Molecular communication in biology is mediated by protein interactions. According to the current paradigm, the specificity and affinity required for these interactions are encoded in the precise complementarity of binding interfaces. Even proteins that are disordered under physiological conditions or that contain large unstructured regions commonly interact with well-structured binding sites on other biomolecules. Here we demonstrate the existence of an unexpected interaction mechanism: the two intrinsically disordered human proteins histone H1 and its nuclear chaperone prothymosin-α associate in a complex with picomolar affinity, but fully retain their structural disorder, long-range flexibility and highly dynamic character. On the basis of closely integrated experiments and molecular simulations, we show that the interaction can be explained by the large opposite net charge of the two proteins, without requiring defined binding sites or interactions between specific individual residues. Proteome-wide sequence analysis suggests that this interaction mechanism may be abundant in eukaryotes.
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We thank S. A. Sjørup and J. H. Martinsen for purification assistance, I. König for a sample of wild-type ProTα, A. Prestel and M. B. Kunze for NMR advice, E. Holmstrom for help with data analysis, D. Mercadante for assistance with electrostatics calculations, R. Sobrino for help with experiments in the early stages of the project, J. Forman-Kay, M. Blackledge, R. Pappu and A. Holehouse for discussions and the Functional Genomics Center Zurich for performing mass spectrometry. This work was supported by the Swiss National Science Foundation (B.S.), the Danish Council for Independent Research (# 4181-00344, B.B.K.), the Novo Nordisk Foundation (B.B.K. and P.O.H.), the Carlsberg Foundation (P.O.H.), and the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health (R.B.B.). This work used the computational resources of the NIH HPC Biowulf cluster (http://hpc.nih.gov).
The authors declare no competing financial interests.
Reviewer Information Nature thanks E. Zuiderweg and the other anonymous reviewer(s) for their contribution to the peer review of this work.
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Extended data figures and tables
a, Titration of 15N-ProTα with zero to sevenfold molar addition of the H1 globular domain followed by 1H–15N HSQC spectra; n = 2 repeats of this measurement yielded consistent results. b, Peak intensity ratios for assigned residues of ProTα relative to the free state induced by zero to 1.7-fold molar addition of the H1 globular domain (n = 2). c, Weighted backbone CSPs per residue of ProTα induced by zero to sevenfold molar addition of the H1 globular domain (n = 2). For comparison, CSPs of ProTα with equimolar addition of H1 are shown in grey (n = 5). In a–c, ‘colour key 1’ applies; grey stars, prolines and unassigned residues. d, ProTα CSPs plotted against concentration and times excess of the H1 globular domain relative to the free state for residues 46–106 upon zero to sevenfold molar addition of the H1 globular domain. Curves corresponding to individual residues are shown in different colours for clarity. e, Far-UV circular dichroism spectrum of the H1 globular domain. f, Thermal denaturation of the H1 globular domain followed by the change in ellipticity at 222 nm (Tm = 320.5 ± 0.3 K, ΔHm = −44 ± 2 kcal mol−1). Inset in f shows fraction of unfolded H1 globular domain (fu) as a function of temperature. g, Titration of 100 μM 13C–15N- H1 globular domain with zero to sevenfold molar addition of ProTα followed by 1H–15N HSQC spectra. Peak intensities gradually decrease during the titration. At 3.5- and 7-fold molar excess ProTα, natural abundance peaks of free ProTα appear. 1H–15N HSQC spectrum of 15N-ProTα is shown in grey for comparison. h, Weighted backbone CSPs of the H1 globular domain plotted against concentration and times excess of ProTα relative to the free state on zero to sevenfold molar addition of ProTα. A total of 66 (unassigned) amide backbone peaks were followed and grouped according to the standard deviation (STD) of the CSPs (1 s.d. = 0.0254 p.p.m.). Of these, 55% had CSPs larger than 1 STD.
