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Protein-Protein Complex Structural Analysis of
Human Fibronectin and Staphylococcus aureus
Fibronectin-binding protein A
Amal Dhivahar S.,
Ist Year M.Tech. Biotechnology,
Department of Biotechnology, School of Agriculture and Bio Sciences,
Vellore Institute of Technology (VIT), Vellore – 632 014, Tamil Nadu, India.
1
Abstract
Staphylococcus aureus is an invasive gram-positive bacteria responsible for an array of infectious
diseases including bacterial keratitis, skin infections, and could even end up in certain malignant
conditions like pneumonia and meningitis. Fibronectins are integrin-binding protein dimers of the
extracellular matrix of vertebrates. They are significant for cellular functions like adhesion, migration,
and growth. The Fibronectin-binding protein A of S. aureus bind to these high molecular weight
glycoproteins on the host cells. This forms a bridge for bacterial invasion and evasion from the host
immuno-surveillance. Since in silico methodologies have become a vital part of drug designing
processes, the present study was designed to analyse the protein-protein interactions between this
protein complex using software such as UCSF Chimera, PyMol, ArgusLab, and other online tools. The
protein complex structures of Fibronectin and Fibronectin-binding protein A were homology
modelled and evaluated using Ramachandran plots and other significant parameters. The
complementarity and interactions between the proteins were observed concerning surface analysis
and various properties. Possible ligands for the proteins were also studied to understand the drug
development trajectory and to develop ideas for new potential drugs.
Key words: Staphylococcus aureus, Fibronectin, Fibronectin-binding protein A, protein interactions, in
silico methodologies, homology modelling
2
Introduction
▪ Fibronectin (FINC) is a glycoprotein from the Extracellular Matrix (ECM) that binds
to proteins on cell membranes called integrins.
▪ It promotes cell-cell and cell-basement membrane adhesions, migration, etc. but
most importantly, the adhesion of pathogens onto host cells.
▪ Fibronectin-binding protein A (FNBA) possesses multiple, substituting FINC binding
regions, each capable of conferring adherence to both soluble and immobilized
forms of FINC.
▪ This confers to Staphylococcus aureus the ability to invade endothelial cells both in
vivo and in vitro, without requiring additional factors, although in a slow and
inefficient way through actin rearrangements in host cells (Maurin et al., 2021).
▪ This invasion process is mediated by integrin alpha-5/beta-1.
3
Host cell
S. aureus FINC
FNBA α5β1 Integrin
Introduction
Figure 1. Some of the minor infections - A. Furuncle, B. Impetigo in a child caused by C. S. aureus
Credits: https://www.msdmanuals.com/home/infections/bacterial-infections-gram-positive-bacteria/staphylococcus-aureus-infections
4
A. B. C.
Introduction
5
▪ S. aureus can cause a range of illnesses, from minor skin infections, such as pimples,
furuncles, abcesses, and impetigo, to malignant diseases such as pneumonia, meningitis,
and endocarditis (David and Daum, 2017).
▪ The identification of Protein-Protein Interactions (PPIs) can lead to a better
understanding of infection mechanisms and the development of several medication
drugs and treatment optimization.
▪ Protein-protein recognition depends on the complementarity between the surface of the
two proteins, i.e., a complementarity in shape and in electrostatic and hydrophobic
interactions (Waiho et al., 2021).
▪ Therefore it’s necessary to analyse the surface of proteins in the complex as several
properties of PPI such as allosteric sites and hotspots, have been incorporated into drug-
design strategies.
Software & Tools + Work Plan
(Materials and Methods)
➔ UniProtKB
➔ RCSB PDB
➔ SWISS-MODEL
➔ STRING database
➔ UCSF Chimera
➔ PyMol
➔ ArgusLab
6
Figure 2. A rough work plan of the present study.
Results
7
Structures of FINC and FNBA proteins via UCSF Chimera
Figure 3. Homology models of FINC and FNBA protein structures using SWISS MODEL.
A. FINC B. FNBA
8
Model of FINC-FNBA Protein Complex
FINC
FNBA
Figure 4. Homology model of
FINC-FNBA protein complex.
9
Homology Model Assessment - FINC
10
Figure 5. Ramachandran Plot for the FINC Homology Model
Homology Model Assessment - FNBA
11
Figure 6. Ramachandran Plot for the FNBA Homology Model
Protein- Protein Intermolecular Interactions - H Bonds
12
Figure 7. H-Bonds at the interface between
the FINC-FNBA proteins.
