Positive cofactor 4 (PC4)
PC4 can act as a cofactor for many different transcription activators.
Domain organization/sequence features
The activity of PC4 is regulated by a disordered NTD (1-60 AA), which consists of a Ser- and acidic- rich region and a Lys-rich
Upon interaction with VP16ad, not significant changes in NOE contacts for PC4 NTD were observed, which suggest that
PC4ntd is highly flexible and mostly unstructured both in the free form and in the complex with the activator. The
15N-T values indicate some enhanced mobility on the pico-nanosecond time scale in the first part of the lysine-
rich region (∼20-29), when compared to the rest of PC4 NTD. The addition of VP16 to PC4 resulted in progressive amide
peak shifts, indicative for fast exchange on the NMR time scale and characteristic of a low-affinity complex.
Significant chemical-shift changes are also observed for the strongly positively charged lysine-rich region (21-48) and the
amino-terminal end (3-8 residues) of PC4 NTD. Upon the interaction with VP16ad, no peak broadening or decrease of signal
intensity is observed for PC4 NTD to the extent of PC4 CTD and NOE contacts are present in comparable proportions as for
PC4 alone. These observations exclude the formation of a tight complex between PC4 NTD and VP16ad bound to PC4 CTD.
The affected site in PC4ntd might be involved in the electrostatic attraction of VP16ad, bringing the activator in proximity of
the PC4 CTD interaction surface, thereby affecting the on/off rate of VP16 toward PC4.
With VP16 Kd is 90±20 nM, for CTD 150±80 nM, while deletion of the amino-terminal domain of PC4 (PC4 NTD) diminishes
the interaction with VP16ad. The N terminal domain (PC4 NTD) region can cooperate with the conserved carboxy-terminal
core of the protein (PC4 CTD) to increase the interaction of PC4ctd with VP16ad. The PC4 NTD alone lacks the ability to
interact with ssDNA, dsDNA, and VP16ad and does not possess any helix-destabilizing activity. The precise role as well as
the mechanism underlying the regulation of the PC4 function by its amino-terminal domain is unclear. The presence of PC4
NTD can either be stimulatory (VP16 interaction), neutral (dsDNA), or inhibitory (ssDNA) on the PC4 CTD functions. The
interaction between the VP16 activation domain (VP16ad) and PC4 are highly salt-dependent and influenced by pH,
indicating that the interactions are predominantly electrostatic in nature.
The flexibility of this part of the lysine-rich region is somewhat reduced upon VP16ad complex formation, indicating that this
region may decrease the VP16ad off rate by stabilizing the PC4ctd-VP16ad interaction.
The modulatory role of PC4 NTD is further underscored by the observed phosphorylation-dependent increase in ssDNA binding
and a decrease in dsDNA and VP16 binding and helix-destabilizing activity of PC4.
Transient and dynamic interdomain interactions between the unstructured PC4 NTD and structured PC4 CTD overlapping with
the ssDNA/VP16ad interaction surface that affects the cofactor activities of PC4. Fuzzy nature of the complex provides further
fine-tuning opportunities by posttranslational modifications.