Team:Lethbridge/Modelling

Introduction

The modeling team is looking into developing molecular dynamics simulations based on canonical peptides found in literature. This first involves locating AMPs of interest that have a crystal structure and a site of interaction. Although not all of our peptides have identified crystal structures, for the current iGEM season we are focusing on those that do to allow for more seamless simulations and effective training for new members to the subgroup. We examined 8 of the peptides looked at by Portell-Buj et al. (2019) looking at their structural characteristics, mode of action, toxicity to humans, antimicrobial properties, thermostability, and if there was a crystal structure.¹⁹

Peptides from Portell-Buj et al (2019)

Mastoparans

Mastoparans are a subcategory of AMPs isolated from social wasp venom and are composed of polycationic peptides with 14 amino acid residues and an amidated C-terminus. They contain 2–4 lysine residues, and have a positive net charge and an alpha-helical conformation. Some mastoparan peptides have been well-studied and are known to possess antifungal activity. These mastoparan AMPs may act by altering membrane permeability or by directly forming pores in the membrane¹².

According to their mode of action, these peptides may be classified into two groups: (i) those acting by cell lysis from pore formation on cell membranes. (ii) and those acting by interaction with G-protein–coupled receptors¹⁰.

Mastoparan melting point: 56°C⁹, ideally stored at -20°C and at a pH between 5.0 and 7.0¹¹. Effects on Humans: In the experiment conducted in the following paper, virtually no hemolytic activity on human erythrocytes was observed at 10-4M. These results indicated that the peptide is rather strongly associated with bacterial cell membranes rather than mammalian cell membranes. There are currently believed to be no other avenues of negative health effects on humans from this peptide⁹.

Mastoparan Sequences:

Mastoparans effective against fungi: Mastoparan M (C. albicans and C. parapsilosis), Mastoparan VT2 (C. albicans and C. parapsilosis), Mastoparan VT3 (C. albicans and C. parapsilosis), Mastoparan VT4 (C. albicans and C. parapsilosis), Mastoparan VT5 (C. albicans), Mastoparan VT6 (C. albicans and C. parapsilosis), Mastoparan VT7 (C. albicans), Mastoparan S (A. niger, A. fumigates, and C. albicans), Mastoparan V1 (C. albicans and C. neoformans). Mastoparan melting point: 56°C⁹

Mastoparan Crystal Structure⁵:

Histatin 5

Sequence: DSHAKRHHGYKRKFHEKHHSHRGY (Length of 24 amino acids residues)

Versatile, cationic AMP originating in the salivary glands of homo sapiens (human beings).

Properties include Anti-Gram+ & Gram- (ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanni, Pseudomonas aeruginosa, and Enterobacter species), Antiviral, Antifungal (C. albicans, candidacidal, Anti-HIV, Anti-MRSA, Enzyme inhibition action. Also, it is essential that Zn2+ ions bind in order for antibacterial activity to occur¹.

The mode of action by which Histatin-5 acts involves multiple intracellular targets. The effects on fungal targets range from non-lytic leakage of ATP and K+ ions to mitochondrial damage and oxidative stress generation; and more recently, potential metal scavenging causing nutritional stress. Specifically, Histatin-5 is believed to exert its anticandidal effect through binding to receptor proteins on the fungal cell membrane. Once internalized, Hst-5 inhibits mitochondrial respiration, thus inducing the formation of reactive oxygen species leading to mitochondrial and cytoplasmic membrane damage, efflux of ATP, and cell death. However, it has been clearly shown that the killing is non-lytic and energy dependent⁴.

Toxicity: Being of human origin, this peptide is not considered to be toxic to humans, even at greater quantities than what is usually found in the human body⁸. Heat-stability: While the exact melting point of this peptide is not known, being of human origin, it follows that the peptide is able to withstand the average human body temperature (~37°C and likely reasonably above that).

Histatin 8

Sequence: KFHEKHHSHRGY (Length of 12 amino acid residues)

This cationic AMP originates in the salivary glands of homo sapiens (human beings) and is a fragment of the aforementioned Histatin-5, it Anti-Gram+ & Gram-, Antifungal and highly Anti-candidacidal (Active against C. albicans and C. tropicalis.)

