Chapter Eight - Characterization of Dynamic IDP Complexes by NMR Spectroscopy
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.
Section snippets
Trimming IDPs to Optimize Complexes for NMR Studies
The first step in the successful characterization of an IDP complex by NMR spectroscopy is to consider if the protein sequence needs to be subdivided to simplify analysis. Sometimes the binding region is part of a longer disordered chain and sometimes it is limited to a smaller region. In this case, several factors need consideration, including the length of the region, the presence of transient structures or folded domains, and whether the interaction sites are known or can be predicted. Due
Selecting and Optimizing NMR Sample Conditions for IDP Complexes
Optimization of NMR sample conditions is critical for the successful acquisition of NMR data. For all proteins, sample condition optimization aims at striking the right balance between capturing the essential characteristics of the native environment that supports functionality and achieving sufficient NMR data quality so that the intensity and number of observable peaks in the NMR spectra are maximized. With the higher average exposure of residues to the surrounding solvent of IDPs compared to
The 1H,15N-HSQC Spectrum—The Protein Fingerprint
Evaluation of different NMR sample conditions is often done with 1H,15N-HSQC spectra, which give rise to a correlation peak for each covalently bonded 1H–15N group (Fig. 2(1)). In the ideal situation, the 1H,15N-HSQC spectrum will contain one peak for each backbone amide (except those of prolines) and peaks for each side-chain amide or indole groups (and, under certain conditions, guanidino groups). Therefore, this spectrum is considered the protein “fingerprint.” It will provide the first
Interpretation of Binding-Induced Changes in the 1H,15N-HSQC Spectrum
A major strength of NMR spectroscopy is that it allows mapping of residue-specific effects of complex formation through relatively fast and simple experiments. Usually, this is achieved by the acquisition of 1H,15N-HSQC spectra in the absence and presence of an interaction partner, simply requiring sufficient amounts of isotope-labeled and unlabeled forms of both partners. To prepare a sample of an IDP in the presence of the interaction partner, some considerations are needed: First, it is
Titration With an Interaction Partner
The 1H,15N-HSQC spectrum of the fully bound state of the IDP can give information about the mode of interaction, but to obtain insight into the dynamical properties of the complex a titration with varying concentrations of interaction partner can be performed. To maximize the level of information, it is advisable to record several concentration ratios until no further changes in the NMR spectrum are detectable. The key to an interpretable 1H,15N-HSQC titration series lies in careful sample
NMR Methods to Study Dynamic IDP Complexes—Aims, Needs, and Pitfalls
In this section, different NMR experiments relevant to the characterization of dynamic IDP complexes are presented along with the expected outcomes, pitfalls, and examples, some of which are shown in Fig. 3.
Conclusions and Outlook
As outlined in this chapter, NMR spectroscopy offers a wide range of experiments allowing residue-specific characterization of interactions, distances, and dynamics in IDP complexes. As recent work has highlighted, the characteristic dynamics of IDPs may be conserved in their complexes (Bah et al., 2015; Borgia et al., 2018; Charlier et al., 2017; Delaforge et al., 2018; Milles et al., 2015; Sparks, Temel, Rout, & Cowburn, 2018), requiring increased focus on methodological developments that
Acknowledgments
We are grateful to all the questions raised by the students at SBiNLab struggling with IDP complexes and for their comments. This work was supported by grants from the Danish Research Councils (#4181-00344) and the Novo Nordisk Foundation (to B.B.K.).
References (135)
- et al.
NMR analysis of protein interactions
Current Opinion in Chemical Biology
(2005) - et al.
The acidic transcription activator Gcn4 binds the mediator subunit Gal11/Med15 using a simple protein interface forming a fuzzy complex
Molecular Cell
(2011) Generating accurate contact maps of transient long-range interactions in intrinsically disordered proteins by paramagnetic relaxation enhancement
Biophysical Journal
(2013)- et al.
Protein interacting with C-kinase 1 (PICK1) binding promiscuity relies on unconventional PSD-95/discs-large/ZO-1 homology (PDZ) binding modes for nonclass II PDZ ligands
The Journal of Biological Chemistry
(2014) - et al.
Novel methods based on 13C detection to study intrinsically disordered proteins
Journal of Magnetic Resonance
(2014) - et al.
IDPs in macromolecular complexes: The roles of multivalent interactions in diverse assemblies
Current Opinion in Structural Biology
(2018) - et al.
