Sortase-mediated segmental labeling: A method for segmental assignment of intrinsically disordered regions in proteins.

Kristina V Boyko,Patrick N Reardon,Micah Z Lund,Derrick M Smith,Melissa Oueld Es Cheikh,Jeffery C Young,Serge L Smirnov,Erin A Rosenkranz,Mark Okon,John M Antos,Heather L Miears,Patrick Van Der Wel

doi:10.1371/journal.pone.0258531

Abstract

A significant number of proteins possess sizable intrinsically disordered regions (IDRs). Due to the dynamic nature of IDRs, NMR spectroscopy is often the tool of choice for characterizing these segments. However, the application of NMR to IDRs is often hindered by their instability, spectral overlap and resonance assignment difficulties. Notably, these challenges increase considerably with the size of the IDR. In response to these issues, here we report the use of sortase-mediated ligation (SML) for segmental isotopic labeling of IDR-containing samples. Specifically, we have developed a ligation strategy involving a key segment of the large IDR and adjacent folded headpiece domain comprising the C-terminus of A. thaliana villin 4 (AtVLN4). This procedure significantly reduces the complexity of NMR spectra and enables group identification of signals arising from the labeled IDR fragment, a process we refer to as segmental assignment. The validity of our segmental assignment approach is corroborated by backbone residue-specific assignment of the IDR using a minimal set of standard heteronuclear NMR methods. Using segmental assignment, we further demonstrate that the IDR region adjacent to the headpiece exhibits nonuniform spectral alterations in response to temperature. Subsequent residue-specific characterization revealed two segments within the IDR that responded to temperature in markedly different ways. Overall, this study represents an important step toward the selective labeling and probing of target segments within much larger IDR contexts. Additionally, the approach described offers significant savings in NMR recording time, a valuable advantage for the study of unstable IDRs, their binding interfaces, and functional mechanisms.

Highlights

Disordered regions (IDRs), often involving hundreds of residues, are widespread in all kingdoms of life and perform many key cellular roles [1,2,3,4,5]
As an extension of our previous work with the villin 4 headpiece domain in isolation [54], we elected to focus our segmental isotopic labeling studies on a construct consisting of the 35-residue acidic intrinsically disordered region (IDR) fragment joined to the headpiece
To render this system compatible with sortase-mediated ligation (SML), it was first necessary to construct an IDR derivative fused at its C-terminus to an LPXTG sortase recognition motif, along with a headpiece derivative displaying an accessible polyglycine unit at its N-terminus (Fig 1)

Summary

Introduction

Disordered regions (IDRs), often involving hundreds of residues, are widespread in all kingdoms of life and perform many key cellular roles [1,2,3,4,5]. These disordered polypeptides are dynamic systems, wherein there exists a set of distinct semi-structured or semi-disordered states for each IDR. The balance between these states can be altered by changes in the environment, posttranslational modifications, and binding to other domains or molecules, which in turn impacts IDR function [6, 7] This inherent complexity makes IDR systems difficult to probe experimentally. There is a growing need for new experimental approaches for characterizing IDRs, including their functions and binding interfaces

Methods

Results

Conclusion