Visualizing the molecular recognition trajectory of an intrinsically disordered protein using multinuclear relaxation dispersion NMR

Despite playing important roles throughout biology, molecular recognition mechanisms in intrinsically disordered proteins remain poorly understood. We present a combination of (1)H(N), (13)C', and (15)N relaxation dispersion NMR, measured at multiple titration points, to map the interaction between the disordered domain of Sendai virus nucleoprotein (NT) and the C-terminal domain of the phosphoprotein (PX). Interaction with PX funnels the free-state equilibrium of NT by stabilizing one of the previously identified helical substates present in the prerecognition ensemble in a nonspecific and dynamic encounter complex on the surface of PX. This helix then locates into the binding site at a rate coincident with intrinsic breathing motions of the helical groove on the surface of PX. The binding kinetics of complex formation are thus regulated by the intrinsic free-state conformational dynamics of both proteins. This approach, providing high-resolution structural and kinetic information about a complex folding and binding interaction trajectory, can be applied to a number of experimental systems to provide a general framework for understanding conformational disorder in biomolecular function

Live-cell three-dimensional single-molecule tracking reveals modulation of enhancer dynamics by NuRD

To understand how the nucleosome remodeling and deacetylase (NuRD) complex regulates enhancers and enhancer–promoter interactions, we have developed an approach to segment and extract key biophysical parameters from live-cell three-dimensional single-molecule trajectories. Unexpectedly, this has revealed that NuRD binds to chromatin for minutes, decompacts chromatin structure and increases enhancer dynamics. We also uncovered a rare fast-diffusing state of enhancers and found that NuRD restricts the time spent in this state. Hi-C and Cut&Run experiments revealed that NuRD modulates enhancer–promoter interactions in active chromatin, allowing them to contact each other over longer distances. Furthermore, NuRD leads to a marked redistribution of CTCF and, in particular, cohesin. We propose that NuRD promotes a decondensed chromatin environment, where enhancers and promoters can contact each other over longer distances, and where the resetting of enhancer–promoter interactions brought about by the fast decondensed chromatin motions is reduced, leading to more stable, long-lived enhancer–promoter relationships

Publisher Correction: Live-cell three-dimensional single-molecule tracking reveals modulation of enhancer dynamics by NuRD

Correction to: Nature Structural & Molecular Biology, published online 28 September 2023. In the version of the article initially published there were some errors in the affiliations. A. Ponjavic’s second and third affiliations have been corrected to Present address: School of Physics and Astronomy, University of Leeds, Leeds, UK and Present address: School of Food Science and Nutrition, University of Leeds, Leeds, UK; and L. Morey now has only two affiliations: Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain and Present address: Sylvester Comprehensive Cancer Center, Department of Human Genetics, University of Miami Miller School of Medicine, Biomedical Research Building, Miami, FL, USA. Additionally, the received date has been corrected to 14 April 2020 from 26 October 2021. These errors have been corrected in the HTML and PDF versions of the article

Live-cell 3D single-molecule tracking reveals how NuRD modulates enhancer dynamics

Abstract Enhancer-promoter dynamics are critical for the spatiotemporal control of gene expression, but it remains unclear how these dynamics are controlled by chromatin regulators, such as the nucleosome remodelling and deacetylase (NuRD) complex. Here, we use Hi-C experiments to show that the intact NuRD complex increases CTCF/Cohesin binding and the probability of the interaction of intermediate-range (∼1Mb) genomic sequences. To understand how NuRD alters 3D genome structure in this way, we developed an approach to segment and extract key biophysical parameters from trajectories of the NuRD complex determined using live-cell 3D single-molecule imaging. Unexpectedly, this revealed that the intact NuRD complex decompacts chromatin structure and makes NuRD-bound sequences move faster, thus increasing the overall volume of the nucleus that these sequences explore. Interestingly, we also uncovered a rare fast-diffusing state of chromatin that exhibits directed motion. The intact NuRD complex reduces the amount of time that enhancers/promoters remain in this fast-diffusing state, which we propose would otherwise re-organise enhancer-promoter proximity. Thus, we uncover an intimate connection between a chromatin remodeller and the spatial dynamics of the local region of the genome to which it binds