The mode of action by which Histatin-8 acts involves multiple intracellular targets. The effects on fungal targets range from non-lytic leakage of ATP and K+ ions to mitochondrial damage and oxidative stress generation; and more recently, potential metal scavenging causing nutritional stress. Specifically, Histatin-8 is believed to exert its anticandidal effect through binding to receptor proteins on the fungal cell membrane. Once internalized, Histatin-8 inhibits mitochondrial respiration, thus inducing the formation of reactive oxygen species leading to mitochondrial and cytoplasmic membrane damage, efflux of ATP, and cell death. However, it has been clearly shown that the killing is non-lytic and energy dependent⁴.

Toxicity: Being of human origin, this peptide is not considered to be toxic to humans, even at greater quantities than what is usually found in the human body⁸. Heat-stability: While the exact melting point of this peptide is not known, being of human origin, it follows that the peptide is able to withstand the average human body temperature (~37°C and likely reasonably above that).

Melittin

Sequence: GIGAVLKVLTTGLPALISWIKRKRQQ (Length of 26 amino acid residues).

This AMP is Anti-Gram+ & Gram-, Antiviral, Antifungal, candidacidal (C. albicans), Antiparasitic, Insecticidal, Anti-HIV, Anti-sepsis, Hemolytic, Anticancer, and is found in Bee venom. It is water-soluble as a tetramer, but it spontaneously integrates into lipid bilayers and is thought to act as a lytic agent¹³. Each melittin chain is composed of two a-helical segments and its overall shape is that of a bent rod, with the bend occurring between residues 11-15. The NH2-terminal 20 residues are arranged asymmetrically about the bent rod according to their polarity. Ten large apolar residues in each chain are on one side of the bent rod, and the opposite face contains four polar side chains, four small apolar side chains, and the prolyl residue. The C-terminal 6 residues of the chain are entirely polar¹³

Mode of action: Melittin can open thermal nociceptor TRPV1 channels via cyclooxygenase metabolites resulting in depolarization of nociceptor cells. The pore forming effects in cells causes the release of pro-inflammatory cytokines. It also activates G-protein-coupled receptor-mediated opening of transient receptor potential channels. Finally melittin up-regulates the expression of Nav1.8 and Nav1.9 sodium channels in nociceptor cell causing long term action potential firing and pain. sensation.nMelittin inhibits protein kinase C, Ca2+/calmodulin-dependent protein kinase II, myosin light chain kinase, and Na+/K+-ATPase (synaptosomal membrane). Mellitin blocks transport pumps such as the Na+-K+-ATPase and the H+-K+-ATPase³.

Toxicity: As it was previously mentioned, melittin is insecticidal, however, in humans studies have shown that it has a relatively low toxicity. It does block transport pumps such as the Na+-K+-ATPase and the H+-K+-ATPase, since it is a component of bee venom, it stimulates the nocioreceptors that are associated with the pain of bee sting³. However, it is currently being studied for its viability as a treatment for Diabetes Mellitus, thus indicating that in reasonable doses, it represents a relatively low risk to human health⁷.

Heat/pH stability: Melittin converts from a monomeric random coil to an alpha-helical tetramer as the pH is raised from 4.0 to 9.5. The melittin tetramer is found to have a temperature of maximum stability ranging from 35.5 to 43 degrees C depending on the pH, unfolding above and below that temperature¹⁴. Melittin has also been shown to maintain its antimicrobial properties at temperatures of 70 degrees C or 90 degrees C for the time intervals 5, 15, and 30 minutes it was tested at¹⁵.

Melittin Crystal Structure⁵:

Indolicidin

Sequence: ILPWKWPWWPWRR-NH2

The Indolicidin peptide comes from the cathelicidin family from Bos taurus, and is 13-residues in length and cationic. This peptide has the highest tryptophan content of any known protein at 39%, and the native peptide has an amidated carboxyl terminus. Structurally, Indolicidin is unique as a result of its high tryptophan and proline content.¹⁶ It has been shown to have antibacterial, antifungal, and antiviral properties.^17,18

Mode of Action: The mode of action of this peptide was explored by Falla et al, (1996).²² The uptake of Indolicidin was shown to occur in a mechanism similar to alpha-helical and beta-sheet AMPs, where binding occurs at the divalent cation binding sites on the lipopolysaccharide (LPS), which is known as the self-promoted uptake pathway. In Gram-Negative bacteria, Indolicidin was shown to interact with both the inner and outer cytoplasmic membranes by permeabilization from the displacement of the divalent cations on the LPS binding site.²²