Protein- Protein Intermolecular Interactions - Contacts
13
Figure 8. Contact Interactions between the
FINC-FNBA proteins.
Protein- Protein Intermolecular Interactions - Clashes
14
Figure 9. Clashes Interactions between the
FINC-FNBA proteins.
Surface Analysis of FINC
15
Figure 10. Surface analysis of FINC:
A - Molecular Surface representation
B - the surfaces are coloured according to the Coulombic potential,
going from negative potential (red) to positive potential (blue).
C - the surfaces are coloured according to amino acid
hydrophobicity: Maroon - More hydrophobicity; Cyan - hydrophilic
residues.
D - The Chain separated.
A B C (b)
D
C (a)
Surface Analysis of FNBA
16
Figure 11. Surface analysis of FNBA:
A - Molecular Surface representation
B - the surfaces are coloured according to the Coulombic potential,
going from negative potential (red) to positive potential (blue).
C - the surfaces are coloured according to amino acid
hydrophobicity: Maroon - More hydrophobicity; Cyan - hydrophilic
residues.
A B
C (b)
C (a)
Analysis of FINC & FNBA’s protein network using STRING
database
Figure 12. Functional interaction network of FINC
and FNBA obtained from STRING Database.
17
A B
Functional enrichment data for FINC and FNBA interaction
network from STRING database.
● FINC
18
● FINC
● FBNA
● FBNA
Structures of FINC and FNBA proteins via PyMol
Figure 13. Homology models of FINC, FNBA protein and FINC-FNBA protein complex structures
using SWISS MODEL.
19
A. C.
B.
Ligand docking with PyMol
20
Figure 14. A. Ligand in the Fibronectin Protein.
B. Bond length between the ligand and the amino acid residues,
1.2Å from T-493 and, 3.2Å and 3.3Å from R-491.
C. Surface of the Protein in display.
Green  Ligand
Blue  H-bonding residues
Red  Non-polar residues
Yellow  Ribbons of rest of the complex
A. C.
B.
Visualization via ArgusLab
21
Figure 15. Homology models of A. FINC, B. FINC – Ligand highlighted and C. FNBA protein
structures using SWISS MODEL.
A. C.
B.
Mutation Analysis for FNBA – Point Mutation
22
A. B.
Figure 16. Mutation analysis of FNBA protein at the amino acid position 222 A. Gly at 222 position
B. Gly  Ala at 222
Mutagenesis Case I:
At 222, G → A = No
change in
elastin/fibrinogen/fibr
onectin binding
(Keane et al., 2007).
Mutation Analysis of FNBA – Deletion Mutation
Figure 17. Mutation analysis of FNBA protein at the amino acid segment 484  511 A. Highlighted segment B.
Highlighted segment in the FINC-FNBA complex C. View from another angle showing the clashes interactions
vital for the FINC-FNBA complex binding.
23
Mutagenesis Case II: Deletion of the amino acid segment 484  511 abolishes interaction with elastin,
fibrinogen, and fibronectin. (Keane et al., 2007).
Discussion
▪ The structure of the FINC-FNBA complex being understudied experimentally, was
predicted by homology modelling with respect to the QMEAN score, coverage,
sequence identity, etc.
▪ The interactions between these two proteins were analysed keeping in view of their
hydrogen bonding, contact interactions, and clashes properties.
▪ The surface analysis of the two proteins revealed their molecular structure,
coulombic potential, levels of hydrophilicity and hydrophobicity, and so on.
▪ Observing each monomers of the proteins separately makes it possible to check if a
pocket in one structure fits with a protrusion in the partner.
▪ Ramachandran plot analysis of the predicted homology models of FINC and FNBA
proteins indicates a good quality model with more than 95% of the residues in the
favoured region of the plot.
24
Discussion
▪ The protein interaction network from STRING database shows the functional
association of the genes in cell adhesion and several other biological pathways.
▪ However secondary structures may also confer certain conformational changes in
the complexes due to H-bonding.
▪ The docking of the ligand in the FINC protein revealed possible polar amino acid
residue interactions and the bond lengths components required for drug designing.
▪ Some of the ligands from RCSB PDB did not bind with the target protein because of
less affinity, unfavourable binding energy and unfavourable structural orientations
(Cd, Co, Cu, ACT, 2K2, etc.).
▪ Mutational analysis of the FNBA protein in two cases showed how in silico
techniques are useful in simplifying tedious in vitro mutation experiments.
