Dynamic post-translational histone modifications studied by NMR spectroscopy

Lead Research Organisation: University College London
Department Name: Structural Molecular Biology

Abstract

DNA molecules of human cells are many times longer than the diameter of the cell and consequently the DNA is packed into a compact structure called the chromatin. The chromatin consists of DNA molecules coiled around histone proteins (sticky pulleys) in a very systematic manner. The cell utilises several mechanisms to control exactly what inheritable information from the DNA molecule that is turned into functional product (cellular machines). One mechanism that the cell exploits is to change the charge of certain histone proteins (weaken or strengthen the stickiness of the pulleys) and thus expose or restrict a specific part of the DNA to the cells gene production apparatus. HDAC, an enzyme that is responsible for changing the charge of histone proteins (a stickiness enhancer) will be the focus of the proposed research project. The HDAC enzyme works as a scissors that strips a negative charge off the histone proteins, thereby rendering the histone tails positively charged which strengthen the interaction with the negatively charged DNA. In particular, the focus of the proposed project is the dynamics and molecular motions of the HDAC enzyme (how does the scissors cut) and the dynamics will be studied primarily with nuclear magnetic resonance (NMR) spectroscopy. Thus, one of the key objectives of the research is to characterise, at atomic resolution, the mechanism by which the HDAC enzyme alter the histone charges. The goal is also to characterise how HDAC enzymes interact with inhibitors (drugs) and histones. HDAC enzymes are involved in cancers where they are believed to suppress the production of tumour suppressors. Inhibitors of HDAC enzymes have shown anti-tumour activity and it is therefore likely that the outcome of the proposed research will lead to the design of specific inhibitors of HDAC enzymes ultimately resulting in more efficient cancer therapy. An understanding of histone modifications requires a detailed picture of the three-dimensional structure of the involved enzymes and an appreciation of how these structures vary and fluctuate with time (a scissors cuts due to its opening and closing motions). Static structures of HDAC enzymes have been determined over the last decade, however, very few studies on the flexibility and dynamics of these regulatory molecules have been published. The proposed project focuses on the use of NMR spectroscopy as the primary biophysical tool to elucidate molecular flexibility and interactions since NMR has the potential to provide a description of the dynamics and interactions at atomic resolution. It is the goal that the NMR measurements together with other experimental techniques and computer simulations will create a coherent characterisation of the enzyme function. Another major objective of the proposed research is to develop new NMR methods to characterise molecular dynamics and flexibility in general. These developments aim at a time-resolved description of enzyme motions, that is, a visualisation of the enzyme motions over time - as a movie - as opposed to previous methods that primarily provides the amplitudes of protein motions. The research will be carried out at the Institute of Structural and Molecular Biology (ISMB), a joint venture between Birkbeck and University College London (UCL). UCL and ISMB provide a state-of-the-art and stimulating research environment with dedicated NMR machines suitable for the proposed project. Also, the highly collaborative environment and world-class expertise at ISMB and UCL open up the possibility for fruitful collaborations. For example, to increase the likelihood of success in the development of new HDAC inhibitors, I have initiated a collaboration with Prof Charles Marson, UCL, who is an expert on the productions of HDAC inhibitors. In my opinion this collaboration will allow the results about the HDAC enzyme dynamics, obtained by NMR spectroscopy, to be taken one important step further towards the design of new medicine.