Toxicity: Indolicidin has received the certification for clinical use, and is currently being used as a pharmaceutical for skin acne and infections.²⁰

Heat/pH Stability: A study examined the thermostability of Indolicidin among other AMPs, and it retained antimicrobial activity at temperatures as high as 70 degrees C or 90 degrees C for 5, 15, and 30 minute time intervals.¹⁵

Crystal Structure (1HR1)21:

Cecropin P1

Sequence:SWLSKTAKKLENSAKKRISEGIAIAIQGGPR

Cecropin was first discovered in pig intestines, and was thought to be a mammalian AMP, but it has been confirmed that this peptide is from the parasitic nematode, Ascaris suum, that resides in the pig intestine. The peptide is active against a significant range of microbes including Gram-positive and Gram-negative bacterial species, and weakly active against some yeasts.²³

Mode of Action: Cecropin P1 acts in a similar manner to the other cecropins, and lyses bacterial cells by membrane permeabilization.²⁴

Heat/pH stability In a study from Ebbensgaard et al. (2015), the thermostability of a range of AMPs including Cecropin P1 and Cecropin B was tested.15 After being incubated for 5, 15, or 30 minutes, both of these peptides were stable at 70 degrees C or 90 degrees C. The results are summarized in the following table:

The pH for this specific peptide has not been examined thoroughly, but a similar peptide (Cecropin AB) was not greatly affected by pH changes within the range of 4.0 to 9.0, although pHs lower than 4.0 did lower the activity.²⁵.

LPS-Bound crystal structure (2N92):

Cecropin A

Sequence:AKWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQA

Cecropin A is a well-characterized antimicrobial peptide that permeabilizes cell membranes. It has antibacterial, antiviral, and antiparasitic properties and comes from the organism Hyalophora cecropia, or the cecropia silkworm. ²⁶

Mode of Action: The mechanism for this peptide was studied in depth against the unpathogenic E. coli (UPEC) biofilm which causes urinary tract infections. Cecropin A acts by outer membrane permeabilization and inhibition of efflux pump activity. It also interacts with nucleic acids extracellularly and intracellularly. ²⁷

The thermostability and pH range has not been examined specifically, but since this peptide is from the cecropin family it would likely exhibit similar trends in stability to Cecropin P1 and Cecropin B.

Crystal Structure

The crystal structure has not been identified, and thus we have included a homology model from SWISS-MODEL:

Cecropin B

Sequence:KWKIFKKIEKVGRNIRNGIIKAGPAVAVL

Cecropin B comes from the giant silk moth (Hyalophora cecropia) and is active against Gram-negative and some Gram-positive bacteria.²⁸

Mode of Action: The mechanism of Cecropin B has been assessed by Gazit et al. (1994) using spectrofluorimetry. Cecropin B and its truncated analogue [3->35]Cecropin B were found to permeate acidic phospholipid vesicles.³⁰

Heat/pH stability: In a study from Ebbensgaard et al. (2015), the thermostability of a range of AMPs including Cecropin P1 and Cecropin B was tested. After being incubated for 5, 15, or 30 minutes, both of these peptides were stable at 70 degrees C or 90 degrees C. The pH for this specific peptide has not been examined thoroughly, but a similar peptide (Cecropin AB) was not greatly affected by pH changes within the range of 4.0 to 9.0, although pHs lower than 4.0 did lower the activity.¹⁵

Crystal Structure

Although there has not been a crystal structure developed, the CB1a peptide is derived from cecropin B and could be used as a template (2IGR):

References

1. Antimicrobial Peptide AP00505 [Internet]. detailed information. [cited 2020Oct23]. Available from: http://aps.unmc.edu/AP/database/query_output.php?ID=00505

2. Antimicrobial Peptide AP00523 [Internet]. detailed information. [cited 2020Oct23]. Available from: http://aps.unmc.edu/AP/database/query_output.php?ID=00523

3. Chen J, Guan S-M, Sun W, Fu H. Melittin, the Major Pain-Producing Substance of Bee Venom [Internet]. Neuroscience bulletin. Springer Singapore; 2016 [cited 2020Oct24]. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5563768/

