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Regulation of the Activity in the p53 Family Depends on the Organization of the Transactivation Domain.
Despite high sequence homology among the p53 family members, the regulation of their transactivation potential is based on strikingly different mechanisms. Previous studies revealed that the activity of TAp63α is regulated via an autoinhibitory mechanism that keeps inactive TAp63α in a dimeric conformation. While all p73 isoforms are constitutive tetramers, their basal activity is much lower compared with tetrameric TAp63. We show that the dimeric state of TAp63α not only reduces DNA binding affinity, but also suppresses interaction with the acetyltransferase p300. Exchange of the transactivation domains is sufficient to transfer the regulatory characteristics between p63 and p73. Structure determination of the transactivation domains of p63 and p73 in complex with the p300 Taz2 domain further revealed that, in contrast to p53 and p73, p63 has a single transactivation domain. Sequences essential for stabilizing the closed dimer of TAp63α have evolved into a second transactivation domain in p73 and p53.The research was funded by the DFG (DO 545/8 and DO 545/13), the Center for Biomolecular Magnetic Resonance (BMRZ), and the Cluster of Excellence Frankfurt (Macromolecular Complexes). M.T. was supported by a fellowship from the Fonds of the Chemical Industry
Molecular Crowding Drives Active Pin1 into Nonspecific Complexes with Endogenous Proteins Prior to Substrate Recognition
Proteins
and nucleic acids maintain the crowded interior of a living
cell and can reach concentrations in the order of 200â400 g/L
which affects the physicochemical parameters of the environment, such
as viscosity and hydrodynamic as well as nonspecific strong repulsive
and weak attractive interactions. Dynamics, structure, and activity
of macromolecules were demonstrated to be affected by these parameters.
However, it remains controversially debated, which of these factors
are the dominant cause for the observed alterations <i>in vivo</i>. In this study we investigated the globular folded peptidyl-prolyl
isomerase Pin1 in Xenopus laevis oocytes
and in native-like crowded oocyte extract by in-cell NMR spectroscopy.
We show that active Pin1 is driven into nonspecific weak attractive
interactions with intracellular proteins prior to substrate recognition.
The substrate recognition site of Pin1 performs specific and nonspecific
attractive interactions. Phosphorylation of the WW domain at Ser16
by PKA abrogates both substrate recognition and the nonspecific interactions
with the endogenous proteins. Our results validate the hypothesis
formulated by McConkey that the majority of globular folded proteins
with surface charge properties close to neutral under physiological
conditions reside in macromolecular complexes with other sticky proteins
due to molecular crowding. In addition, we demonstrate that commonly
used synthetic crowding agents like Ficoll 70 are not suitable to
mimic the intracellular environment due to their incapability to simulate
biologically important weak attractive interactions