Analysis and comparison of insulin-IR interactions
An important approach to confirm if the pdam receptor we identified with
hhblits really does what the corresponding human receptor does, is to
check for conservation of amino acids crucial for function, here ligand
(insulin) binding. Similar to the human insulin-IR complex, we expect
two distinct insulin binding sites per IR monomer (4 insulin molecules
that can be bound in total). To verify if these binding sites are
conserved in our coral homology model, we used ClusPro to dock coral
insulin to coral IR (see Methods). For comparison, we also docked human
insulin to coral IR and vice versa (Figure 4 ). Our
protein-protein docking clearly showed the binding of coral insulin
protein to the coral insulin receptor at two sites (similar to human
insulin-IR complex). In addition, we found some insulin molecules docked
to additional sites in the lower parts of the EC structure. Because
these are expected to be located close to the membrane, these structures
may not be fully reliable and these putative insulin binding sites
(labeled 5 and 6 in Figure 4 ) have been excluded from further
analysis.
To systematically compare the interactions between insulin and IR at the
two binding sites, we identified all amino acids within 5 Angstrom
distance of either protein (insulin or IR). We find that site-1 of coral
insulin binding to the IR consists of Arg532, Gly533, Ser531, Val530,
Asp479, Asn529, Val528, Asp480, Tyr481, Arg482, Gln103, Leu71, Asp692,
Gln691, Tyr695, Met43, Arg45, Lys46, Lys696, Phe699, Leu698, Thr697,
Asn20, Ser19, Lys701, Thr700, Lys17, and Thr70 (Figure 5, top ).
Site-1 was found to be interacting with chain-A and chain-B residues of
insulin. Site-2 was found at a similar position to the human insulin
site-2 (Figure 5, bottom , and also see Figure 4 ). The
site-2 residues in coral IR consist of Cys463, Asn464, Pro465, Arg461,
Lys547, Pro460, Leu459, Lys546, Cys467, Thr458, Ile457, Thr544, Lys469,
Glu456, Arg543, Val470, Glu542, Glu471, Thr522, Ile541, Val523, Lys540,
Arg539, Pro524, Pro525, Asn159, Asn160, and Asp161.
Within these close-by residues, we identified polar contacts and
hydrogen bonds in the human insulin-IR co-structure (Figure 6 )
and the coral insulin-IR homology models (Figure 7 ). We can see
that there is overlap between the residues in both binding pockets,
although the human interface is more extensive than the coral interface
for both binding sites. This suggests that the human insulin-IR pair is
likely a higher affinity complex than the coral one. Presumably,
evolution has gradually improved and expanded the network of
interactions between insulin and its receptor over the 700 million years
in between human and pdam species.