Site-specific modification and segmental isotope labelling of HMGN1 reveals long-range conformational perturbations caused by posttranslational modifications

Interactions between histones, which package DNA in eukaryotes, and nuclear proteins such as the high mobility group nucleosome-binding protein HMGN1 are important for regulating access to DNA. HMGN1 is a highly charged and intrinsically disordered protein (IDP) that is modified at several sites by posttranslational modifications (PTMs) - acetylation, phosphorylation and ADP-ribosylation. These PTMs are thought to affect cellular localisation of HMGN1 and its ability to bind nucleosomes; however, little is known about how these PTMs regulate the structure and function of HMGN1 at a molecular level. Here, we combine the chemical biology tools of protein semi-synthesis and site-specific modification to generate a series of unique HMGN1 variants bearing precise PTMs at their N- and C-termini with segmental isotope labelling for NMR spectroscopy. This study demonstrates the power of combining protein semi-synthesis for introduction of site-specific PTMs with segmental isotope labelling for structural biology, allowing us to understand the roles of PTMs with atomic precision, from both structural and functional perspectives.<br>

Download Full-text

Site-Specific Modification and Segmental Isotope Labelling of HMGN1 Reveals Long-Range Conformational Perturbations Caused by Posttranslational Modifications

10.26434/chemrxiv.12950942 ◽

2020 ◽

Author(s):

Gerhard Niederacher ◽

Debra Urwin ◽

David J. Tremethick ◽

K. Johan Rosengren ◽

Christian F. W. Becker ◽

...

Keyword(s):

Posttranslational Modifications ◽

High Mobility ◽

Intrinsically Disordered Protein ◽

Site Specific ◽

Isotope Labelling ◽

Specific Modification ◽

Intrinsically Disordered ◽

Cellular Localisation ◽

And Function ◽

Nucleosome Binding

Interactions between histones, which package DNA in eukaryotes, and nuclear proteins such as the high mobility group nucleosome-binding protein HMGN1 are important for regulating access to DNA. HMGN1 is a highly charged and intrinsically disordered protein (IDP) that is modified at several sites by posttranslational modifications (PTMs) - acetylation, phosphorylation and ADP-ribosylation. These PTMs are thought to affect cellular localisation of HMGN1 and its ability to bind nucleosomes; however, little is known about how these PTMs regulate the structure and function of HMGN1 at a molecular level. Here, we combine the chemical biology tools of protein semi-synthesis and site-specific modification to generate a series of unique HMGN1 variants bearing precise PTMs at their N- and C-termini with segmental isotope labelling for NMR spectroscopy. This study demonstrates the power of combining protein semi-synthesis for introduction of site-specific PTMs with segmental isotope labelling for structural biology, allowing us to understand the roles of PTMs with atomic precision, from both structural and functional perspectives.<br>

Download Full-text

Segmental and site specific isotope labelling strategies for structural analysis of posttranslationally modified proteins

RSC Chemical Biology ◽

10.1039/d1cb00045d ◽

2021 ◽

Author(s):

Dominik P Vogl ◽

Anne C. Conibear ◽

Christian F.W. Becker

Keyword(s):

Nuclear Magnetic Resonance ◽

Raman Spectroscopy ◽

Structural Analysis ◽

Posttranslational Modifications ◽

Protein Structures ◽

Spectroscopic Techniques ◽

Site Specific ◽

Isotope Labelling ◽

Infrared And Raman Spectroscopy ◽

Modified Proteins

Posttranslational modifications can alter protein structures, functions and localisation, and are important cellular regulatory and signalling mechanisms. Spectroscopic techniques such as nuclear magnetic resonance, infrared, and Raman spectroscopy, as well...

Download Full-text

Site-specific ubiquitylation acts as a regulator of linker histone H1

Nature Communications ◽

10.1038/s41467-021-23636-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Eva Höllmüller ◽

Simon Geigges ◽

Marie L. Niedermeier ◽

Kai-Michael Kammer ◽

Simon M. Kienle ◽

...

Keyword(s):

Posttranslational Modifications ◽

Histone H1 ◽

Linker Histone ◽

Active Chromatin ◽

Site Specific ◽

Linker Histone H1 ◽

Fundamental Process ◽

Core Histones ◽

Wide Scale

AbstractDecoding the role of histone posttranslational modifications (PTMs) is key to understand the fundamental process of epigenetic regulation. This is well studied for PTMs of core histones but not for linker histone H1 in general and its ubiquitylation in particular due to a lack of proper tools. Here, we report on the chemical synthesis of site-specifically mono-ubiquitylated H1.2 and identify its ubiquitin-dependent interactome on a proteome-wide scale. We show that site-specific ubiquitylation of H1 at position K64 modulates interactions with deubiquitylating enzymes and the deacetylase SIRT1. Moreover, it affects H1-dependent chromatosome assembly and phase separation resulting in a more open chromatosome conformation generally associated with a transcriptionally active chromatin state. In summary, we propose that site-specific ubiquitylation plays a general regulatory role for linker histone H1.

Download Full-text