Double first! First synchrotron user from the University of Zululand solves partial structure of the Schistosomiasis (Bilharzia) G4LZI3 universal stress protein

In a ‘double first’, Dr Priscilla Masamba, has become the first University of Zululand student to use the UK’s National Synchrotron Light Source, Diamond, and solve the partial structure of a protein from Schistosoma mansoni. With access to the synchrotron made possible by GCRF START, Priscilla employed sophisticated robotic instruments and macromolecular X-ray crystallography techniques remotely from South Africa to solve the partial structure of the G4LZI3 universal stress protein – a protein regarded as a target for novel medicines for treating the disease Schistosomiasis. The experiments took place in February 2020, using the Diamond’s I04-1 beamline.

Dr Priscilla Masamba in the laboratory at the University of Cape Town.
Photo credit Rebekka Stredwick. ©Diamond Light Source

Schistosomiasis is an acute and chronic disease caused by parasitic worms (schistosomes) endemic in more than 78 countries with an estimated 4 million people infected in South Africa alone. The disease requires an intermediate host, the freshwater snail Bulinus africanus, and occurs most often in rural areas where people become infected during routine agricultural, domestic, occupational, and recreational activities which expose them to infested water.

Only one drug, Praziquantel, is available to treat Schistosomiasis leaving people vulnerable to schistosome resistance and this treatment is only partially effective in treating adults.  The aim of Priscilla ’s research is therefore to generate insights for the design of alternative treatment regimen targeting specific stages during the developmental cycle of the schistosome.

Describing the experiments at Diamond as “close to a cool sci-fi movie,” Dr Masamba was able to control the sophisticated instruments on I04-1 beamline and collect data in real time from the University of Cape Town’s (UCT’s) Aaron Klug Centre for Imaging and Analysis – established as a GCRF START Centre of Excellence for structural biology research.

“Remote data collection at Diamond was so exciting!” Dr Masamba explains, “I could literally control and see a robot that was thousands of miles away on the other side of the world, mount a microscopic crystal (sample) within the firing line of a powerful X-ray beam, and determine the amount of energies released by light emitted from the sample caused by incident X-ray beams, and all of this while working from the laboratory in Cape Town. I didn’t need to get in a plane to achieve the one of the most imperative steps in the crystallography process! The whole experience provided me with rare exposure to the world of X-ray crystallography, impacting my view of science in a spectacular way.”

Proteins are thermodynamically and kinetically responsible for all biochemical processes that occur, and are therefore responsible for coordinating, regulating and dictating numerous metabolic functions. Exposure of the Schistosome parasite to extreme conditions during its developmental stage triggers the expression of heat shock proteins and universal stress proteins, of which the G4LZI3 USP has been identified as a potential druggable target for the development of alternative treatments (schistosomicides). Techniques like X-ray crystallography can provide insight, not only into the composition of these biomolecules, but also into their various interactions with other compounds and their roles in biological mechanisms, an imperative foundation for rational drug design and development.

Before the experiments took place, diffraction of the crystals was first checked at UCT using a diffractometer. Crystals from these conditions were then flash-frozen in liquid nitrogen and shipped to the Diamond synchrotron to be used as samples.

The BART robot and sample holder on beamline I04-1. The drum (Dewar) contains liquid nitrogen, and space for 37 pucks, each containing 16 pins, so 592 samples. These pins and pucks are shipped in a Dewar from South Africa. The robotic arm is grey and is shown ready to pick up the next sample. When it selects the next sample, this is placed onto the goniometer, which holds the sample and rotates it for data collection.
©Diamond Light Source.
The goniometer on beamline I04-1 holds the microscopic crystal on a pin with the sample on the end of it which rotates in the firing line of the powerful X-ray beam.
©Diamond Light Source.

The solved structure of the S. mansoni G4LZI3 is a success story for the University of Zululand, a small resource-constrained university in the rural part of KwaZulu-Natal Province of South Africa. The University of Zululand lacked the resources required for Dr Masamba to achieve all her objectives for her PhD, which meant the collaboration through START in order to carry out the experiments needed was imperative both professionally and personally.