Quantitative analysis of protein-ligand interactions by NMR
Progress in Nuclear Magnetic Resonance Spectroscopy
(2016) - et al.
A comparison between the sulfhydryl reductants tris(2-carboxyethyl)phosphine and dithiothreitol for use in protein biochemistry
Analytical Biochemistry
(1999) - et al.
Direct detection of carbon and nitrogen nuclei for high-resolution analysis of intrinsically disordered proteins using NMR spectroscopy
Methods
(2018) - et al.
Structural basis of ribosomal S6 kinase 1 (RSK1) inhibition by S100B protein: Modulation of the extracellular signal-regulated kinase (ERK) signaling cascade in a calcium-dependent way
The Journal of Biological Chemistry
(2016)
Predicting the energetics of conformational fluctuations in proteins from sequence: A strategy for profiling the proteome
Structure
Structure of the C-terminal region of p21(WAF1/CIP1) complexed with human PCNA
Cell
Structure determination of protein–protein complexes with long-range anisotropic paramagnetic NMR restraints
Current Opinion in Structural Biology
A phosphorylation-motif for tuneable helix stabilisation in intrinsically disordered proteins—Lessons from the sodium proton exchanger 1 (NHE1)
Cellular Signalling
Quantitative determination of the conformational properties of partially folded and intrinsically disordered proteins using NMR dipolar couplings
Structure
Disordered proteins studied by chemical shifts
Progress in Nuclear Magnetic Resonance Spectroscopy
An introduction to NMR-based approaches for measuring protein dynamics
Biochimica et Biophysica Acta
NMR contributions to structural dynamics studies of intrinsically disordered proteins
Journal of Magnetic Resonance
Faithful estimation of dynamics parameters from CPMG relaxation dispersion measurements
Journal of Magnetic Resonance
Random-phase-approximation theory for sequence-dependent, biologically functional liquid-liquid phase separation of intrinsically disordered proteins
Journal of Molecular Liquids
Plasticity of an ultrafast interaction between nucleoporins and nuclear transport receptors
Cell
Detection of initiation sites in protein folding of the four helix bundle ACBP by chemical shift analysis
FEBS Letters
Altered flexibility in the substrate-binding site of related native and engineered high-alkaline Bacillus subtilisins
Journal of Molecular Biology
Structures and short linear motif of disordered transcription factor regions provide clues to the interactome of the cellular hub protein radical-induced cell death 1
The Journal of Biological Chemistry
A dynamic look backward and forward
Journal of Magnetic Resonance
Multiple weak linear motifs enhance recruitment and processivity in SPOP-mediated substrate ubiquitination
Journal of Molecular Biology
Diffusion nuclear magnetic resonance spectroscopy detects substoichiometric concentrations of small molecules in protein samples
Analytical Biochemistry
TROSY and CRINEPT: NMR with large molecular and supramolecular structures in solution
Trends in Biochemical Sciences
Overview of probing protein-ligand interactions using NMR
Liquid-liquid phase separation of the microtubule-binding repeats of the Alzheimer-related protein Tau
Nature Communications
Sequence-specific determination of protein and peptide concentrations by absorbance at 205 nm
Protein Science
Conformational propensities of intrinsically disordered proteins influence the mechanism of binding and folding
Proceedings of the National Academy of Sciences of the United States of America
The unique domain forms a fuzzy intramolecular complex in Src family kinases
Structure
Folding of an intrinsically disordered protein by phosphorylation as a regulatory switch
Nature
A small molecule causes a population shift in the conformational landscape of an intrinsically disordered protein
Journal of the American Chemical Society
Utilization of site-directed spin labeling and high-resolution heteronuclear nuclear magnetic resonance for global fold determination of large proteins with limited nuclear overhauser effect data
Biochemistry
Structural assembly of molecular complexes based on residual dipolar couplings
Journal of the American Chemical Society
Extreme disorder in an ultrahigh-affinity protein complex
Nature
NMR methods for the study of instrinsically disordered proteins structure, dynamics, and interactions: General overview and practical guidelines
Advances in Experimental Medicine and Biology
Structure of radical-induced cell death1 hub domain reveals a common αα-scaffold for disorder in transcriptional networks
Structure
Protein NMR spectroscopy. Principles and practice
Structure and dynamics of an intrinsically disordered protein region that partially folds upon binding by chemical-exchange NMR
Journal of the American Chemical Society
Theory, practice, and applications of paramagnetic relaxation enhancement for the characterization of transient low-population states of biological macromolecules and their complexes
Chemical Reviews