25
Conclusion
The protein-protein interactions in the homology modelled protein complex (FINC-
FNBA) was analysed through various software and tools. The possible ligands were
explored and their surface properties were studied. The molecular surface and
interaction analysis made it possible to understand some of the key features for
protein-protein recognition, and it could give interesting hints about the cases that
involve single point mutations. Further analysis of the complex function with
respect to the various pathways it is involved, will enable developing a better drug
that specifically targets to treat the associated diseases.
E.g. Cefazolin - adverse effects: loss of appetite, nausea, vomiting, diarrhoea, headache, swelling,
redness, pain /soreness at the site of injection.
26
References
▪ David, M. Z., & Daum, R. S. (2017). Treatment of Staphylococcus aureus infections. Staphylococcus
aureus, 325-383.
▪ Keane, F. M., Loughman, A., Valtulina, V., Brennan, M., Speziale, P., & Foster, T. J. (2007). Fibrinogen and
elastin bind to the same region within the A domain of fibronectin binding protein A, an MSCRAMM of
Staphylococcus aureus. Molecular microbiology, 63(3), 711-723.
▪ Maurin, C., Courrier, E., He, Z., Josselin, R., Frédéric, L., Thuret, G., ... & Verhoeven, P. (2021). Key role of
fibronectin for Staphylococcus aureus adherence to ex vivo corneal epithelium. Acta Ophthalmologica,
99.
▪ The PyMOL Molecular Graphics System (2021). Version 1.2r3pre, Schrödinger, LLC. 2021.
▪ UCSF Chimera--a visualization system for exploratory research and analysis (2004). Pettersen EF, Goddard
TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE. J Comput Chem. 25(13):1605-12.
▪ Waiho, K., Afiqah‐Aleng, N., Iryani, M. T. M., & Fazhan, H. (2021). Protein–protein interaction network:
an emerging tool for understanding fish disease in aquaculture. Reviews in Aquaculture, 13(1), 156-177.
27
Thank You!
Amal Dhivahar S.,
Ist year M.Tech. Biotechnology,
Department of Biotechnology,
School of Agriculture and Bio Sciences,
Vellore Institute of Technology (VIT),
Vellore – 632 014, Tamil Nadu, India.
28

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Protein-Protein Complex Structural Analysis of Human Fibronectin and Staphylococcus aureus Fibronectin-binding protein A

  • 1. Protein-Protein Complex Structural Analysis of Human Fibronectin and Staphylococcus aureus Fibronectin-binding protein A Amal Dhivahar S., Ist Year M.Tech. Biotechnology, Department of Biotechnology, School of Agriculture and Bio Sciences, Vellore Institute of Technology (VIT), Vellore – 632 014, Tamil Nadu, India. 1
  • 2. Abstract Staphylococcus aureus is an invasive gram-positive bacteria responsible for an array of infectious diseases including bacterial keratitis, skin infections, and could even end up in certain malignant conditions like pneumonia and meningitis. Fibronectins are integrin-binding protein dimers of the extracellular matrix of vertebrates. They are significant for cellular functions like adhesion, migration, and growth. The Fibronectin-binding protein A of S. aureus bind to these high molecular weight glycoproteins on the host cells. This forms a bridge for bacterial invasion and evasion from the host immuno-surveillance. Since in silico methodologies have become a vital part of drug designing processes, the present study was designed to analyse the protein-protein interactions between this protein complex using software such as UCSF Chimera, PyMol, ArgusLab, and other online tools. The protein complex structures of Fibronectin and Fibronectin-binding protein A were homology modelled and evaluated using Ramachandran plots and other significant parameters. The complementarity and interactions between the proteins were observed concerning surface analysis and various properties. Possible ligands for the proteins were also studied to understand the drug development trajectory and to develop ideas for new potential drugs. Key words: Staphylococcus aureus, Fibronectin, Fibronectin-binding protein A, protein interactions, in silico methodologies, homology modelling 2
  • 3. Introduction ▪ Fibronectin (FINC) is a glycoprotein from the Extracellular Matrix (ECM) that binds to proteins on cell membranes called integrins. ▪ It promotes cell-cell and cell-basement membrane adhesions, migration, etc. but most importantly, the adhesion of pathogens onto host cells. ▪ Fibronectin-binding protein A (FNBA) possesses multiple, substituting FINC binding regions, each capable of conferring adherence to both soluble and immobilized forms of FINC. ▪ This confers to Staphylococcus aureus the ability to invade endothelial cells both in vivo and in vitro, without requiring additional factors, although in a slow and inefficient way through actin rearrangements in host cells (Maurin et al., 2021). ▪ This invasion process is mediated by integrin alpha-5/beta-1. 3 Host cell S. aureus FINC FNBA α5β1 Integrin
  • 4. Introduction Figure 1. Some of the minor infections - A. Furuncle, B. Impetigo in a child caused by C. S. aureus Credits: https://www.msdmanuals.com/home/infections/bacterial-infections-gram-positive-bacteria/staphylococcus-aureus-infections 4 A. B. C.