Intrinsic disorder in measles virus nucleocapsids

The genome of measles virus is encapsidated by multiple copies of the nucleoprotein (N), forming helical nucleocapsids of molecular mass approaching 150 Megadalton. The intrinsically disordered C-terminal domain of N (NTAIL) is essential for transcription and replication of the virus via interaction with the phosphoprotein P of the viral polymerase complex. The molecular recognition element (MoRE) of NTAIL that binds P is situated 90 amino acids from the folded RNA-binding domain (NCORE) of N, raising questions about the functional role of this disordered chain. Here we report the first in situ structural characterization of NTAIL in the context of the entire N-RNA capsid. Using nuclear magnetic resonance spectroscopy, small angle scattering, and electron microscopy, we demonstrate that NTAIL is highly flexible in intact nucleocapsids and that the MoRE is in transient interaction with NCORE. We present a model in which the first 50 disordered amino acids of NTAIL are conformationally restricted as the chain escapes to the outside of the nucleocapsid via the interstitial space between successive NCORE helical turns. The model provides a structural framework for understanding the role of NTAIL in the initiation of viral transcription and replication, placing the flexible MoRE close to the viral RNA and, thus, positioning the polymerase complex in its functional environment

Live-cell three-dimensional single-molecule tracking reveals modulation of enhancer dynamics by NuRD.

Acknowledgements: We thank T. Kretschmann for preparing the figures for publication, L. Lavis (Howard Hughes Medical Institute, Janelia Farm) for providing the JF549 dye, J. Wysocka (Stanford) for the Tbx3 constructs used for 2D enhancer tracking, A. Riddell for flow cytometry and the CSCI imaging (P. Humphreys and D. Clements) and DNA sequencing (M. Paramor and V. Murray) facilities. We thank K. Bowman, G. Brown and A. Crombie for preliminary computational analysis of NuRD-regulated genes and 2D enhancer tracking experiments, respectively. We thank the EU FP7 Integrated Project ‘4DCellFate’ (277899 E.D.L., B.D.H., I.B., C.S. and L.D.C.), the Medical Research Council (MR/P019471/1 E.D.L.) and the Wellcome Trust (206291/Z/17/Z E.D.L.) for program funding. We also thank the MRC (MR/R009759/1 B.D.H., and MR/M010082/1 E.D.L.), the Wellcome Trust (106115/Z/14/Z I.B. and 210701/Z/18/Z C.S.) and the Isaac Newton Trust (17.24(aa) B.D.H.) for project grant funding, and we thank the Wellcome Trust/MRC for core funding (203151/Z/16/Z) to the Cambridge Stem Cell Institute (including a starter grant to S.B.).To understand how the nucleosome remodeling and deacetylase (NuRD) complex regulates enhancers and enhancer-promoter interactions, we have developed an approach to segment and extract key biophysical parameters from live-cell three-dimensional single-molecule trajectories. Unexpectedly, this has revealed that NuRD binds to chromatin for minutes, decompacts chromatin structure and increases enhancer dynamics. We also uncovered a rare fast-diffusing state of enhancers and found that NuRD restricts the time spent in this state. Hi-C and Cut&Run experiments revealed that NuRD modulates enhancer-promoter interactions in active chromatin, allowing them to contact each other over longer distances. Furthermore, NuRD leads to a marked redistribution of CTCF and, in particular, cohesin. We propose that NuRD promotes a decondensed chromatin environment, where enhancers and promoters can contact each other over longer distances, and where the resetting of enhancer-promoter interactions brought about by the fast decondensed chromatin motions is reduced, leading to more stable, long-lived enhancer-promoter relationships