Research carried out at Diamond has given new insight into the mechanism of histone deacetylase (HDAC) complexes, which are key regulators of the human genome. Due to their role as gene expression regulators, HDAC complexes are potential drug targets for a variety of diseases, including cancers, Alzheimer’s, and HIV infection. Revealing their mechanisms is therefore invaluable for the development of novel therapeutics.
Previous work at Diamond led to the discovery that HDAC complexes are regulated by a small molecule called inositol phosphate. Now researchers have determined the stereochemical requirements for inositol phosphate activation and discovered that the molecule acts to enhance substrate binding and catalytic turnover through allosteric communication.
The study published in Nature Communications, detailing experiments on the Microfocus MX beamline (I24), identifies a novel peptide inhibitor for HDAC complexes, opening up a potential avenue to design novel drugs to modulate the activity of these complexes. A second study published in eLife used both I24 and the Solution State SAXS beamline (B21) to understand the structure of one of the core complexes in which HDAC1 is assembled.
Histone deacetylase (HDAC) enzymatic complexes are well known regulators of the human genome, playing a key role both during development and to maintain homeostasis. Made up of an HDAC enzyme bound to associated proteins, there are a number of different complexes found in vivo each with related but distinct functions.
HDAC complexes act to repress gene expression by catalysing the removal of acetyl groups from histones that make up chromatin, condensing the genome in the targeted area and repressing transcription. While HDAC complexes are mainly known for transcriptional repression there is empirical evidence showing the HDAC complexes have a role in gene activation.
Previous work carried out at Diamond1,2 revealed a surprising finding – that the catalytic activity of the HDAC complexes could be activated by a small molecule called inositol phosphate.
Now the research group, led by the University of Leicester, with chemists from the University of Bath have built on this, further exploring the mechanism of activation of HDAC complexes as described in a paper published in Nature Communications.
The new paper details the stereochemical requirements for an inositol phosphate to activate the HDAC3:SMRT complex. Chemical biology and crystallographic studies of the inositol phosphate binding site determined that for activation to occur three adjacent phosphate groups are needed on the inositol ring. Phosphates, or other bulky groups, at the remaining three positions can be tolerated, but are not essential for activation.
The researchers then extended this work to the HDAC1:MTA1 complex (HDAC 1 bound to a metastasis-associated protein 1) which has a related but distinct function compared to HDAC3:SMRT in gene regulation. They determined that it too could be regulated by inositol phosphate, with the same principles holding with regards to phosphate groups on the inositol ring.
“There are three positions on the inositol that are important for activation,” said Professor John Schwabe from the University of Leicester. “Because of that, we know that only inositol phosphates, made by one particular enzyme called inositol phosphate multikinase, will activate the complexes.”
Figure 1: The structure of the HDAC1:MTA1 dimeric complex. The enlarged panels shows the peptide inhibitor bound to the active site with the inositol phosphate bound at the interface of HDAC1 and MTA1 at the allosteric site. HDAC1 is shown in grey, MTA1 in blue, the peptide inhibitor in mageneta, and the inositol phosphate in green/orange.
Following the investigation into inositol phosphates, the team designed an inhibitor peptide for HDAC1:MTA1, to test the mechanism of action of the complex (Fig. 1).
To design their peptide inhibitor, the researchers took a peptide from a histone tail – the natural substrate for the HDAC complex – and substituted the acetyllysine with a hydroxamic acid. The inhibitor was able to mimic the natural substrate, illustrating the binding mechanism to the catalytic site on the enzyme. X-ray diffraction of the complex also demonstrated cross stabilisation between the inositol phosphate and peptide inhibitor binding sites, showing that if an inositol phosphate is bound to the HDAC complex it enhances substrate binding and vice versa – a phenomenon known as allosteric communication.
For both aspects of the investigation the researchers needed to obtain crystal structures from very small crystals, a problem that Diamond was able to help them overcome.
“We used I24, which is a microfocus beamline,” said Prof Schwabe. “It has not only been crucial for this study but it was crucial for our previous studies because these protein complexes, made in human cells, only give very tiny crystals. I24 gives us good diffraction from very tiny crystals where other beamlines wouldn’t give us any useful diffraction.”
Thanks to the work at Diamond, the researchers now have a better understanding of the mechanism through which the binding of inositol phosphates results in a hugely increased activation of these enzymes. They also now have an understanding of how substrates are expected to interact with HDAC complexes and showed that hydroxamic acid substituted peptides can potentially be used as potent inhibitors.
Figure 2: The SAXS scattering curve that revealed the positioning of the RBBP4 chromatin binding module relative to the HDAC1:MTA1 complex. RBBP4 is shown in green, MTA1 in blue and HDAC1 in grey.
Combining crystallography and SAXS
The team have already extended their work by investigating the structure of HDAC1:MTA1 bound to another protein, RBBP4 (Figs. 2,3). How the HDAC1:MTA1:RBBP4 complex interacts with chromatin is unknown and initial steps to understand this problem were undertaken at Diamond using X-ray diffraction and Solution State X-ray Scattering (SAXS) techniques, at I24 and B21 respectively. Published in eLife, the SAXS experiments provided the first structural model of the complex, demonstrating the arrangement of the proteins in the complex. B21 introduced new SAXS capabilities to the UK science community and the beamline staff have provided invaluable technical support to the research group enabling analysis of their SAXS experiments.
Figure 3: Details of the crystal structure of the MTA1:RBBP4 complex. RBBP4 is shown in green and MTA1 in blue.
The researchers now plan to continue exploring the assembly and function of these complexes and have embarked upon a project to make more specific inhibitors that will specifically target individual HDAC complexes rather than the enzymes in all HDAC complexes.
To find out more about the I24 or B21 beamlines, or to discuss potential applications, please contact Principal Beamline Scientists Robin Owen (I24) email@example.com or Robert Rambo (B21) firstname.lastname@example.org.
Diamond Light Source is the UK's national synchrotron science facility, located at the Harwell Science and Innovation Campus in Oxfordshire.
Copyright © 2020 Diamond Light Source
Diamond Light Source Ltd
Harwell Science & Innovation Campus
Diamond Light Source® and the Diamond logo are registered trademarks of Diamond Light Source Ltd
Registered in England and Wales at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom. Company number: 4375679. VAT number: 287 461 957. Economic Operators Registration and Identification (EORI) number: GB287461957003.