  • 5. Introduction 5 ▪ S. aureus can cause a range of illnesses, from minor skin infections, such as pimples, furuncles, abcesses, and impetigo, to malignant diseases such as pneumonia, meningitis, and endocarditis (David and Daum, 2017). ▪ The identification of Protein-Protein Interactions (PPIs) can lead to a better understanding of infection mechanisms and the development of several medication drugs and treatment optimization. ▪ Protein-protein recognition depends on the complementarity between the surface of the two proteins, i.e., a complementarity in shape and in electrostatic and hydrophobic interactions (Waiho et al., 2021). ▪ Therefore it’s necessary to analyse the surface of proteins in the complex as several properties of PPI such as allosteric sites and hotspots, have been incorporated into drug- design strategies.
  • 6. Software & Tools + Work Plan (Materials and Methods) ➔ UniProtKB ➔ RCSB PDB ➔ SWISS-MODEL ➔ STRING database ➔ UCSF Chimera ➔ PyMol ➔ ArgusLab 6 Figure 2. A rough work plan of the present study.
  • 8. Structures of FINC and FNBA proteins via UCSF Chimera Figure 3. Homology models of FINC and FNBA protein structures using SWISS MODEL. A. FINC B. FNBA 8
  • 9. Model of FINC-FNBA Protein Complex FINC FNBA Figure 4. Homology model of FINC-FNBA protein complex. 9
  • 10. Homology Model Assessment - FINC 10 Figure 5. Ramachandran Plot for the FINC Homology Model
  • 11. Homology Model Assessment - FNBA 11 Figure 6. Ramachandran Plot for the FNBA Homology Model
  • 12. Protein- Protein Intermolecular Interactions - H Bonds 12 Figure 7. H-Bonds at the interface between the FINC-FNBA proteins.
  • 13. Protein- Protein Intermolecular Interactions - Contacts 13 Figure 8. Contact Interactions between the FINC-FNBA proteins.
  • 14. Protein- Protein Intermolecular Interactions - Clashes 14 Figure 9. Clashes Interactions between the FINC-FNBA proteins.
  • 15. Surface Analysis of FINC 15 Figure 10. Surface analysis of FINC: A - Molecular Surface representation B - the surfaces are coloured according to the Coulombic potential, going from negative potential (red) to positive potential (blue). C - the surfaces are coloured according to amino acid hydrophobicity: Maroon - More hydrophobicity; Cyan - hydrophilic residues. D - The Chain separated. A B C (b) D C (a)
  • 16. Surface Analysis of FNBA 16 Figure 11. Surface analysis of FNBA: A - Molecular Surface representation B - the surfaces are coloured according to the Coulombic potential, going from negative potential (red) to positive potential (blue). C - the surfaces are coloured according to amino acid hydrophobicity: Maroon - More hydrophobicity; Cyan - hydrophilic residues. A B C (b) C (a)
  • 17. Analysis of FINC & FNBA’s protein network using STRING database Figure 12. Functional interaction network of FINC and FNBA obtained from STRING Database. 17 A B
  • 18. Functional enrichment data for FINC and FNBA interaction network from STRING database. ● FINC 18 ● FINC ● FBNA ● FBNA
  • 19. Structures of FINC and FNBA proteins via PyMol Figure 13. Homology models of FINC, FNBA protein and FINC-FNBA protein complex structures using SWISS MODEL. 19 A. C. B.
  • 20. Ligand docking with PyMol 20 Figure 14. A. Ligand in the Fibronectin Protein. B. Bond length between the ligand and the amino acid residues, 1.2Å from T-493 and, 3.2Å and 3.3Å from R-491. C. Surface of the Protein in display. Green  Ligand Blue  H-bonding residues Red  Non-polar residues Yellow  Ribbons of rest of the complex A. C. B.
  • 21. Visualization via ArgusLab 21 Figure 15. Homology models of A. FINC, B. FINC – Ligand highlighted and C. FNBA protein structures using SWISS MODEL. A. C. B.