Technical Summary

Histone deacetylases (HDACs) are involved in the regulation of gene expression by catalysing the deacetylation of histone proteins, which in turn leads to a condensed chromatin at point of modification. The physiological importance of HDACs is stressed by the fact that aberrant recruitment of HDACs has been linked to lymphomas and inhibitors of HDACs have shown anti-tumour activity in preclinical trials. The X-ray structures of HDAC:inhibitor complexes are available, however, very few reports on the molecular dynamics of HDACs have been published. It is suggested that structural flexibility of the HDACs is one of the factors that control the formations of HDAC:substrate and HDAC:inhibitor complexes, thus, experimental determinations of such flexibilities become important. A major goal of the proposed research is to characterise the molecular dynamics and interactions of the HDAC isoform HDAC8. This includes a characterisation of the formation of HDAC8:substrate and HDAC8:inhibitor complexes, including those interactions that are transient. Experimental determinations of the dynamics and structural changes of regions that surround the entrance to the active site (inhibitor binding site) will likely facilitate the design of isoform-specific inhibitors. Another goal of the proposed research is to develop new NMR methods to characterise molecular dynamics. Methods will be developed to determine the dynamics of active sites of metalloproteins by exploiting the strong interactions between unpaired electrons and nuclear spins. Moreover, methods to determine the dynamics of flexible proteins (histone tails and C-terminal domains of HDACs) will be developed. The new methods will together with theoretical molecular dynamics simulations allow for a time-resolved description of the molecular motions including models that visualize the molecular motions as a function of time.

Planned Impact

I strongly believe that the outcome of the proposed research will be beneficial for both the private sector (commercial drug discovery), the public sector, and in general beneficial for enhancing the health of society. The description of the histone deacetylase (HDAC) structural dynamics can be used readily by commercial drug discovery for molecular docking purposes and the new NMR methods that will be developed during the project will be useful for the general research community to characterise protein dynamics. Moreover, the outcome of the research will most likely facilitate the design of HDAC isoform specific inhibitors that will be beneficial for therapy of certain cancers. A major objective of the proposed research is to provide a description of the HDAC structures that encompasses dynamics and heterogeneity. Such a representation of the structure that surrounds the entrance to the active site will provide an accurate and veracious template to facilitate computer-assisted molecular docking and screenings for new HDAC inhibitors relevant for cancer and lymphoma therapy. In general, the development of methodology to provide a time-resolved description of protein dynamics will greatly assist in silico predictions of drugs and inhibitor binding, which in turn reduces the number of tests to perform in the laboratory. Consequently drug design will be faster and cheaper, which is of considerable economic and societal relevance. I therefore believe that both the new methods that will be developed and the characterisations of the HDAC dynamics will be beneficial for commercial drug design. The scientific results will be made available by publications in peer-reviewed journals and by presentations at national and international conferences. Furthermore, the software that will be developed during the project will be made available online, as I have done previously during my postdoctoral and graduate studies. I expect that cancer patients will benefit ultimately from the outcome of the proposed research. One of the goals of the proposed project is to facilitate the design of HDAC isoform specific inhibitors to improve therapy of certain cancers, possibly as suggested previously by using HDAC inhibitors as single agents or in combination with a chemotherapeutic agent (e.g. dexamethasone) or radiation therapy. In order to increase the likelihood of success in the development of isoform specific inhibitors, I have initiated a collaboration with Prof C Marson, UCL, who is an expert on the synthesis of HDAC inhibitors. In this future collaboration, I will provide the group of Prof Marson with dynamical descriptions of the HDAC structure and characterisations of binding interfaces obtained via NMR spectroscopy. New inhibitors will thereafter be designed collaboratively and synthesised in the lab of Prof Marson for further investigations. In my opinion this collaboration with a synthetic organic chemist will allow the results obtained by NMR spectroscopy to be taken one important step further towards the design of new drugs. One of the first HDAC inhibitors (pan-inhibitor) took ca. one decade from discovery to FDA approval, thus giving an approximate timeline. Overall the outcome of the proposed project will likely impact the research fields of chromatin remodelling and proteins dynamics. For example, the design of isoform specific HDAC inhibitors will provide a valuable tool to elucidate the individual functions of the HDAC isoforms. Moreover, the proposed research will provide the growing research community that utilises NMR spectroscopy with several new NMR methods and tools to investigate macromolecular dynamics and interactions. The new methods will be generally applicable to study molecular dynamics and interactions, i.e., not specific to HDAC proteins. I expect that the new methods will be particularly beneficial to the growing group of researchers that are studying intrinsically disordered proteins.

