshold set to 1, BLOSUM62, low complexity filter enabled, query coverage filter set to 20 percent) against LhKNOT. 

The following transcriptomes were similarly searched to identify L. boulardi knottins: L. boulardi female abdominal transcriptome GAJA [72] and the whole-body transcriptomes from Ground GITC (GenBank Accession: GITC00000000; TSA: Leptopilina boulardi strain Lb17, transcriptome shotgun), Space GISX (GenBank Accession: GISX00000000; TSA: Leptopilina boulardi strain Lb17, transcriptome shotgun), and GGGI00000000 (female abdomen/head) [73]. In addition, targeted searches for Ganaspis spp. homologs were carried out. For G. hookeri, the GAIW00000000 TSA: Ganaspis spp. 

G1 dataset [74] was searched (tblastn, e-value threshold set to 1, BLOSUM45, low complexity filter enabled, query coverage filter set to 20 percent) using the LhKNOT query. The Expasy tool (web.expasy.org/translate/; accessed on 9 December 2022) was used to translate these TSA sequences [75]. The resulting predicted peptide sequences were manually curated based on the presence of the cysteine motif. For G. brasiliensis, AUGUSTUS-based (http://bioinf.uni-greifswald.de/augustus; accessed on 9 April 2020) [76] gene predictions from the partially assembled genomic sequence (Gen-Bank: GCA_009823575.1) were made using the Nasonia vitripenis genome training module. 

The resulting predicted proteins were searched using blastp (e-value threshold set to 1, low complexity filter enabled, query coverage filter set to 20 percent) against LhKNOT. Alignments and visualization were performed using EBI’s Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo; accessed on 9 December 2022) [77] to confirm the presence of the knottin motif and for further curation of the sequences. 

The set of identified sequences was aligned to LhKNOT to create a multiple sequence alignment using T-COFFEE (https://www.ebi.ac.uk/Tools/msa/tcoffee; accessed on 9 December 2022) [78] and visualized using ESPript3 (https://espript.ibcp.fr/ESPript/ESPript/index.php; accessed on 9 December 2022) [79] to identify any conserved residues and/or motif(s) in the aligned sequences.

4.2. LhKNOT Primary Structure Analysis

The LhKNOT sequence was also analyzed by the domain/motif detection database Conserved Domain Database (CDD; https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd. shtml; accessed on 6 April 2018) and Pfam (pfam.xfam.org; accessed on 6 April 2018) to identify any domains or sequence signatures [80,81]. 

The sequence was further analyzed using the signal sequence prediction software SignalP-5.0 (https://services.healthtech.dtu. dk/service.php?SignalP-5.0; accessed on 6 April 2018) [82] and Phobius (https://www. ebi.ac.uk/Tools/pfa/phobias; accessed on 6 April 2018) [83] to identify the location of the signal sequence and allow for more accurate characterization of the peptide. The mature peptide of 36 amino acids (with the predicted signal sequence, residues 1–24, removed) was used for further analysis. 

The secondary structure of the peptide was predicted using the meta-server Sympred (https://www.ibi.vu.nl/programs/sympredwww; accessed on 6 April 2018) [84] and analyzed to confirm its correspondence to the expected secondary structure for a knottin fold: an initial alpha-helical segment (predicted signal sequence) followed by three antiparallel beta strands.

4.3. Tertiary Structure Prediction and Evaluation of LhKNOT and Its Homologs

The programs I-Tasser (https://zhanggroup.org/I-TASSER; accessed on 16 April 2018) [85], Modeller (https://salilab.org/modeller; accessed on 16 April 2018) [86], and HHPred (https://toolkit.tuebingen.mpg.de/tools/hhpred; accessed on 6 April 2018) [37] were used to model the tertiary structure of LhKNOT. Three structural homologs with the knottin-like fold Alo-3 (PDB: 1Q3J) [30], omega-agatoxin-IVA (PDB: 1IVA) [28], and PAFP-S (PDB: 1DKC) [25] were identified as top candidates and used as templates in model building programs. 

The predicted model for LhKNOT was evaluated by Verify3D (https://www.doe-mbi.ucla.edu/verify3d; accessed on 18 April 2018) [39], VoroMQA (https://bioinformatics.lt/wtsam/voromqa; accessed on 18 April 2018) [40], and Prosaweb (https://prosa.services.came.sbg.ac.at/prosa.php; accessed on 18 April 2018) [38]. Evaluating small peptides with standard evaluation tools can have limitations. 

