The first full-length structures of the human angiotensin-converting enzyme (ACE) have been determined by researchers from the University of Cape Town (UCT) using cryo-electron microscopy (cryo-EM). ACE is a blood pressure-regulating protein that is critical for heart health.
Published in The EMBO Journal [https://www.embopress.org/doi/10.15252/embj.2021110550] on 12 July 2022, the cryo-EM structures of ACE in two different conformations have the potential to improve drug design for cardiovascular disease - the leading cause of death worldwide.
The research was part of START (Synchrotron Techniques for African Research and Technology) - a £3.7million project funded by a Global Challenges Research Fund grant, provided by the UK Research and Innovation’s Science and Technology Facilities Council (STFC).
Dr Lizelle Lubbe, Dr Jeremy Woodward, Professor Ed Sturrock, and Professor Trevor Sewell performed the study. The ACE protein was produced in UCT’s Sturrock Laboratory and prepared for high-resolution imaging at UCT’s Electron Microscope Unit (EMU). It was transported to the electron Bio-Imaging Centre (eBIC) at the UK’s national synchrotron, Diamond Light Source (Diamond) for high-resolution imaging. Image processing took place at South Africa’s CSIR Centre for High Performance Computing (CHPC) and the EMU.
ACE is a key target for the treatment of hypertension (elevated blood pressure) and cardiovascular disease since it produces the hormone Angiotensin II, which constricts blood vessels and raises blood pressure. Hypertension is a major risk factor for heart failure, heart attack, kidney disease, stroke, and loss of vision. It often shows no symptoms and is known as ‘the silent killer’.
The monomeric form (one copy of the protein) of ACE is intriguing because it is made up of two structurally similar but functionally distinct domains that are linked together. It also exists in a functionally relevant dimeric form (two interacting copies of the protein) that was seen in the study. Communication between the different parts of ACE influences its function and drug binding properties, which are vital for therapeutic drug design.
“Clinically, ACE inhibitors are recommended as one of the first-line treatments for hypertension, but they non-selectively target both ACE domains and thereby trigger side effects in some patients,” says Principal Investigator of the study, Professor Sturrock. “It is really important to understand the structure and dynamics of these newly seen forms of ACE because this could help identify novel sites for the design of domain-selective inhibitors that avoid such side effects.”
The study’s findings uniquely reveal ACE’s highly dynamic nature, and the mechanisms by which dimerization and communication occur between its different domains. “By changing from an active-site centred to a holistic view of this vital protein, we obtained valuable new insights into how ACE works,” says first author of the study, Dr Lubbe.
“The dynamic nature of ACE prevents crystal formation, which meant that X-ray crystallography studies over the past two decades could only solve parts of the structure,” explains study co-author, Professor Sewell. “We discovered many clues using this method but were unable to solve the full puzzle until we had access to high-resolution cryo-EM.”
To obtain the complete structure, the protein was rapidly cooled to -180 degrees Celsius, trapping the different conformations in a very thin, glass-like film of water at the EMU. After this, an advanced Titan Krios microscope at eBIC was used for imaging. “Even with high-resolution imaging, the unique shape, small size, and dynamic nature of ACE posed many challenges,” says study co-author, Dr Woodward.
“Recently developed cryo-EM image processing methods were crucial to solving the structures,” Dr Lubbe explains. “We had to separate the images computationally through extensive classification, amounting to ‘digital purification’ because biochemical methods failed to separate the monomeric and dimeric forms of ACE. We could then solve both ACE structures by focusing the 3D refinement on different parts of the structure in turn.”
“We are delighted with the findings of this study achieved by a brilliant team of scientists in Africa, using eBIC’s advanced cryo-EM at Diamond,” says Professor Chris Nicklin, Diamond Science Group Leader, and Principal Investigator in the GCRF START grant project. “This is an excellent example of UK and African research partnerships and global impact through the very successful GCRF START grant. The world urgently needs sustainable solutions for killer heart diseases and other chronic health conditions. We are very excited that the study’s structural insights could pave the way for improved antihypertensive drug design.”