  • 22. Mutation Analysis for FNBA – Point Mutation 22 A. B. Figure 16. Mutation analysis of FNBA protein at the amino acid position 222 A. Gly at 222 position B. Gly  Ala at 222 Mutagenesis Case I: At 222, G → A = No change in elastin/fibrinogen/fibr onectin binding (Keane et al., 2007).
  • 23. Mutation Analysis of FNBA – Deletion Mutation Figure 17. Mutation analysis of FNBA protein at the amino acid segment 484  511 A. Highlighted segment B. Highlighted segment in the FINC-FNBA complex C. View from another angle showing the clashes interactions vital for the FINC-FNBA complex binding. 23 Mutagenesis Case II: Deletion of the amino acid segment 484  511 abolishes interaction with elastin, fibrinogen, and fibronectin. (Keane et al., 2007).
  • 24. Discussion ▪ The structure of the FINC-FNBA complex being understudied experimentally, was predicted by homology modelling with respect to the QMEAN score, coverage, sequence identity, etc. ▪ The interactions between these two proteins were analysed keeping in view of their hydrogen bonding, contact interactions, and clashes properties. ▪ The surface analysis of the two proteins revealed their molecular structure, coulombic potential, levels of hydrophilicity and hydrophobicity, and so on. ▪ Observing each monomers of the proteins separately makes it possible to check if a pocket in one structure fits with a protrusion in the partner. ▪ Ramachandran plot analysis of the predicted homology models of FINC and FNBA proteins indicates a good quality model with more than 95% of the residues in the favoured region of the plot. 24
  • 25. Discussion ▪ The protein interaction network from STRING database shows the functional association of the genes in cell adhesion and several other biological pathways. ▪ However secondary structures may also confer certain conformational changes in the complexes due to H-bonding. ▪ The docking of the ligand in the FINC protein revealed possible polar amino acid residue interactions and the bond lengths components required for drug designing. ▪ Some of the ligands from RCSB PDB did not bind with the target protein because of less affinity, unfavourable binding energy and unfavourable structural orientations (Cd, Co, Cu, ACT, 2K2, etc.). ▪ Mutational analysis of the FNBA protein in two cases showed how in silico techniques are useful in simplifying tedious in vitro mutation experiments. 25
  • 26. Conclusion The protein-protein interactions in the homology modelled protein complex (FINC- FNBA) was analysed through various software and tools. The possible ligands were explored and their surface properties were studied. The molecular surface and interaction analysis made it possible to understand some of the key features for protein-protein recognition, and it could give interesting hints about the cases that involve single point mutations. Further analysis of the complex function with respect to the various pathways it is involved, will enable developing a better drug that specifically targets to treat the associated diseases. E.g. Cefazolin - adverse effects: loss of appetite, nausea, vomiting, diarrhoea, headache, swelling, redness, pain /soreness at the site of injection. 26
  • 27. References ▪ David, M. Z., & Daum, R. S. (2017). Treatment of Staphylococcus aureus infections. Staphylococcus aureus, 325-383. ▪ Keane, F. M., Loughman, A., Valtulina, V., Brennan, M., Speziale, P., & Foster, T. J. (2007). Fibrinogen and elastin bind to the same region within the A domain of fibronectin binding protein A, an MSCRAMM of Staphylococcus aureus. Molecular microbiology, 63(3), 711-723. ▪ Maurin, C., Courrier, E., He, Z., Josselin, R., Frédéric, L., Thuret, G., ... & Verhoeven, P. (2021). Key role of fibronectin for Staphylococcus aureus adherence to ex vivo corneal epithelium. Acta Ophthalmologica, 99. ▪ The PyMOL Molecular Graphics System (2021). Version 1.2r3pre, Schrödinger, LLC. 2021. ▪ UCSF Chimera--a visualization system for exploratory research and analysis (2004). Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE. J Comput Chem. 25(13):1605-12. ▪ Waiho, K., Afiqah‐Aleng, N., Iryani, M. T. M., & Fazhan, H. (2021). Protein–protein interaction network: an emerging tool for understanding fish disease in aquaculture. Reviews in Aquaculture, 13(1), 156-177. 27
  • 28. Thank You! Amal Dhivahar S., Ist year M.Tech. Biotechnology, Department of Biotechnology, School of Agriculture and Bio Sciences, Vellore Institute of Technology (VIT), Vellore – 632 014, Tamil Nadu, India. 28