The top four residues obtained with the JS-Distance metric are identified as either allosteric or orthosteric (Figure ), and the two most distal allosteric residues (R312 and D357) are both among the top ranked residues when compared to other residues with similar minimum heavy atom distances to the peptide (Figure ). The residue E373 is the lowest ranked residue when compared to other residues with a similar minimum distance (Figure ), and was consistently identified as unimportant by all five approaches used (Figure ). This residue's experimentally observed mutational sensitivity towards peptide binding is perhaps unsurprising given the location of the N-terminal lysine on the peptide (Figure ). It may therefore be more accurate to characterize this residue as "orthosteric" as opposed to "allosteric" (the original authors characterized this residue as allosteric due to its distance from the peptide 87 ). Alongside E373, several orthosteric residues (such as L323, G324 and A376) are identified to have a relatively low per residue score given their close proximity to the binding site (Figure ). It is important to consider that the features/interactions we used to build our models contain no protein-peptide interactions. Our results would therefore suggest that whilst these residues of course play a key role in regulating the binding of the peptide (through direct interactions with the peptide), they have a limited role in any subsequent allosteric signaling to the remainder of the PDZ3 domain. In contrast, H372 is an orthosteric residue that is consistently identified as one of the most important residues present in the PDZ3 domain across all 5 models (Figure ). H372 is identified as such due to its interactions with five residues on the neighboring β2-β3 loop (see Table ), suggesting a more substantial role in allosteric signaling for this residue compared to the other binding site residues, as discussed in detail in e.g. ref. .