Molecular Crowding Drives Active Pin1 into Nonspecific Complexes with Endogenous Proteins Prior to Substrate Recognition

Abstract

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 in vivo. 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.

Physicochemical code for quinary protein interactions inEscherichia coli

“…S3), suggesting that the intracellular environment remains unperturbed. However, it is apparent, from the NMR spectral features, that the underlying quinary encounters are short-lived and diffusive, in agreement with previous observations (5,37,38,50). As the correlations in Fig.…”

Physicochemical Properties of Cells and Their Effects on Intrinsically Disordered Proteins (IDPs)

“…When folded proteins engage in transient intracellular interactions they often respond as single entities and display uniform degrees of signal attenuations. 177,182,183,826 By contrast, transient interactions of disordered proteins often elicit line broadening of few residues only. 151,184,185 In many instances, these effects identify weakly interacting proteins regions.…”

A Unique Tool for Cellular Structural Biology: In-cell NMR

“…In X. laevis oocytes and extracts, Luh et al (45) showed that a peptidyl-prolyl isomerase (Pin1) interacts nonspecifically with the environment through the N-terminal Trp-Trp-binding module (WW) domain. Interestingly, upon substrate recognition, the nonspecific interactions between the WW domain and the environment are lost, and both specific and nonspecific interactions are abrogated when Pin1 contains a mutation that mimics phosphorylation of the WW domain (Fig.…”