Confident that biologically there was strong evidence for cooperativity between the two binding sites, we utilized hydrogen exchange mass spectrometry to try to understand how this synergy worked mechanistically.
For search of putative binding sites we utilized a PWM that is associated with the reference regulog and was used for its original reconstruction.
To identify the critical binding sites, we performed a comprehensive binding assay that utilized mutants containing four random amino acid mutations spanning nearly all of the loop regions.
Moreover, for the TF binding site predictions, we utilized experimental data retrieved from ChIP-Seq experiments.
To estimate distances between binding sites, we construct a distance matrix for the 22 amino acid residues of each binding site, utilizing Grantham indices [26].
Molecular modeling in combination with X-ray crystallographic information was employed to identify a region of the kinesin spindle protein (KSP) binding site not fully utilized by our first generation inhibitors.
The HIR has three sites for phosphorylation including autophosphorylation sites (YxxxYY1190), which are the key step in IR kinase activation [54]; an ATP binding site, which is utilized by many proteins which perform functions requiring an input of energy (from ATP); and a Mg2+ binding site, which plays a critical and conserved role in kinase function [52], [55].
Transcription factor binding site (TFBS) analysis utilized the Clover program [ 9] and the TRANSFAC professional database.
However, if HNF4α binding to MMP15 is independent of ELK1, when ELK1 is knocked down, one would expect to see no change in the binding of MMP15 in the ChIP assay, this is not the case as shown in Figure 9. Furthermore, we found that ELK1 can directly bind to its potential ELK1 binding site in MMP15 utilizing electrophoretic mobility shift analysis (data not shown).
We have presented an algorithm that infers binding site patterns by utilizing local similarity among active site sub-cavities.