Publications

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Gopalan AB (2018) CPMG Experiments for Protein Minor Conformer Structure Determination. in Methods in molecular biology (Clifton, N.J.)

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Juen MA (2016) Excited States of Nucleic Acids Probed by Proton Relaxation Dispersion NMR Spectroscopy. in Angewandte Chemie (International ed. in English)

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Sauerwein A (2015) Protein NMR

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Werbeck ND (2014) Using ¹5N-ammonium to characterise and map potassium binding sites in proteins by NMR spectroscopy. in Chembiochem : a European journal of chemical biology

 
Description We have developed new NMR-based methods to characterise the motion of protein molecules. We have also applied our new methods to probe the regulation and function of key enzymes, such as the histone deacetylases (involved in the progression of cancers) and the von Willebrand Factor (involved in bleeding disorders).
Exploitation Route Our new NMR-based methods are already being used in our own research and by other research groups around the world.
Sectors Chemicals,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

URL http://www.rsc.org/ScienceAndTechnology/Awards/MarlowAward/2015-Winner-Hansen.asp
 
Description - Our investigations and our determination of the major coagulation factor VIII binding site on the von Willebrand factor are already being used by a biotech company to improve the shelf-life and the stability of their drugs. It is therefore anticipated that our findings soon will improve the quality of life and health, in particular of those with a bleeding disorder such as von Willebrand disease and haemophilia A. - We have attracted highly skilled individuals to the UK and trained these further during the project. Some of these individuals have attracted their own funding from outside the UK and are thereby not only contributing intellectually but also economically to the UK.
Sector Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Societal

 
Description Committee member of NMRDG
Geographic Reach National 
Policy Influence Type Membership of a guidance committee
URL http://www.rsc.org/Membership/Networking/InterestGroups/NMR/?e=1
 
Description Committee member; Faraday division
Geographic Reach Multiple continents/international 
Policy Influence Type Membership of a guideline committee
URL http://www.rsc.org/ScienceAndTechnology/Awards/FaradayLectureshipPrize/
 
Description Fellow of the Royal Society of Chemistry, UK
Geographic Reach Multiple continents/international 
Policy Influence Type Membership of a guideline committee
URL http://www.rsc.org
 
Description Leverhulme Trust project grant
Amount £198,000 (GBP)
Funding ID RPG-2016-268 
Organisation The Leverhulme Trust 
Sector Academic/University
Country United Kingdom
Start 01/2017 
End 01/2020
 
Description Novo Nordisk A/S, Industrial support
Amount £299,900 (GBP)
Organisation Novo Nordisk 
Sector Public
Country Denmark
Start 01/2015 
End 12/2016
 
Description Rosetrees Trust
Amount £14,000 (GBP)
Funding ID CM247 
Organisation Rosetrees Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2012 
End 09/2013
 
Description UCB Pharma
Amount £122,500 (GBP)
Organisation UCB Pharma 
Sector Private
Country United Kingdom
Start 06/2017 
End 09/2021
 
Description Wellcome Trust NMR spectrometer
Amount £600,000 (GBP)
Funding ID 063393 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2014 
End 11/2019
 
Title A method to probe potassium binding in proteins. 
Description We have in a series of recent publications developed a method to probe potassium binding in proteins by using 15N-ammonium chloride as a proxy. 
Type Of Material Data analysis technique 
Year Produced 2014 
Provided To Others? Yes  
Impact Since the method is still very new the impact is still quite low and hard to judge. 
 