The citation for the article is: ‘Lubbe, L., Sewell, B.T., Woodward, J.D., Sturrock, E.D. (2022) Cryo‐EM reveals mechanisms of angiotensin I‐converting enzyme allostery and dimerization. EMBO J https://www.embopress.org/doi/10.15252/embj.2021110550. DOI: 10.15252/embj.2021110550’
Notes to Editors
UCT Press Officer: Nombuso Shabalala [Media Liaison Head, University of Cape Town Communications and Marketing Department] email: email@example.com; Tel: + 27 21 650 4190
Diamond Press Office: Isabelle Boscaro-Clarke, [Head of Communications, Engagement & Impact], email: firstname.lastname@example.org; Tel: +44 1235 778130
First and co-corresponding author: Dr Lizelle Lubbe, email: email@example.com
Study co-author: Professor Trevor Sewell, Tel +27 (0) 827161790, email: firstname.lastname@example.org
Images and Captions
Image 1: Figure 1, Cryo-EM structures of the hourglass-shaped ACE monomer (one protein copy; left) and dimer (two interacting protein copies; middle). A flexible loop (yellow; protein centre) acts as a hub for communication between different areas of the protein. Dimerization is like a ‘kiss of death’ since it triggers conformational changes in the protein core that likely inactivate it. These novel insights into the structure of ACE light the way to better drugs against heart disease. © Lizelle Lubbe
Image 2: Dr Lizelle Lubbe working with cryo-Electron Microscopy grids of the human angiotensin-converting enzyme (ACE) in the electron Bio-Imaging Centre (eBIC) at the UK’s national synchrotron, Diamond Light Source. Photo credit: Dr Jeremy D Woodward, ©Diamond Light Source Ltd
Image 3: The Titan Krios microscope in the electron Bio-Imaging Centre (eBIC) at the UK’s national synchrotron, Diamond Light Source. ©Diamond Light Source Ltd
UK Global Challenge Research Fund grant details: Grant ref. ST/R002754/1. START [https://start-project.org/] is a collaborative project that fosters the development of Synchrotron Techniques for African Research and Technology (START). It builds partnerships between world leading scientists in Africa and the UK, working together on Structural Biology and Energy Materials using synchrotron science through research, mentoring and training. START was awarded a three year grant of £3.7M in 2018 by the UK’s Science and Technology Facilities Council (STFC) – UKRI from the Global Challenges Research Fund (GCRF).
About the findings of the study
The first crystal structure of an engineered form of a single ACE domain was solved almost two decades ago in 2003. Since then, 56 structures of the truncated domains were reported, but the complete structure remained a mystery, until it was recently solved using cryo-EM. Cryo-EM offers the advantage of visualising the protein in a more biologically relevant (near-native) state which has not been possible using X-ray crystallography, thus revealing both ACE domains in an open conformation for the first time.
Spontaneous opening and closing of the molecule (shapeshifting) was also observed. Since closure is required to produce Angiotensin II, this structure offers opportunities for the design of drugs that bind to novel pockets and lock ACE in an open conformation. Regions of the protein involved in closure have less similarity between the domains, thus drugs with this mechanism of action may be more domain-selective and less likely to trigger side effects.
Dimerization (interaction of two copies of ACE with each other) occurs near a small surface cavity and changes the conformation of core amino acids that are critical for ACE function. This means that dimerization could be like an ‘off switch’, which triggers changes in the core of the protein and potentially inhibit it. If a drug-like molecule could be designed to bind in this cavity and elicit the same effect, it could provide a novel means to allosterically (indirectly) inactivate the enzyme and thus prevent the side-effects of current ACE inhibitors. Dimerization has long been thought to cause beneficial intracellular effects but since it is a very short-lived and dynamic event, it has been difficult to study in the past. The structure of the dimerization interface provides the opportunity for designing small molecules that stabilise the dimer to enable further research into its intracellular function.
Leading causes of global deaths
According to the World Health Organization cardiovascular disease and stroke accounted for 27% of all deaths globally in 2019. Ischaemic heart disease (the world’s biggest killer) caused 8.9 million deaths in 2019 (16% of the world’s total deaths). Also in 2019, stroke caused 11% of the world’s total deaths; kidney diseases caused 1.3 million deaths [https://www.who.int/news-room/fact-sheets/detail/the-top-10-causes-of-death]. In the same year, an estimated 1.28 billion adults world-wide were living with hypertension, two thirds of whom reside in low- and middle-income countries. [https://www.who.int/news-room/fact-sheets/detail/hypertension]
Collaborating Institutes / laboratories
- The Sturrock Laboratory at the University of Cape Town’s Institute for Infectious Disease and Molecular Medicine (IDM) for protein expression and purification. The IDM is located at the Faculty of Health Sciences: http://www.idm.uct.ac.za/
- The Electron Microscope Unit at the University of Cape Town for grid preparation, grid screening, sample shipping and image processing: http://www.emu.uct.ac.za/contact-the-unit
- Diamond Light Source: https://www.diamond.ac.uk/Home/About.html
- The UK’s electron Bio-Imaging Centre (eBIC) for cryo-EM embedded at Diamond Light Source: https://www.diamond.ac.uk/Instruments/Biological-Cryo-Imaging/eBIC.html
- The CSIR Centre for High Performance Computing (CHPC) in Cape Town for image processing, https://www.chpc.ac.za/
Acknowledgements – the authors extend thanks to:
- CHPC for providing computational resources for image processing.
- UCT’s Advanced Computing Committee for the purchase of a GPU/TPU based computer for image processing at the Electron Microscope Unit.
- Diamond Light Source for access to and support of cryo-EM facilities at eBIC [proposal BI24039], funded by the Wellcome Trust, the UK’s Medical Research Council (MRC) and Biotechnology and the UK’s Biological Sciences Research Council (BBSRC).
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Cryo‐EM reveals mechanisms of angiotensin I‐converting enzyme allostery and dimerization.
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