Priscilla is thankful for the guidance and mentoring from her PhD supervisor, Professor Abidemi Paul Kappo, who heads up the Biotechnology and Structural Biology (BSB) Research Group in the Department of Biochemistry and Microbiology at the University of Zululand, and from START Principal Investigator, Professor Trevor Sewell, of UCT’s Aaron Klug Centre for Imaging and Analysis, both of whom helped Priscilla overcome various challenges.

“I have been able to learn and cultivate scarce, critical and sought-after skills here in Africa in the fields of bioinformatics and drug discovery, molecular biology and especially, structural biology,” says Dr Masamba. “These include gene cloning, recombinant protein expression and purification, as well as characterisation of proteins. This has not been an easy task because I am from an underrepresented group in science as a black female and study at a historically-disadvantaged and resource-constrained institution.”

Professor Abidemi Paul Kappo, (left) head of the Biotechnology and Structural Biology (BSB) Research Group in the Department of Biochemistry and Microbiology at the University of Zululand, and START Principal Investigator, Professor Trevor Sewell (right), from the University of Cape Town’s Aaron Klug Centre for Imaging and Analysis.
©Diamond Light Source.

An important objective of the START programme is to increase the number of structural biologists in similar less developed universities in South Africa and across the continent. This can present complex challenges, not least because many students are ill-equipped for work in the field of structural biology.

“A key concept behind the creation of the START Centre of Excellence at UCT’s Aaron Klug Centre for Imaging and Analysis, for example, is to provide the necessary infrastructure to enable senior students and staff at South Africa’s historically disadvantaged universities to access both the human and material resources necessary to overcome the difficulties and determine protein structures,” Professor Sewell says. “We count the collaboration with Professor Paul Abidemi Kappo and Dr Masamba as a major success in this respect.”

This collaboration between Prof. Kappo and Prof. Sewell was enabled by GCRF START with Prof. Sewell providing the technological resource for the G4LZI3 structural biology project, as well as the linkage to Diamond.

“Above all, Professor Sewell’s enthusiasm to train and develop a “critical mass” of students in Structural Biology is second to none,” Prof. Kappo says. “This has been a joint effort and a model of national and international collaboration. In addition to the technological resources through UCT and linkage with Diamond in the UK, funding for this project was provided by the National Research Foundation (NRF) of South Africa through a doctoral bursary awarded Dr Masamba. It is expected that structure-guided drug discovery for schistosomiasis will be the concluding part the project.”

Dr Masamba and Prof. Trevor Sewell with colleagues collaborating with GCRF START at the Aaron Klug Centre for Imaging and Analysis at the University of Cape Town. In the picture from left to right: Dr Priscilla Masamba, Dr Jeremy Woodward, Melissa Marx, Dr Mulelu, Dr Philip Venter, Dr Lizelle Lubbe, Professor Trevor Sewell
Photo Credit: Rebekka Stredwick. ©Diamond Light Source.

About Dr Priscilla Masamba

Born to Congolese parents in the DR Congo, Dr Masamba lived in the UK and Zimbabwe as a child, before moving to South Africa where she matriculated and studied for her first degree in Biological Sciences at Walter Sisulu University, Mthatha. Thereafter, Priscilla joined the Biotechnology and Structural Biology (BSB) Research Group in the Department of Biochemistry and Microbiology at the University of Zululand headed by Prof Abidemi Paul Kappo and registered under his tutelage for a BSc (Hons) degree, followed by an MSc and later a PhD in Biochemistry. Priscilla’s desire is to continue in the path of macromolecular X-ray crystallography of proteins through the NRF Postdoctoral Fellowship in Structural Biology at the University of Johannesburg.

Acknowledgements

Dr Priscilla Masamba extends a special thanks to Dr Brandon Weber (UCT), Dr Phillip Venter (UCT), Kaylene Baron (UCT), and Ndibonani Qokoyi (University of Zululand) who were involved in different ways in the production, purification and crystallisation of the G4LZI3 protein, as well as in data collection.

New collaboration opportunities for computational insights into catalysis

A successful secondment by GCRF START computational scientist, Dr Michael Higham, has led to exciting new computational modelling collaborations involving leading catalysis institutes in South Africa and the UK.

These opportunities range from investigating adsorption induced magnetisation changes in nickel catalysts, to research into bimetallic catalysts for CO2 hydrogenation of environmental and industrial importance in the search for sustainable, clean energy sources to tackle climate change.

