In the case of the two serine/threonine kinases, Gly200 PKA) or Gly405(Pak2) is binding directly to the backbone of the P-site residue that is to be phosphorylated.
To verify that PLN was binding directly to COP, we cloned COP, PLN and PLN RΔ14 into a His-tagged bacterial expression vector (pET28-MHL Vector; GenBank accession #EF456735), a kind gift from Dr Sirano Dhe-Paganon (U of Toronto).
Given that the immunoglobulin-like repeats are highly similar [45], it is possible that the nature of their activity, whether it is binding directly to fibronectin or fibrinogen or imparting conformational changes that modulate binding, depends on their interaction with other repeats.
As shown in Figure 8a, a portion of the myc-tagged OEP9, Toc33 and Toc34 added to the incubations was recovered in gradient fractions containing chloroplast-like liposomes (fractions 1 and 2, solid arrowheads), indicating that all three proteins were binding directly to this lipid bilayer.
Thus these early models of down-regulation by nuclear receptors and other later studies [17] concluded that the liganded receptor was binding directly to DNA and interfering with the process of transcriptional activation that would normally take place in the absence of liganded nuclear receptor protein.
To test whether the intracellular portion of TfR1 was binding directly to IKK, we cloned the intracellular 64 residues of TfR1 into a bacterial expression construct.
While it is unclear whether the ATP is competing directly at the C-terminal site or is acting allosterically by binding to the N-terminus and thus preventing accessibility at the C-terminal pocket, this data demonstrates that KU174 is binding directly to Hsp90.
It is also possible that in many instances MYCN is not binding directly to DNA sequences, but instead is interacting with another DNA binding partner, such as a methyl binding protein.
