Characterization of Dynamic IDP Complexes by NMR Spectroscopy

Abstract

NMR spectroscopy has proven to be a key method for studying intrinsically disordered proteins (IDPs). Nonetheless, traditional NMR methods developed for solving structures of ordered protein complexes are insufficient for the full characterization of dynamic IDP complexes, where the energy landscape is broader and more rugged. Furthermore, due to their high sensitivity to environmental changes, NMR studies of IDP complexes must be conducted with extra care and the observed NMR parameters thoroughly evaluated to enable disentanglement of binding events from ensemble distribution changes. In this chapter, written for the non-NMR expert, we start out by outlining sample preparation for IDP complexes, guide through the recording and evaluation of diagnostic ¹H,¹⁵N-HSQC spectra, and delineate more sophisticated NMR strategies to follow for the particular type of complex. The most relevant experiments are then described in terms of aims, needs, pitfalls, analysis, and expected outcomes, with references to recent examples.

Introduction

Intrinsically disordered proteins (IDPs) or regions (IDRs) (here collectively termed IDPs) are proteins that are functional while existing in a broad ensemble of near isoenergetic conformations. Despite their lack of tertiary structure, IDPs are involved in communication with other molecules by forming all kinds of associations ranging from binary, discrete complexes to large multicomponent assemblies. IDP complexes form with affinities similar to those of globular folded proteins, and are on average only 2.5 kcal mol^− 1 less stable due to a conformational entropy loss (Teilum, Olsen, & Kragelund, 2015). Similar to globular proteins their complexes serve structural, functional, and regulatory roles (Uversky, 2018; Wright & Dyson, 2015), but due to their dynamic nature, they expand the types of possible complexes (Miskei, Antal, & Fuxreiter, 2016; Mittag, Kay, & Forman-Kay, 2010; Motlagh, Wrabl, Li, & Hilser, 2014). The fast dynamics characteristic of IDPs may persist in their complexes and the degree of disorder can vary greatly. In one end of the scale, IDPs may completely fold upon binding to form globular-like complexes with little disorder (Rogers, Steward, & Clarke, 2013; Wright & Dyson, 2009), while at the other end disorder may persist and result in complexes where both binding partners stay disordered (Borgia et al., 2018) (Fig. 1). Furthermore, some IDP regions have limited sequence diversity which under certain conditions can lead to large-scale associations involving an undefined number of molecules forming liquid–liquid phase-separated states referred to as condensates (Fung, Birol, & Rhoades, 2018) (see also chapter “Visualization and quantitation of phase-separated droplet formation by human HP1α” by Keenen et al.

Solution-state NMR spectroscopy has proven to be a valuable tool in the studies of IDPs, as this method allows data acquisition in the absence of structure formation. The dynamical properties of IDPs can even be advantageous in NMR studies, as this may result in narrow line widths. Where X-ray crystallography may solve structures of IDP complexes in cases where folding occurs (Qi et al., 2017) or when the binding region can be contained within a small peptide (Gulbis, Kelman, Hurwitz, O’Donnell, & Kuriyan, 1996), NMR spectroscopy allows studying complexes retaining disorder. Sometimes NMR data are integrated with computation (Delaforge et al., 2018), or X-ray crystallography (Gógl et al., 2016; Leyrat et al., 2011). Yet, the dynamics inherent to IDPs pose both conceptual and technical challenges to the study of their complexes by NMR spectroscopy. From the technical side, line broadening from dynamics on unfavorable timescales, lack of intermolecular nuclear Overhauser effects (NOEs), peak overlap from disordered regions, and weak affinities challenge information retrieval. Conceptually, for designing the study and interpreting the data it must be taken into account that the complex may not exist in a single state.

This chapter has been written for the non-NMR expert and focuses on the use of NMR spectroscopy to study IDP complexes where disorder is at least partly retained. Thus, we will not go into details in cases where complete folding upon binding is achieved, as relevant and well-written reviews and textbooks are available (see Bonvin, Boelens, & Kaptein, 2005; Gronenborn & Clore, 1995; Hass & Ubbink, 2014; Schieborr et al., 2005; Thompson, Beck, & Campbell, 2015). Likewise, NMR methods for the study of free IDPs have been well described (Brutscher et al., 2015; Jensen et al., 2009; Jensen, Zweckstetter, Huang, & Blackledge, 2014; Konrat, 2014). The reader will be taken from the sample preparation of the complex through the recording of diagnostic 2D ¹H,¹⁵N-heteronuclear single quantum coherence (HSQC) spectra that may reveal important information on the system under investigation and help define further possible NMR strategies for the specific type of complex. NMR experiments of particular relevance for IDP complexes are then suggested, including aims, needs, pitfalls, analysis, and expected outcomes, as well as references to relevant examples.