Dr Higham, who is a START-funded Postdoctorate Research Associate at Cardiff Catalysis Institute working with the UK’s national synchrotron light source, Diamond Light Source and the UK Catalysis Hub, spent two months from December 2019 to January 2020 at the University of Cape Town meeting researchers, undertaking initial computational work, and getting to know the projects.

Now back in the UK, Dr Higham’s aim is to provide theoretical inputs through computational modelling in order to support findings from experimental results.

One of these projects focusses on bimetallic alloy catalysts for methanol synthesis and conversion involving Dr Mohamed Fadlalla and Christopher Mullins from the University of Cape Town’s Centre of Catalysis and c*Change, South Africa’s DST-NRF Centre of Excellence in Catalysis Research.

“In early December 2019, our team from the University of Cape Town and Southampton University (UK) visited the Diamond Light Source synchrotron and used the B18 beamline to study the influence of substituents in the ferrite structure on the reduction behaviour and carbon dioxide hydrogenation reaction to valuable products (e.g. fuels),” Dr Fadlalla explains. “The next step is Michael’s computational work to help us to calculate certain insights from the results such as how the catalyst looks and how the reactant is interacting with the catalyst.”

The computational calculations will examine the catalytic product distributions which provide detailed insights into possible explanations for observed catalyst selectivity, as Dr Higham explains,

“Through modelling adsorption energies, activation energies and reaction energies we hope to shed light on what happens on the catalyst’s surface. We want to compare the observed, experimental data with the computational calculations.”

Michael Higham (L) & Mohamed (R) Fadlalla working on bimetallic alloy catalysts for methanol synthesis and conversion using computational studies.
Photo credit: Rebekka Stredwick; ©Diamond Light Source Ltd.

Another project focuses on the rationalisation of experimentally observed adsorption-induced changes in magnetisation of Ni particles. Working together with Dominic de Oliveira from UCT, Dr Higham’s intention is that the computational results, in conjunction with the experimental work, will pave the way for possible new techniques to employ magnetism to probe surface area and composition.

“The secondment was a great opportunity to meet some really enthusiastic scientists doing some excellent work on catalysis,” Dr Higham reports. “The collaborations represent not only a trajectory for progress in solving real-world energy problems but also a fundamental knowledge foundation that can inform future studies”.

“Computational experts like Michael can predict how certain catalysts perform and we can try to confirm that with our techniques which is a reciprocal learning process,” says Professor Michael Claeys, Director of c*Change. “This expertise informs us and we inform them through the results which is something a single group can’t do because very specific expertise is needed. Collaborating provides some of the most powerful learning opportunities which really shape your people.”

Such collaborations through START not only enable shared learning, they increase opportunities for publications and – in Dr Fadlalla’s words – “give Africa a bigger footprint in the wider catalyst society”, as Co-I on the START grant, Dr Peter Wells, explains,

“START is a fantastic opportunity for the UK to work in concert with our African partners to tackle shared challenges. It is clear that the excitement generated can be inspirational and helps to further integrate research partnerships.”

More about the contributors

Dr Michael Higham works with Professor Richard Catlow’s group at Cardiff University and is supported by GCRF START to provide computational insights for experimental work utilising the synchrotron facilities at Diamond Light Source Ltd. Michael’s current research project concerns Cu-based catalysts for methanol synthesis from CO2.

Dr Mohamed Fadllala is a Post Doctorate Fellow at the University of Cape Town’s Catalysis Institute in the Department of Chemical Engineering.

Dr Peter Wells is an Associate Professor and a Co-I on the START grant. He currently has a joint appointment between Diamond Light Source Ltd and the University of Southampton.

Professor Michael Claeys is the Director of DST-NRF Centre of Excellence in Catalysis, c*change, hosted by the Catalysis Institute in the Department of Chemical Engineering at the University of Cape Town, South Africa

The START of great things!

Visualising the structure of an intact helical filament at close-to-atomic resolution for the first time

“We are seeing critical scientific discoveries and the emergence of a new generation of experts that have resulted directly from our training programmes in advanced methods and the use of synchrotron facilities and tools.” Dr Gwyndaf Evans, START Principal Life Sciences Principal Investigator and Principal beamline scientist on Diamond’s VMXm beamline.