Thus, scores from solved tertiary structures of NMR and crystallography-derived peptides of similar size and shape (PDB: 1Q3J, 1IVA, and 1DKC) were used to interpret the evaluation scores of our LhKNOT models. The top-ranked model was created using HHPred [37], a modeling program that uses the underlying model-building program, Modeller [86] using a restraint-based comparative modeling approach. In addition, the KNOTTIN database’s KNOTER3D tool (https://www.dsimb.inserm.fr/KNOTTIN/knoter3d.php; accessed on 15 May 2020) [36] was used to evaluate the model’s tertiary structure, confirming LhKNOT’s adoption of the Knottin fold. All the identified homologs with no known structures were modeled using the top template identified in HHPred [37].

4.4. CPC Clip Motif Identification

Superposition of LhKNOT’s structural model with the known structures of a CPC clip containing knottin peptide from the cactus Pereskia bleo (PDB: 5XBD) [26] using the programs Superpose (http://superpose.wishartlab.com; accessed on 12 September 2018) [87] and MultiProt (http://bioinfo3d.cs.tau.ac.il/MultiProt; accessed on 12 September 2018) [88] suggested the presence of a heparin-binding CPC clip motif. To confirm the presence of the CPC clip in LhKNOT, docking analysis using the program, ClusPro 2.0 (which offers parameters specific to heparin as ligand; https://cluspro.org; accessed on 2 March 2019) [89], and subsequent analysis of predicted docking scenarios in the visualization program, PyMOL (https://pymol.org; accessed on 10 March 2019) [90] was performed. 

All potential docking scenarios were analyzed for identifying residues forming the putative CPC clip motif using the following steps. All polar interactions between LhKNOT and heparin were visualized in PyMOL and lists of participating residues were compiled. Predictions that contained the necessary Cation–Polar–Cation interactions were then further scrutinized for adherence to the CPC clip motif parameter by measuring distances between alpha carbons of participating residues as well as distances between the center of mass of participating residues. PyMOL’s “Measurements” Wizard tool was used to measure distances between α-carbons of the participating residues. 

To evaluate the distances between centers of mass, a user-generated script available on the Pymolwiki website (Henschel, https://pymolwiki.org/index.php/Center_of_mass; accessed on 15 March 2019) was used to generate pseudo-atoms at the calculated sidechain center of mass. PyMOL’s APBS Electrostatics Plugin [91] was used to render the surface electrostatics for the modeled LhKNOT. Similar steps were carried out to identify the CPC clip motif in each of the remaining knottin proteins in Supplementary Table S5.

4.5. Association of LhKNOT with Other GAGs

To assess whether the CPC clip would facilitate binding with additional GAGs, further docking was performed with hyaluronic acid (PDB: 1HYA) and keratan sulfate (PDB: 1KES). In the case of hyaluronic acid, extraneous atoms (O, Na) were removed from the space surrounding the molecule. Keratan sulfate’s model was used as is. Docking was performed using AutoDock (https://autodock.scripps.edu; accessed on 7 April 2020) [92], and results were visualized in PyMOL. Results were then input into PDBSum (http://www.ebi.ac. uk/thornton-srv/databases/pdbsum/Generate.html; accessed on 10 April 2020) [93] to generate Ligplots to confirm GAG-CPC clip binding.

4.6. Protein-Heparin Docking Analysis for Mutated CPC Clip in LhKNOT

To investigate the importance of the CPC clip in heparin binding, mutated models of LhKNOT were generated using PyMOL’s mutagenesis tool, substituting the CPC clip residues with alanine. Models were created piecewise, first substituting just R14, then R1 and R14, and finally R14, R1, and S3. Heparin docking of all models was performed using ClusPro, and analysis was performed as described previously.

4.7. Protein-Heparin Docking Analysis in Structural Homologs and Drosomycin

To further evaluate the presence of the CPC clip motif in the knottin peptide family, the previous methods were further expanded to (a) structural homologs of LhKNOT (PDB: 5XBD, 1Q3J, 1IVA, and 1DKC) [25,26,28,30]; (b) Drosophila Drosomycin (PDB: 1MYN) [43], a peptide with known antifungal activity; (c) modeled homologous knottin proteins and (d) representative knottins from different categories in the KNOTTIN database. Results were considered to adhere to the motif if measurements fell within 3Å of the CPC clip parameters, to allow for some flexibility of protein loop conformations.