a, 1H–15N HSQC spectrum of 11 μM free 15N-ProTα with assigned residues labelled (left) and titrated with zero to fourfold molar addition of H1 (right); n = 5 individual repeats of this measurement yielded consistent results. b, Weighted backbone CSPs of ProTα (residues 46–106) relative to the free state on zero to fourfold molar addition of H1, plotted against concentration and times excess of H1. Curves corresponding to individual residues are shown in different colours for clarity. c, d, CSPs (c) and peak intensity ratios (d) for assigned residues of ProTα induced by zero to fourfold molar addition of H1 (bar colours correspond to key); n = 5 for both. e, T115N relaxation times of free (red) and H1-bound (purple) 15N-ProTα. 〈T1〉 = 610 ms (free) and 636 ms (complex); n = 2 individual repeats of this measurement yielded consistent results. f , T215N relaxation times of free (red) and H1-bound (purple) 15N-ProTα. 〈T2〉 = 302 ms (free) and 217 ms (complex). In c–f, light grey stars, prolines and unassigned residues; dark grey stars, overlap and/or insufficient data quality. Circles in e and f are mean values from n = 3 consecutive data acquisitions on the same samples, errors are s.d.
a, 1H–15N HSQC spectra of the free 13C–15N-H1 globular domain (dark green) and free 13C–15N-H1 (orange). The majority of the amide peaks of the H1 globular domain overlap with the more dispersed peaks from H1, indicating the similarity in structure of the H1 globular domain in isolation and within H1. b, Titration followed by 1H–15N HSQC spectra of 13C–15N-H1 with zero to fourfold molar addition of ProTα. Data acquired on His6-tagged H1; n = 2 individual repeats of this measurement yielded consistent results. c, CSPs relative to free H1 of 11 traceable H1 amide backbone peaks from the intrinsically disordered region (based on overlay with 1H–15N HSQC spectra of the H1 globular domain (in a)) on zero to fourfold molar addition of ProTα plotted against concentration and times excess. Curves corresponding to individual residues are shown in different colours for clarity. d, CSPs plotted against peak intensity ratios relative to the free state of H1 of the 11 H1 amides at 1× excess of ProTα. Colours correspond to those in c. e, Overlay of the Cα–Hα region from 1H–13C HSQC spectra of free 13C–15N-H1 (blue) and the 13C–15N-H1 globular domain (green). The H1 1H–13C HSQC spectrum is dominated by intense clusters of peaks not present in the H1 globular domain spectrum, consistent with the large fraction of residue repeats in the H1 disordered regions. f, Cα–Hα region of 13C–15N-H1 on titration with ProTα. The lack of detectable changes in Cα–Hα resonances is consistent with the absence of secondary structure induction in the disordered regions of H1 on binding.
a, RH of free and bound 15N-ProTα (100 μM) determined with pulsed-field gradient NMR at 283 K. The signal decays of free 15N-ProTα (red), with H1 at a 1:1 molar ratio (purple) and with the H1 globular domain at a 1:7 molar ratio (green) as a function of gradient strength, together with corresponding fits and a table of the diffusion coefficients and resulting RH values. b, RH measured by two-focus fluorescence correlation spectroscopy at 295 K. Lines show the mean RH from n = 2 independent measurements of H10 (blue) and ProTα2 (red) labelled with Alexa 594 in the absence of the binding partner. Symbols represent the mean RH from n = 2 independent measurements of labelled ProTα (5 nM) in the presence of equimolar concentrations of unlabelled ProTα and unlabelled H1. Error bars or shaded bands, s.d. c, Stoichiometry ratio71 versus transfer efficiency plots from intermolecular single-molecule FRET measurements of ProTα2 + H1194 (top), ProTα56 + H1194 (middle), and ProTα110 + H1194 (bottom); pictograms in panels indicate labelling locations. A stoichiometry ratio of 0.5 indicates a 1:1 complex. The peaks at E ≈ 0 originate from molecules or complexes that lack an acceptor dye and remain after filtering for donor-only fluorescence bursts based on pulsed-interleaved excitation. d, e, Transfer efficiency changes at a large excess of unlabelled binding partner for FRET-labelled ProTα56/110 (d) and H1104/194 (e). See Methods for further information on statistics.