A seminal work of Dr Jeremy Woodward, Dr Andani Mulelu and Angela M.Kirykowicz from the University of Cape Town (UCT), South Africa, has provided novel and exciting insights into the structure and inner workings of nitrilase enzymes with the potential to address key health, food security and environmental challenges within Africa and beyond.

The results were published on the 17 July 2019 in Nature Communications Biology 2:260 (2019)4 and made possible through the UK’s national synchrotron light source, Diamond Light Source, which has integrated facilities for life sciences users as a ‘one stop shop’ for structural biology.

Dr Mulelu and Dr Woodward in front of the cryo-electron microscope
at the University of Cape Town.
Photo credit: Rebekka Stredwick; ©Diamond Light Source Ltd.
The first high resolution visualisation of a Cryo-EM6 protein structure in Africa

Nitrilases are a class of plant enzymes that play an important role in the synthesis of a broad range of chemicals. Although have specificity for a small range of substrates they have a large potential to create products of biotechnological significance.

Building on more than a decade of structural biology research, and exploiting knowledge sharing and synchrotron access opportunities through the Global Challenges Research Fund’s GCRF START Programme, Dr Woodward, Dr Mulelu and Angela Kirykowicz were able to visualise the structure of an intact helical filament at close-to-atomic resolution for the first time – the first high resolution visualisation of a Cryo-EM6 protein structure ever to be produced in Africa (Fig 1).

The scientists achieved this at the UK’s national Electron Bio-Imaging Centre (eBIC), using Cryo-Electron Microscopy, on the Titan Krios III (Beamline M06) – one of Diamond’s ‘super microscopes’ – to observe how the maximum size of a bound substrate is limited by a loop which shifts with helical twist after mutating a single amino acid.

Photo credit: Rebekka Stredwick; ©Diamond Light Source Ltd.

Observing the ‘loop’, ‘lock’ and ‘lid’: Dr Woodward describes their observations in terms of a ‘loop’, ‘lock’ and ‘lid’: the size of the binding pocket seems to be limited by a ‘lid’, which prevents long substrates from being converted. This lid is formed by a ‘loop’ which is held in position by electrostatic force. A single amino acid is responsible for maintaining this interaction – this is referred to as the ‘lock’. Other amino acids within the binding pocket interact with various chemical groups of the substrate and either allow it to bind or prevent it from binding. Two regions in particular are important for this effect. The three amino acids are: “the lock”, which defines the overall size of the substrate that can bind (either by tightening or loosening the lock by altering its chemical properties); and two others within the binding pocket, which interact with the substrate directly.

The insights gained allowed the team to semi-rationally design a new mutant nitrilase enzyme that produces biotechnologically interesting products, whether pharmaceuticals, fine chemicals or even food.

“We did this by identifying ‘hot spot’ amino acids for directed evolution and selecting them by coupling the survival of bacteria to the successful conversion of a library of substrates,” explains Dr Woodward, who had identified the most important of these residues ten years ago.

“Previously, at low low-resolution, we had seen large-scale changes,” Dr Mulelu adds “but we needed to answer the question: “How did this work? How did these translate into differences that could account for the ability of these enzymes to distinguish between substrates that differ by only one carbon atom? The Titan Krios III (beamline M06) housed at eBIC enabled us to answer our questions and see the enzyme at atomic scale resolution and to achieve these wonderful results.”

Dr Mulelu setting up the Vitrobot ® for preparing grids for cryo-Electron Microscopy. Photo credit: Rebekka Stredwick; ©Diamond Light Source Ltd.
Designer nitrilase enzymes

These results pave the way for further exciting research opportunities. Going forward, Dr Woodward’s ultimate aim is to produce a ‘catalogue’ of ‘designer’ nitrilases’ in a quest to find solutions through biotechnology which could lead to sustainable transformations in the lives and livelihoods of populations across Africa.

“My vision is to create a ‘catalogue’ of ‘designer nitrilases’ for any substrate by making appropriate changes to the helical twist as well as the binding pocket,” Dr Woodward says. “To achieve this, we would like to visualise a collection of key nitrilases with a range of different helical states (and substrate specificities). We are currently using computer modelling to predict binding energies and correlate these with known substrate specificities of various binding pocket mutants we have.”

