Molecular Details of a Coupled Binding and Folding Reaction between the Amyloid Precursor Protein and a Folded Domain.

Thomas M T Jensen,O Andreas Karlsson,Per Jemth,Linda M Haugaard-Kedström,Kristian Strømgaard,Emma Åberg,Christian R O Bartling

doi:10.1021/acschembio.1c00176

Thomas M T Jensen, O Andreas Karlsson + Show 5 more

Open Access

https://doi.org/10.1021/acschembio.1c00176

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Abstract

Intrinsically disordered regions in proteins often function as binding motifs in protein–protein interactions. The mechanistic aspects and molecular details of such coupled binding and folding reactions, which involve formation of multiple noncovalent bonds, have been broadly studied theoretically, but experimental data are scarce. Here, using a combination of protein semisynthesis to incorporate phosphorylated amino acids, backbone amide-to-ester modifications, side chain substitutions, and binding kinetics, we examined the interaction between the intrinsically disordered motif of amyloid precursor protein (APP) and the phosphotyrosine binding (PTB) domain of Mint2. We show that the interaction is regulated by a self-inhibitory segment of the PTB domain previously termed ARM. The helical ARM linker decreases the association rate constant 30-fold through a fast pre-equilibrium between an open and a closed state. Extensive side chain substitutions combined with kinetic experiments demonstrate that the rate-limiting transition state for the binding reaction is governed by native and non-native hydrophobic interactions and hydrogen bonds. Hydrophobic interactions were found to be particularly important during crossing of the transition state barrier. Furthermore, linear free energy relationships show that the overall coupled binding and folding reaction involves cooperative formation of interactions with roughly 30% native contacts formed at the transition state. Our data support an emerging picture of coupled binding and folding reactions following overall chemical principles similar to those of folding of globular protein domains but with greater malleability of ground and transition states.

Highlights

Cellular regulation is highly reliant on protein−protein interactions (PPIs).[1−3] Often, such PPIs are mediated by a globular well-folded protein interaction domain, which binds to an intrinsically disordered region of an interacting protein.[4]Upon binding to the folded domain, the intrinsically disordered region typically adopts an ordered extended conformation, such as an α-helix, a β-strand, a coil, or a combination of secondary structures.[5]
NM-WTTer(mkoinna=l dansyl labeling of the APPWT peptide to increase the change in the fluorescence intensity upon binding did not affect the binding kinetics (Figure S1)
In the case of Alzheimer’s disease, the role of munc-18 interaction protein (Mint)[2] is established,[21] and very recently, we investigated the Mint2−amyloid precursor protein (APP) interaction in great detail

Summary

Introduction

Cellular regulation is highly reliant on protein−protein interactions (PPIs).[1−3] Often, such PPIs are mediated by a globular well-folded protein interaction domain, which binds to an intrinsically disordered region of an interacting protein.[4]Upon binding to the folded domain, the intrinsically disordered region typically adopts an ordered extended conformation, such as an α-helix, a β-strand, a coil, or a combination of secondary structures.[5]. One general conclusion is that the binding involves an initial encounter complex, which rearranges into the native complex.[6,10] The intrinsically disordered protein (IDP) can be viewed as a large ensemble of interconverting structures of similar Gibbs free energies, many of which can bind and form the encounter complex. In this model, some conformations may be preferred over others, and depending on the concentration of the interacting proteins and their conformations, the rate (or flux) via different parallel binding pathways is modulated.[11] For example, a helical conformation in an IDP may bind with a rate constant that is higher than those of disordered conformations, but the latter are present at much higher concentrations such that binding predominantly occurs via a disordered conformation. Recent studies have found evidence of templated folding, whereby the structure and dynamics of the interaction partner influence the coupled binding and folding pathway of IDPs.[12−15]

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