a–f, Plots of the fluorescence lifetimes of Alexa 488 donor () and Alexa 594 acceptor () normalized by the intrinsic donor lifetime () versus the ratiometric transfer efficiency E (calculated from the number of donor and acceptor photon counts), as a diagnostic for the presence of a broad distance distribution rapidly sampled during the time of a fluorescence burst28,33,34. If fluctuations in transfer efficiency occur on a timescale between the donor fluorescence lifetime (∼4 ns) and the burst duration (∼1 ms), the normalized donor lifetimes cluster above—and the acceptor lifetimes below—the solid diagonal line expected for a single fixed distance, as previously observed for intrinsically disordered proteins34,72. The large deviation from the diagonal observed for both unbound and bound ProTα and H1 supports the presence of broad and rapidly sampled distance distributions. a, ProTα56/110; b, ProTα56/110 + unlabelled H1; c, H10/113; d, H10/113 + unlabelled ProTα; e, ProTα2 + H1194; and f, ProTα110 + H1194. All variants labelled with Alexa 488 (green) and/or Alexa 594 (red) as indicated by the pictograms in the figure panels.
a, Decision graph using the Rodriguez–Laio clustering algorithm69, showing only a single density maximum distant from other density maxima (that is, a single distinct cluster). b, Free energy of association from simulation for ProTα and H1 along the distance between their centres of mass, RPH, yielding a Kd of 7 fM (black curve). Blue and red curves are the free energies for addition of a second H1 or a second ProTα, respectively, to an existing H1–ProTα complex. c, Principal component vectors shown as contact maps. Colours indicate the increase or decrease in each pair distance for that principal component, relative to the other distances. ProTα and H1 residue numbers are indicated in red and blue, respectively. Each principal component describes a feature of the chain arrangement: principal component 1, for example, captures the presence or absence of interactions between the ProTα N terminus and H1. d, Intramolecular (top row) and intermolecular (bottom three rows) distributions of distances corresponding to FRET labelling sites, within the H1–ProTα complex. P and H numbers refer to ProTα and H1 residues, respectively. Filled distributions, simulations without explicit chromophores; green lines, simulations with explicit chromophores.
FRET-labelled ProTα56/110 was mixed rapidly with unlabelled H1 in TBS buffer, and the resulting increase in acceptor fluorescence was monitored. Inset, example at 10 nM H1 with single-exponential fit and residuals shown above (see Methods for details). Decay rates were obtained from single-exponential fits, with an instrument dead time of 3 ms. Standard errors for each H1 concentration were obtained using bootstrapping. The observed rate, kobs, is shown as a function of H1 concentration (cH1); for H1 concentrations between 10 and 100 nM—for which pseudo-first order conditions apply (ProTα concentration after mixing was 2 nM)—the observed rates were fit with kobs = koncH1 + koff = koncH1 + konKd, using the independently determined Kd of 2.1 pM (Extended Data Table 2). The fit yields a bimolecular association rate coefficient of kon = 3.1 ± 0.1 × 109 M−1 s−1 and an apparent dissociation rate coefficient of koff = 6.5 ± 3.1 × 10−3 s−1. The grey area represents the 95% confidence band.
Electrospray ionization mass spectrum of H1(T161C) labelled with Alexa 488 (calculated mass 21,800 Da) and preparative reversed-phase HPLC (Vydac C4) chromatogram (inset) showing absorption at 280 nm (red) and 488 nm (blue) and the elution gradient from solvent A (H2O + 0.1% TFA) to solvent B (100% acetonitrile) (black), illustrating the high purity of the sample. The peak at approximately 5.5 min corresponds to free Alexa 488, and the peak at approximately 16.8 min to H1(T161C) labelled with Alexa 488.
This file contains histograms data for source data Extended Data table 2. (XLSX 39 kb)
A short fragment of the coarse-grained simulation of the ProT⍺-H1 complex. ProT⍺ and H1 are shown in red and blue, respectively. The disordered regions are displayed as stick representation with the folded domain of H1 shown as a smoothed surface. The N-terminal C⍺ carbon of each protein is indicated by a sphere. The duration of the trajectory is approximately 5 times the typical autocorrelation time for pair distances within the complex. The total length of the simulation used for FRET calculations was ∼103 times longer than the fragment shown in the video. (MP4 23668 kb)
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Borgia, A., Borgia, M., Bugge, K. et al. Extreme disorder in an ultrahigh-affinity protein complex. Nature 555, 61–66 (2018). https://doi.org/10.1038/nature25762
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