Methods
Sequence sources. The FASTA sequence files for human and coral
proteins used in this study were acquired from the UniProt database
website at www.uniprot.org.
Sequence analysis. In-depth sequence analysis of coral was
performed as (Ye et al.,
2017). The 3D reconstruction was made to provide detailed, domain wise
information of the coral insulin receptor. Full coral receptor model
(left, right, top, and bottom view) was represented as surface
representation with color coded description of individual domain.
Hhblits alignment and selection . Protein sequences were
imported to the online hhblits coral protein remote homology search tool
atwww.hhblits.cs.tufts.edubased on (Remmert et al.,
2011). The sequences were provided as a FASTA sequence file format and
the results of the remote homology detection were received via email.
Coral protein sequence pdam_00006633-RA showed homology with human
insulin protein and pdam_00013976-RA protein sequence with human
insulin receptor.
Structural mapping of conservation. The ConSurf2016 server atwww.consurf.tau.ac.il was
used to identify sequence conservation and plot it onto the structure of
insulin and insulin receptors. ConSurf is a bioinformatics application
for estimating the evolutionary conservation of amino acid position in a
protein molecule between homologous sequences based on the phylogenetic
relations.
Insulin signaling information: To uncover the coral insulin
signaling we use the literature information from KEGG: Kyoto
Encyclopedia of Genes and Genomes atwww.genome.jp/kegg. KEGG
database consists of high-level functions of molecular information from
large-scale datasets of organisms generated by high-throughput
sequencing techniques.
Swiss modeling (Coral IR model construction): The coral insulin
receptor homolog model was constructed using Swiss Model atwww.swissmodel.expasy.org.
Swiss-Model is an integrated web-based service dedicated to homology
modelling of proteins. We used the target-template alignment function of
swiss model and provided the full structure of the human insulin
receptor (6pxv) as a template to model pdam_00013976-RA. From the same
structure we used insulin structure to model pdam_00006633-RA as coral
insulin. The model was downloaded and analyzed using PyMOL software
(Version-2.3.4, Schrodinger, LLC).
ClusPro protein-protein docking: Protein-protein docking
studies were performed using Clus-Pro 2.0 protein-protein docking tool
at www.cluspro.bu.edu. We
performed the docking of coral insulin protein models with coral IR
model (dimer). The docked poses were downloaded and analyzed using PyMOL
software. We have analyzed the insulin binding site-1 and site-2 in
detail and provided the information of interacting residues between
coral insulin and coral IR. The information was extracted using PyMOL.
Autodock Vina (protein-ligand docking): Molecular docking of
selected ligands with coral Insulin receptor EC and TK domain was
performed using Autodock Vina
(Trott and Olson, 2009). We
constructed a grid box covering all desired residues using Autodock
vina/Autodock tools 1.5.6 atvina.scripps.edu(Trott and Olson, 2009).
The gridbox parameters for full protein include: center_x= -11.318,
center_y= -15.45, center_z= 18.207, size_x=126, size_y=126, and
size_z=126. We also used the full TK structure for identification of
putative allosteric ligands. We used the latest inhibitor-bound
structure (5HHW) for standardizing our docking experiments. Results are
expressed as binding affinity for the top pose with polar contacts for
the same.
Coral Insulin Receptor Kinase domain inhibitor binding site
analysis: The most recent structures of human kinase domains with their
inhibitors were analyzed and essential residues forming polar contacts
with inhibitors were identified as the inhibitor pocket (see details in
text). The identified residues were also mapped in the coral TK domain
model and represented in the coral TK domain using PyMOL.