Title Structure of Methyl-Bearing side-chains 
Description We have over a series of publications generated a method to start to characterise the structure and dynamics of methyl-bearing side-chains in proteins from the NMR 13-C chemical shifts. This method is because of its sensitivity applicable to large protein complexes and thermally excited states of proteins, where previous methods failed. 
Type Of Material Data analysis technique 
Year Produced 2010 
Provided To Others? Yes  
Impact Based on the number our papers are cited and now also used in a wide range of protein NMR applications, both in liquid state and in solid state, we judge that the impact has been quite high. For example, other research groups and our own group have used the new method for more accurate structure determinations of proteins by NMR spectroscopy. 
URL http://www.biochem.ucl.ac.uk/hansen/sider/
 
Description Collaboration with biotech company UCB Pharma 
Organisation UCB Pharma
Country United Kingdom 
Sector Private 
PI Contribution One PhD student working on NMR method developments
Collaborator Contribution Full PhD studentship, laboratory consumables, costs towards NMR spectroscopy
Impact Nil
Start Year 2016
 
Description Collaboration with the biotech company Novo Nordisk A/S 
Organisation Novo Nordisk
Country Denmark 
Sector Public 
PI Contribution We have used NMR spectroscopy and molecular dynamics simulations at UCL to characterise the structure and dynamics of domains of the von Willebrand Factor (vWF), its binding to Factor VIII, and the molecular mechanism of type 2N von Willebrand disease.
Collaborator Contribution Novo Nordisk A/S contributed initially funding towards a PhD student and also contributed material in the form of recombinant Factor VIII for investigations of FVIII:vWF interactions. Novo Nordisk has also contributed knowledge of the purification of FVIII for binding studies and general protein characterisations. Subsequently, Novo Nordisk has supported our research with a two-year research grant (£299,000; 1 postdoc, materials, and overheads). The aim of this two-year grant is to further characterise the FVIII:vWF interaction with the long-term goal of increasing the shelf-life and half-life of N8 drug used to treat haemophilia.
Impact The publication: "Solution structure of the major factor VIII binding region on von Willebrand factor". Blood, 2014, 123(26), p. 4143-51 Novo Nordisk has recently obtained a patent, which follows our ideas and protocols, that is, produce in e.coli. domains of vWF which includes the TIL'E' domains and use these for stabilisation of N8. (WO2014198699)
Start Year 2012
 
Title Characterising potassium binding in proteins 
Description We have over a series of publications developed an NMR technique to probe potassium binding in proteins by using 15N-ammonium chloride. Binding characteristics and the environment of the macromolecular-binding site can be probed using simple tools available in most laboratories that use NMR spectroscopy. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2014 
Impact This method is still very new and there has not yet been notable impacts, apart from our publications and citations of these. 
 
Title NMR pulse sequences to characterise arginine side-chains 
Description We have developed new NMR radio-frequency pulse sequences to probe arginine side-chains of proteins. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2013 
Impact The new pulse sequences have been used by our group members to characterise a group of proteins called histone deacetylases. Because of our new method we have, among others, established a collaboration with a group at the Max-Planck institute for medical research. This collaboration deals with the characterisation of kinases; a group of enzymes involved in several cellular mechanisms. 
URL http://onlinelibrary.wiley.com/doi/10.1002/anie.201209385/abstract;jsessionid=A02E50FD97F99213528A5A...
 
Title Software to determine protein side-chain structure from NMR chemical shfits 
Description The software, which both runs on our webserver and can be downloaded as a stand-alone package, is used to determine the structure and dynamics of protein side-chains from 13C NMR chemical shifts. As of now, the software is only able to analyse methyl-bearing side-chains, but we are planning on implementing more side-chains soon. 
Type Of Technology Software 
Year Produced 2011 
Impact Other researchers have downloaded our software, used our webserver, and used our models and techniques. The results of the calculations of the software are, among others, used in protein structure calculations as evident from publications citing our work. 
URL http://www.biochem.ucl.ac.uk/hansen/sider/
 
Description High School Students 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact We have had high-school students visiting our laboratory to shadow our research. Some of these students are now undergraduate biochemistry students at UCL.
Year(s) Of Engagement Activity 2014,2015