Meeting the global challenges

The results achieved by the UCT team could play an important part in providing sustainable solutions in the future to meet the UN’s Sustainable Development Goals, from novel drug design and manufacturing to tackle communicable and non-communicable diseases, to pioneering ‘green’ biotechnology options for agricultural food security, industrial and mining waste treatment using enzyme-catalysed products.

Medical drug discovery and manufacturing

Dr Woodward is convinced that investments in new drug manufacturing methods may have a bigger impact on health on the African continent than the development of entirely new drugs,

”Africa has the worst burden of life-threatening communicable diseases in the world,” explains Dr Woodward. “According to the UN, 1.6 million people died from a combination of preventable and treatable diseases in 2015 – malaria, tuberculosis and HIV-related disease. A big problem in Africa, however, is access to medicines and not necessarily the development of new medicines. Part of the problem is that only 2% of the medicines used in Africa are manufactured in Africa, which has implications for the cost and accessibility of medicines.”

This is where nitrilase enzymes could provide solutions. Nitrilase enzymes are attractive biocatalysts for the synthesis of amides and carboxylic acids for use in the manufacture of drugs for major life-threatening communicable diseases, such as malaria, tuberculosis and HIV-related disease.

Drug discovery and testing for these diseases would also benefit from the economies of scale of an ‘off the shelf’ catalogue of nitrilases manufactured in Africa, making drugs for these diseases much more accessible and affordable.

“The ability to manipulate how nitrilases work,” Dr Mulelu points out, “could enable more cost-effective manufacturing of drugs for such diseases, leading to cheaper and more accessible medicines and this is welcome news for us in Africa and in other developing countries.

In addition, manufacturing locally has a variety of advantages including lowering costs of transport and improving local economic development. Nitrilases can offer ‘green’ alternatives because the reaction occurs at (close to) room temperature, neutral pH and atmospheric pressures so require less energy to produce. They are also very specific and so produce less waste.”

‘Green biotechnologies’ for agriculture and waste treatment

Looking beyond pharmaceuticals, nitrilases have a host of potential ‘green technology’ solutions to offer for cleaning up environmental pollution. One example is the breaking down of cyanide, which could revolutionise the way we clean hazardous mining dumps, reduce water pollution and improvements in the treatment of industrial waste and green manufacturing using enzyme-catalysed products.

“The nitrilase 4 mutant we have produced (described in the latest paper) can break down cyanide, forming products that can be further broken down by bacteria,” Dr Woodward explains. “This also applies to farming and food security. These compounds release cyanide and animals, especially ruminants, can suffer cyanide poisoning as a result but nitrilases with specificity towards cyanide could be used in a genetically modified strain of sorghum, which could break down the cyanide and make it safer.”

Developing a new generation of research leaders

A significant focus of the START programme focuses on capacity building and investing in the development of existing and future science talent across the continent, as well as funding Post-Doctoral Research Assistants and Fellows, and resourcing laboratories. For Dr Andani Mulelu, the impact of being a ‘Synchrotron Techniques for African Research and Technology (START) Postdoctoral Research Fellow’ has been significant, particularly in achieving results,

“During my honours year I became fascinated by the structure of helical nitrilases, especially those that detoxified cyanide, and I joined the group of Professor Trevor Sewell for my Masters and later my PhD. Working with Dr Jeremy Woodward and GCRF START enabled me to finally to realise my dream of visualising a nitrilase at atomic resolution and to solve the mystery of substrate selectivity in these enzymes.”

Dr Mulelu was subsequently offered a job as a research scientist at the H3D Drug Discovery and Development Centre (UCT) working on Malaria and Tuberculosis target-based drug discovery programmes, a move he attributes to the experience he gained as a START-funded post-doc.

Dr Andani Mulelu
Photo credit: Rebekka Stredwick; ©Diamond Light Source Ltd.

Ms Kirykowicz was a Masters student at the time the team achieved the successful results described in the Nature paper. Interested in applying the directed evolution techniques from her Honours year, Ms Kirykowicz’s role in the team involved working on nitrilase specificity, which gave her the skills she needed her future studies,

“The START-funded project gave me a good understanding of the methods needed to produce a well-sampled mutant library. I liked structural biology and ended up completing my Master’s on solving Mycobacterial protein complexes with Dr. Woodward. I am currently doing a PhD at the University of Cambridge in the UK working on solving the cryo-EM structure of a protein toxin transporter.”

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