Conformational Dynamics of Asparagine at Coiled-Coil Interfaces.

Franziska Thomas,Gail J Bartlett,Derek N Woolfson,Ai Niitsu,Alain Oregioni

doi:10.1021/acs.biochem.7b00848

Franziska Thomas, Gail J Bartlett + Show 3 more

Open Access

https://doi.org/10.1021/acs.biochem.7b00848

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Abstract

Coiled coils (CCs) are among the best-understood protein folds. Nonetheless, there are gaps in our knowledge of CCs. Notably, CCs are likely to be structurally more dynamic than often considered. Here, we explore this in an abundant class of CCs, parallel dimers, focusing on polar asparagine (Asn) residues in the hydrophobic interface. It is well documented that such inclusions discriminate between different CC oligomers, which has been rationalized in terms of whether the Asn can make side-chain hydrogen bonds. Analysis of parallel CC dimers in the Protein Data Bank reveals a variety of Asn side-chain conformations, but not all of these make the expected inter-side-chain hydrogen bond. We probe the structure and dynamics of a de novo-designed coiled-coil homodimer, CC-Di, by multidimensional nuclear magnetic resonance spectroscopy, including model-free dynamical analysis and relaxation–dispersion experiments. We find dynamic exchange on the millisecond time scale between Asn conformers with the side chains pointing into and out of the core. We perform molecular dynamics simulations that are consistent with this, revealing that the side chains are highly dynamic, exchanging between hydrogen-bonded-paired conformations in picoseconds to nanoseconds. Combined, our data present a more dynamic view for Asn at CC interfaces. Although inter-side-chain hydrogen bonding states are the most abundant, Asn is not always buried or engaged in such interactions. Because interfacial Asn residues are key design features for modulating CC stability and recognition, these further insights into how they are accommodated within CC structures will aid their predictive modeling, engineering, and design.

Highlights

Coiled coils (CCs) are some of the most abundant protein folds in nature.[1]
(“inside−inside”) as the Asn side chains would clash in full quaternary models. To test if this heterogeneity of Asn−Asn interactions was representative of Asn-containing CC dimers more generally, we inspected such structures in the RCSB Protein Data Bank (PDB).[15]
We used CC+16 to mine the PDB for parallel CC dimers containing pairs of Asn residues at a positions

Summary

Introduction

Coiled coils (CCs) are some of the most abundant protein folds in nature.[1]. They play key roles in many biological processes, directing and stabilizing protein structures and protein−protein interactions. In CCs, two or more α-helices combine to form bundles with a left-handed supercoil Underlying this is a regular seven-residue or heptad-repeat sequence of hydrophobic (h) and polar (p) residues, hpphppp, commonly denoted abcdefg.[2,5] When configured into an α-helix, this pattern brings together the a and d positions. The resulting hydrophobic face drives association of multiple helices to form the bundles These helix−helix interfaces are cemented by intimate side-chain interactions termed knobs-into-holes (KIH) packing.[6]. Despite this apparent simplicity, CCs show a diversity of oligomeric states, the helices can be parallel or antiparallel, and the complexes can be homo- or heteromeric. More commonly buried Asn residues occur at the a sites of CC dimers, for example, the bZip transcription factors,[25,26] where the residues are often required for dimer specificity.[8,27,28] Asn-at-a is almost an obligatory design feature in the specification of completely de novo-designed homo- and heterodimeric CCs, and in the construction of orthogonal CC pairs.[13,17,29−31] as a cautionary note and to add additional nuance to this, the Asn-at-a has to be located centrally to specify oligomer state; otherwise, alternative and unintended CC assemblies can be observed.[32]

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