4. Du H, Puri S, McCall A, Norris HL, Russo T, Edgerton M. Human Salivary Protein Histatin 5 Has Potent Bactericidal Activity against ESKAPE Pathogens [Internet]. Frontiers. Frontiers; 2017 [cited 2020Oct23]. Available from: https://www.frontiersin.org/articles/10.3389/fcimb.2017.00041/full

5. Gribskov M, Wesson L, Eisenberg D. 2MLT [Internet]. To be Published. [cited 2020Oct24]. Available from: http://www.rcsb.org/pdb/explore/jmol.do?structureId=2MLT

6. Hori Y, Demura M, Iwadate M, Ulrich AS, Niidome T, Aoyagi H, et al. Interaction of mastoparan with membranes studied by 1H-NMR spectroscopy in detergent micelles and by solid-state 2H-NMR and 15N-NMR spectroscopy in oriented lipid bilayers. [Internet]. Eur.J.Biochem. [cited 2020Oct23]. Available from: http://www.rcsb.org/pdb/explore/jmol.do?structureId=1D7N

7. Hossen MS, Gan SH, Khalil MI. Melittin, a Potential Natural Toxin of Crude Bee Venom: Probable Future Arsenal in the Treatment of Diabetes Mellitus [Internet]. Journal of Chemistry. Hindawi; 2017 [cited 2020Oct24]. Available from: https://www.hindawi.com/journals/jchem/2017/4035626/

8. Kong EF, Tsui C, Boyce H, Ibrahim A, Hoag SW, Karlsson AJ, et al. Development and In Vivo Evaluation of a Novel Histatin-5 Bioadhesive Hydrogel Formulation against Oral Candidiasis [Internet]. Antimicrobial agents and chemotherapy. American Society for Microbiology; 2015 [cited 2020Oct23]. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4750700/

9. Konno K, Kazuma K, Rangel M, Stolarz-de-Oliveira J, Fontana R, Kawano M, et al. New Mastoparan Peptides in the Venom of the Solitary Eumenine Wasp Eumenes micado [Internet]. Toxins. MDPI; 2019 [cited 2020Oct23]. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468405/

10. Mastoparan [Internet]. Mastoparan - an overview | ScienceDirect Topics. [cited 2020Oct23]. Available from: https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/mastoparan

11. Sigma-Aldrich.com. [Internet]. MASTOPARAN Product Information. Sigma-Aldrich.com; [cited 2020]. Available from: https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/2/m212pis.pdf

12. Singulani Jde L, Galeane MC, Ramos MD, Gomes PC, dos Santos CT, de Souza BM, et al. Antifungal Activity, Toxicity, and Membranolytic Action of a Mastoparan Analog Peptide [Internet]. Frontiers. Frontiers; 2019 [cited 2020Oct23]. Available from: https://www.frontiersin.org/articles/10.3389/fcimb.2019.00419/full

13. Terwilligert TC, Eisenberg D. [Internet]. The Structure of Melittin 2. INTERPRETATION OF THE STRUCTURE. THE JOURNAL OF BIOLOGICAL CHEMISTRY ; 2003 [cited 2020Oct23]. Available from: https://www.jbc.org/content/257/11/6016.full.pdf

14. Wilcox W, Eisenberg D. Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding [Internet]. Protein science : a publication of the Protein Society. Cold Spring Harbor Laboratory Press; 1992 [cited 2020Oct24]. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2142234/

15. Ebbensgaard A, Mordhorst H, Overgaard MT, Nielsen CG, Aarestrup FM, Hansen EB. Comparative Evaluation of the Antimicrobial Activity of Different Antimicrobial Peptides against a Range of Pathogenic Bacteria [Internet]. PloS one. Public Library of Science; 2015 [cited 2020Oct24]. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4684357/

16. Vergis J, Malik S.S., Pathak R, Kumar M, Ramanjaneya S, Kurkure N.V., Barbuddhe S.B., Rawool D.B. Antimicrobial Efficacy of Indolicidin Against Multi-Drug Resistant Enteroaggregative Escherichia coli in a Galleria mellonella Model. [Internet]. Frontiers in Microbiology. [cited 2020Oct23]. Available from: https://www.frontiersin.org/article/10.3389/fmicb.2019.02723.

