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 200–400 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

DNA damage in oocytes induces a switch of the quality control factor TAp63α from dimer to tetramer

TAp63a, a homolog of the p53 tumor suppressor, is a quality control factor in the female germline. Remarkably, already undamaged oocytes express high levels of the protein, suggesting that TAp63a’s activity is under tight control of an inhibitory mechanism. Biochemical studies have proposed that inhibition requires the C-terminal transactivation inhibitory domain. However, the structural mechanism of TAp63a inhibition remains unknown. Here, we show that TAp63a is kept in an inactive dimeric state. We reveal that relief of inhibition leads to tetramer formation with ~20-fold higher DNA affinity. In vivo, phosphorylation-triggered tetramerization of TAp63a is not reversible by dephosphorylation. Furthermore, we show that a helix in the oligomerization domain of p63 is crucial for tetramer stabilization and competes with the transactivation domain for the same binding site. Our results demonstrate how TAp63a is inhibited by complex domain-domain interactions that provide the basis for regulating quality control in oocytes

Mechanism of TAp73 inhibition by ΔNp63 and structural basis of p63/p73 hetero-tetramerization

Members of the p53 tumor suppressor family are expressed as multiple isoforms. Isoforms having an N-terminal transactivation domain are transcriptionally active while those ones lacking this domain often inhibit the transcriptional activity of other family members. In squamous cell carcinomas, the high expression level of ΔNp63α inhibits the tumour suppressor function of TAp73β. This can in principle be due to blocking of the promotor or by direct interaction between both proteins. P63 and p73 can hetero oligomerize through their tetramerization domains and a hetero-oligomer consisting of two p63 and two p73 molecules is thermodyna mically more stable than both homo tetramers. Here we show that hetero tetramer complexes exist also in differentiating keratinocytes. Through structure determination of the hetero tetramer we reveal why this hetero tetramer is the thermodynamically prefer red species. Based on this structure we have created mutants that either enable the formation of only heterotetramers or only homotetramers, allowing to investigate the function of these heterotetramers. Using these tools we show that inhibition of TAp73β in squamous cell carcinomas is due to promotor squelching and not direct interaction

DNA Damage in Oocytes Induces a Switch of the Quality Control Factor TAp63α from Dimer to Tetramer

TAp63α, a homolog of the p53 tumor suppressor, is a quality control factor in the female germline. Remarkably, already undamaged oocytes express high levels of the protein, suggesting that TAp63α's activity is under tight control of an inhibitory mechanism. Biochemical studies have proposed that inhibition requires the C-terminal transactivation inhibitory domain. However, the structural mechanism of TAp63α inhibition remains unknown. Here, we show that TAp63α is kept in an inactive dimeric state. We reveal that relief of inhibition leads to tetramer formation with ∼20-fold higher DNA affinity. In vivo, phosphorylation-triggered tetramerization of TAp63α is not reversible by dephosphorylation. Furthermore, we show that a helix in the oligomerization domain of p63 is crucial for tetramer stabilization and competes with the transactivation domain for the same binding site. Our results demonstrate how TAp63α is inhibited by complex domain-domain interactions that provide the basis for regulating quality control in oocytes