Unfolding phosphorylation

Coupled folding and binding is a frequent theme in the field of intrinsically disordered proteins (see Chap. 6). One of the earliest examples of this phenomenon was the interaction of the phosphorylated kinase-inducible domain (pKID) of the transcription factor CREB with the KIX domain of the transcriptional coactivator CBP. Free pKID is unfolded in solution [21], but folds into an orthogonal pair of helices, aA and aB, upon binding to the folded KIX domain (Fig. 1.5) [23]. We have recently posed the question, what is the... 

Fig. 6.4. Illustration of the unfolded nature of the phosphorylated kinase-inducible domain (pKID) of CREB (left) and its conformation after folding upon binding to the KIX domain of CBP (right). The mechanism of this process has recently been elucidated by NMR [28] and is discussed more fully in Chap. 1 (Wright). Adapted by permission from [5] (Macmillan Publishers Ltd., copyright 2005)...

Shortly after the discovery of calcineurin as the mechanistic key for FK506 and CsA action, the mechanism of action of rapamycin began to unfold. It had already been noted that rapamycin blocked the IL-2 stimulated G1 to S phase transition in T-cells, inhibiting cell division. Treatment of T-cells with rapamycin was found to result in decreased enzymatic activity of several kinases, including p70 S6 kinase (a 70 kDa protein which phosphorylates the S6 protein of the small ribosomal subunit),27-29 and cyclin-dependent kinases of 33 and 34 kDa.30,31 However, in vitro experiments demonstrated that these kinases were not directly inhibited by the FKBP-rapamycin complex. In 1993, two yeast proteins were identified that appeared to be involved in the mechanistic pathway and mutations in these proteins conferred resistance to rapamycin-induced cytotoxicity.32 These proteins were named TORI and TOR2 (targets of rapamycin). 

Phosphorylation of S19, by addition of negative charge, must disrupt these ionic interactions and cause unfolding. This interpretation is supported by the observation that the unfolding of the bent monomer by phosphorylation can be mimicked by mutation of T18 and S19 to negatively charged amino acids (D or E) (Sweeney et al., 1994 Kamisoyama et al., 1994). 

Figure 13.14 Schematic representation of RNA Interference. Small interfering RNA (siRNA) is generated by Dicer (RNase) cleavage of a short hairpin RNA (shRNA) into small double-stranded RNA (dsRNA) of 21-25 nucleotide lengths, or transfected into the cell. The transfected siRNA is phosphorylated at 5 -ends by an endogenous kinase. The 5 -phosphorylated siRNA is incorporated into RNA-induced silencing complex (RISC) and unfolded. The antisense strand targets the RISC to homologous mRNA (sequence complementary to the siRNA guide) which is then cleaved by an endonuclease in the RISC complex (termed Sheer). The mRNA initially cleaved by Sheer is degraded by exonucleases and thus silenced...

---