17. Selsted M.E., Novotny M.J., Morris W.L., Tang Y.Q., Smith W, Culler J.S. Indolicidin, a novel bactericidal tridecapeptide amide from neutrophils. [Internet]. The Journal of Biological Chemistry. [cited 2020Oct23]. Available from: https://www.jbc.org/content/267/7/4292.abstract?ijkey=342b896882b2e9b41e1050782922382ddd6880e3&keytype2=tf_ipsecsha.

18. Ahmad I, Perkins W.R., Lupan D.M., Selsted M.E., Janoff A.S. Liposomal entrapment of the neutrophil-derived peptide indolicidin endows it with in vivo antifungal activity. [Internet]. Biochimica et Biophysica Acta (BBA) - Biomembranes. [cited 2020Oct23]. Available from: https://www.sciencedirect.com/science/article/pii/000527369500087J.

19. Portell-Buj E, Vergara A​, Alejo I​, López-Gavín A​, Monté M.R.​, San Nicolás L​, González-Martín J​, Tudó G. I​n vitro activity of 12 antimicrobial peptides against Mycobacterium tuberculosis and Mycobacterium avium clinical isolates. [Internet]. Journal of Medical Microbiology [cited 2020Oct23]. Available from: https://www.microbiologyresearch.org/content/journal/jmm/10.1099/jmm.0.000912.

20. Zannella C, Shinde S, Vitiello M, Falanga A, Galdiero E, Fahmi A, Santella B, Nucci L, Gasparro R, Galdiero M, Boccellino M, Franci G, Domenico M.D. Antibacterial Activity of Indolicidin-Coated Silver Nanoparticles in Oral Disease. [Internet]. Applied Sciences. [cited 2020Oct23]. Available from: https://www.mdpi.com/2076-3417/10/5/1837.

21. Friedrich C.L., Rozek A, Patrzykat A, Hancock R.E.W.Structure and Mechanism of Action of an Indolicidin Peptide Derivative with Improved Activity against Gram-positive Bacteria. [Internet]. Applied Sciences. [cited 2020Oct23]. Available from: https://www.jbc.org/content/276/26/24015.

22. Falla TJ, Karunaratne DN, Hancock RE. Mode of action of the antimicrobial peptide indolicidin. J Biol Chem. 1996 Aug 9;271(32):19298-303. doi: 10.1074/jbc.271.32.19298. PMID: 8702613.

23. Pillai A, Ueno S, Zhang H, Lee JM, Kato Y. Cecropin P1 and novel nematode cecropins: a bacteria-inducible antimicrobial peptide family in the nematode Ascaris suum. Biochem J. 2005;390(Pt 1):207-214. doi:10.1042/BJ20050218

24. Boman HG, Agerberth B, Boman A. Mechanisms of action on Escherichia coli of cecropin P1 and PR-39, two antibacterial peptides from pig intestine. Infect Immun. 1993;61(7):2978-2984. doi:10.1128/IAI.61.7.2978-2984.1993

25. Chen X, Zhu F, Cao Y, Qiao S. Novel expression vector for secretion of cecropin AD in Bacillus subtilis with enhanced antimicrobial activity. Antimicrob Agents Chemother. 2009;53(9):3683-3689. doi:10.1128/AAC.00251-09

26. Silvestro L, Weiser JN, Axelsen PH. Antibacterial and antimembrane activities of cecropin A in Escherichia coli. Antimicrob Agents Chemother. 2000;44(3):602-607. doi:10.1128/aac.44.3.602-607.2000.

27. Kalsy, M., Tonk, M., Hardt, M. et al. The insect antimicrobial peptide cecropin A disrupts uropathogenic Escherichia coli biofilms. npj Biofilms Microbiomes 6, 6 (2020). https://doi.org/10.1038/s41522-020-0116-3

28. Jan P, Huang H, Chen H. Expression of a Synthesized Gene Encoding Cationic Peptide Cecropin B in Transgenic Tomato Plants Protects against Bacterial Diseases. Applied and Environmental Microbiology 76, 3 (2010); DOI: 10.1128/AEM.00698-09

29. Chen X, Zhu F, Cao Y, Qiao S. Novel expression vector for secretion of cecropin AD in Bacillus subtilis with enhanced antimicrobial activity. Antimicrob Agents Chemother. 2009;53(9):3683-3689. doi:10.1128/AAC.00251-09

30. Gazit, E. et al. “Mode of action of the antibacterial cecropin B2: a spectrofluorometric study.” Biochemistry 33 35 (1994): 10681-92.