Page 81 - ITPS-7-1

INNOSC Theranostics and
Pharmacological Sciences PfHSP and polyamines interactions

cerevisiae were retrieved from the NCBI. These sequences Put, Spd, and Spn were prepared using the TIP4P solvent
together with the PfHsps sequences were compiled to a txt model, 15 Å3 Orthorhombic boxes, OPLS4 force field,
file using Notepad++ (the HSP sequences were compiled and each system were neutralized with an appropriate
separately according to their molecular weight). The number of Na or Cl counterions (Supplementary S2).
-
+
sequences, together with the P. falciparum sequences, A 0.15 M NaCl solution was created to mimic physiological
were aligned using the Bio-edit tool using ClustalW and conditions. All MD simulations were done with the NPT
[14]
BLOSSUM62 matrix. ensemble at a temperature of 300 K and pressure of 1.01325
bar. Due to wall time limitations on the high-performance
2.3. Homology modeling and structure validation computer being used for the simulations, we had to run the
Phyre2 , a protein structure prediction database, was MD in steps of 50 ns for HSP20, HSP40, and HSP60, 30
[15]
used to generate 3D model structures using sequences of ns for HSP70-1, and 25 ns for HSP90. On completion of
the Pf3D7 selected HSPs under intensive modeling mode. the simulations, the various intervals were combined into a
The modeled structures were visualized with PyMol. The single 200 ns trajectory for analysis.
structures were then validated using PROCHECK on the
online tool PDBSum found on the EBI database. 3. Results and discussion
[16]
2.4. Protein preparation for docking and molecular 3.1. Sequence retrieval and genomic analysis
dynamics (MD) Genomic analysis showed that HSP20 and HSP70-1
All the heat shock proteins (PfHsp20, PfHsp40, PfHsp60, are located on the same chromosome (chromosome 8),
PfHsp70-1, and PfHsp90) obtained from homology while HSP40 is located on chromosome 13, HSP60
modeling were prepared with the aid of the protein on chromosome 10, and HSP90 on chromosome 7
preparation wizard provided in Schrodinger 2022-1 . (Supplementary S3). The bioinformatics analysis of Pf3D7
[17]
heat shock proteins showed that these major HSPs have a
2.5. Site mapping few exons and introns in them, with HSP40 and HSP70
Since the proteins were obtained through homology having 1 exon and no introns, HSP60 and HSP90 having 2
modeling, there were no active sites to work from. Instead exons and 1 intron, and HSP20 having 3 exons and 1 intron.
of making use of a blind docking approach, we made use of 3.2. Multiple sequence alignments of PfHsps and
the SiteMap [18,19] , the tool provided in Schrodinger 2022-1. their homologs
The tool allows for the identification of binding sites
whose size, functionality, and extent of solvent exposure All the heat shock proteins were aligned with S. cerevisiae and
meet certain criteria. The OPLS4 force field was used for E. coli (Supplementary S4) in BioEdit. The similarity index
docking, at least 15 site points per reported site, a more between P. falciparum and E. coli was HSP20, HSP40, HSP60,
limited definition of hydrophobicity, a fine grid, crop site HSP70, and HSP 90 at 20.63%, 19.34%, 67.41%, 58.30%, and
maps at four from the nearest site point, and reporting up 54.15%, respectively. While the similarity index between
to five sites were all necessary for this study. P. falciparum and S. cerevisiae HSP40, HSP60, HSP70, and
HSP90 was higher at 32.90%, 71.92%, 79.32%, and, 75.70%,
2.6. Molecular docking respectively, except for HSP20 which was 12.61%. This is due
All ligands were obtained directly from PubChem. Due to to S. cerevisiae’s small HSP (HSP20 family) being present as
the size of the ligand library, we decided to make use of the HSP26, a 26 kDa protein instead of 20 kDa.
QM Conformer and Tautomer Predictor instead of using 3.3. Homology modeling and structure validation
[20]
the conventional ligand preparation of Schrodinger [21,22] . This
was done to ensure that we end up with quantum mechanical- All PDB structures (Figure 1) were generated from Phyre2.
based minimum energy conformers before docking to the The projected structure of PfHSP20 comprises two sheets,
active site of the proteins of interest . The method generates four beta hairpins, seven strands, six helices, 15 beta
[23]
the lowest energy tautomers or conformers for a set of turns, and six gamma turns, according to motif analysis.
structures with optional protonation or deprotonation. Due This demonstrated that the created structure was suitable
to the computational cost of this method, it is advised that for further investigation. The 3D structure of PfHSP40
[23]
this only be applied to small datasets . reveals that the protein has five helices, nine helix-helix
interactions, and two beta twists. The Ramachandran plot
2.7. MD revealed that all of the residues were in core areas. As a
MD simulations were conducted with the aid of Desmond . result, the structure merits additional investigation. The 3D
[24]
The free protein structures as well as those complexed to structure of HSP60 revealed five sheets, two beta-alpha-beta

Volume 7 Issue 1 (2024) 3 https://doi.org/10.36922/itps.1228