Supplementary Materials:

The following supporting information can be downloaded at https:// www.mdpi.com/article/10.3390/pathogens12010143/s1. Table S1. Identified homologs of LhKNOT using different sequence-based approaches. Table S2. Putative L. heterotoma homologs of LhKNOT. Sequences were identified via tblastn from three Lh14 transcriptome datasets (see Methods for accession numbers). 

Transcripts grouped by shaded cells encode identical knottin peptides. Transcript accession numbers shown in bold encode two distinct peptides. Results were retrieved from the Description Table available from NCBI blast results. Table S3. Putative L. boulardi knottin homologs of LhKNOT. Sequences were identified via tblastn from four L. boulardi transcriptome datasets (see Methods for accession numbers). Identical knottin peptides derived from different transcripts are grouped by color. Results were retrieved from the Description Table from NCBI blast results. Table S4. 

Putative knottin sequences from Ganaspis spp., homologous to LhKNOT. G. hookeri sequences were identified with tblastn, while G. brasiliensis sequences were identified with a blast. Results for G. hookeri and G. brasiliensis were retrieved from the Description Table available from NCBI search results. Shaded sequences encode identical peptides. Table S5. 

A list of amino acid residues in the CPC clips of examined known knottin, modeled knottin peptides, and Drosomycin. Distance measurements (Å) of alpha-carbon and sidechain center of mass (via pseudo-atom) of participating residues of identified CPC clip motifs are shown. Key: + Known offensive knottins (toxins), Φ in silico models, * non-knottin fold, ±alternate putative peptide, highlighted in green and yellow to demarcate peptides arising from the same transcript. Figure S1: (A) Prosa-web evaluation profile. The Z-score of the model is represented as a black dot on a backdrop of Z-scores of known structures from the PDB (left) and visualization of the three-dimensional model LhKNOT with residues colored from blue to red in the order of increasing residue energy (right). (B) Verify evaluation profile. The plot shows the averaged and raw 1D-3D scores for LhKNOT and the panel on top lists the percentage of residues that scored an average of 0.2 or above. 

A passing model has at least 80% of its residues scoring above 0.2. (C) VoroMQA evaluation profile. VoroMQA global scores in the context of high-quality X-ray structure scores (left) and plot of the local score and overall score for LhKNOT (right). Figure S2. Ligplot showing interactions of LhKNOT with keratan sulfate (PDB: 1KES) include the CPC clip residues (R1-S3-R14). Green dashed lines depict hydrogen bond interactions. Figure S3. Ligplot showing interactions of LhKNOT with hyaluronic acid (PDB: 1HYA) include the CPC clip residues (R1-S3-R14). 

Green dashed lines depict hydrogen bond interactions. Figure S4. (A) Ligplot showing interactions of wild-type LhKNOT interacting with heparin via the primary CPC clip (R1-S3-R14). (B) Ligplot showing interactions of mutant R14A LhKNOT interacting with heparin via a secondary CPC clip (R1-S3-R30). Green dashed lines depict hydrogen bond interactions. References [94–111] are cited in the Supplementary Materials.

Author Contributions:

Conceptualization, S.S., S.G., and J.A.; methodology, J.A., and S.S.; validation, S.S., S.G., J.A., J.L., and L.V.C.; formal analysis, J.A., J.L. and L.V.C.; investigation, J.A., J.L., and L.V.C.; resources, S.S. and S.G.; data curation, J.A., J.L., and L.V.C.; writing—original draft preparation, J.A.; writing—review and editing, S.S., S.G., J.A., J.L. and L.V.C.; visualization, J.A., and J.L.; supervision, S.S. and S.G.; project administration, S.S. and S.G. All authors have read and agreed to the published version of the manuscript.

Funding:

This research received no funding.

Institutional Review Board Statement:

Not applicable.

Informed Consent Statement:

Not applicable.

Data Availability Statement:

The data presented in this study are available in the article and Supplementary Materials.

Acknowledgments:

We are grateful to Brian Wey for their help with G. brasiliensis gene predictions and S.A. Tasnim Ahmed for his help with formatting the figures.

Conflicts of Interest:

The authors declare no conflict of interest.

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