Translational diffusion measured by PFG-NMR on full length and fragments of the Alzheimer Aβ(1-40) peptide. Determination of hydrodynamic radii of random coil peptides of varying length
Magnetic Resonance in Chemistry
Two-site binding of beta-cyclodextrin to the Alzheimer Abeta(1-40) peptide measured with combined PFG-NMR diffusion and induced chemical shifts
Biochemistry
The intrinsically disordered RNR inhibitor Sml1 is a dynamic dimer
Biochemistry
Short linear motifs—Ex nihilo evolution of protein regulation
Cell Communication and Signaling: CCS
Proliferating cell nuclear antigen (PCNA) interactions in solution studied by NMR
PLoS One
Structure of p15(PAF)-PCNA complex and implications for clamp sliding during DNA replication and repair
Nature Communications
Mapping long-range interactions in alpha-synuclein using spin-label NMR and ensemble molecular dynamics simulations
Journal of the American Chemical Society
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Machine-learning-based methods to generate conformational ensembles of disordered proteins
2024, Biophysical JournalAssessment of models for calculating the hydrodynamic radius of intrinsically disordered proteins
2023, Biophysical JournalSolvent paramagnetic relaxation enhancement as a versatile method for studying structure and dynamics of biomolecular systems
2022, Progress in Nuclear Magnetic Resonance SpectroscopyCitation Excerpt :Protein phase separation is an emerging important concept in cellular signaling, underlies the formation of membraneless organelles, and can be regulated by binding of ligands and chaperones or by post-translational modifications (PTMs) [266]. Due to their structural heterogeneity, IDPs are usually not amenable to structural characterization by means of X-ray crystallography or cryo-electron microscopy, except for IDP complexes in which folding-upon-binding occurs or where protein disorder is limited to a small confined peptide region [245,267]. In addition to this, conformational analysis of IDPs and IDRs is complicated by their higher average solvent accessibility compared to folded proteins and their higher sensitivity to solvent characteristics.
In-cell NMR: Why and how?
2022, Progress in Nuclear Magnetic Resonance SpectroscopyCitation Excerpt :Nonetheless, NMR remains a very useful technique, particularly for studying smaller proteins that do not crystallize, as well as relatively weak interactions in solution; it has important capabilities in characterizing the conformational dynamics of many macromolecules (for instance, see [1144-1147]). NMR is thus extremely helpful in understanding the function of folded proteins, but it is indispensable for studying disordered proteins [1148-1158]. NMR is also useful to study cellular processes that occur at slower timescales than those of the internal motions, such as protein maturation events, cofactor binding, and post-translational modifications [1159-1166], all of which can contribute to regulation of protein function.
The armadillo-repeat domain of plakophilin 1 binds the C-terminal sterile alpha motif (SAM) of p73
2021, Biochimica et Biophysica Acta - General SubjectsCitation Excerpt :It is not clear whether the different poses of SAMp73 detected in the docking are equivalent to alternative binding modes on the same binding location of ARM-PKP1 (a situation that is more common for small ligands in promiscuous binding sites [73]), or to distinct conformations associated to spatially close but mutually exclusive binding sites (again possibly associated with the promiscuity of a protein towards different ligands [74]), or else they correspond to metastable states that may interconvert on a timescale much longer than the one accessible to MD simulations. At the moment, we do not know yet which of these alternative views is correct: the fact that several residues of SAMP73 showed a different behaviour in their conformational exchange in our NMR experiments could be in agreement with the possible presence of different binding modes observed in the simulations, although for other proteins such different conformational exchange for residues belonging to the same protein has been related to the presence of distinct binding sites [63,64]. In spite of these difficulties, additional information could be obtained about some structural details in the interaction of the two proteins.
Probing Surfaces in Dynamic Protein Interactions
2020, Journal of Molecular BiologyCitation Excerpt :Assignment of chemical shifts is a pre-requisite for most NMR-based approaches, but is increasingly difficult to achieve unambiguously with highly overlapping signals. Especially proton-detected NMR experiments tend to provide limited chemical shift dispersion [118]. To increase signal dispersion of IDPs, heteronuclear direct-detected, especially 13C direct-detected, NMR experiments have gained popularity recently, due to excellent chemical shift dispersion and narrow linewidths.