Atomistic and Systems-level Modeling of Phosphate Catalysis

Lead Research Organisation: King's College London
Department Name: Chemistry


Phosphate processing enzymes are crucial to the majority of biochemical processes that all living cells depend on, including signal transduction, gene regulation, metabolism and energy transfer. The active sites of these enzymes are remarkably similar, in most cases requiring the presence of a catalytic Mg2+ ion bound to the phosphate groups, as the biological cofactor. However, there are distinct, specific differences in the coordination geometry of these enzymes that contain hidden information about the corresponding function.

To gain insight towards constructing a general, unifying theory of how phosphate processing enzymes modulate the rates of their catalytic reactions, we propose to study two important systems. We will study the RAF kinase catalytic reaction. RAF kinases are part of the ERK signalling pathway, which is aberrantly activated in >30% of all cancers, with RAS and RAF being the main oncogenic factors.

We will also study the catalytic reaction of a bacterial ligase, Ddl, from M. tuberculosis, the causative agent of TBC. Ddl is an essential enzyme in the biosynthesis of bacterial cell wall. It is one of the validated drug targets of the antibiotic drug Seromycine, and a major drug target against TBC. Seromycine is used as a second line of treatment, for multiple drug-resistant and extensively drug-resistant strains of M. tuberculosis.

Using our novel computational methods that can provide accurate free energies and kinetic information about the dynamics of the systems, we will identify coupled proton transfer steps that occur together with the phosphate cleavage reactions. We will use our calculated structures and mechanism to develop and test novel inhibitor design strategies.

Technical Summary

We will carry out computational studies of BRAF kinase and Ddl catalytic reactions using novel enhanced sampling algorithms, building on our recently developed accurate free energy calculation method that coupled Hamiltonian replica exchange with the finite temperature string method. We will continue to use MD and QM/MM calculations to study phosphate cleavage and transfer reactions that occur together with rate determining proton transfer processes. We will analyse the results from biased and unbiased dynamical trajectories using our newly developed Markov chain-based kinetic analysis method, DHAM. We will focus on the role of Mg2+ ions in the active site, and identify general features of this essential Mg2+ cofactor - using a systems-level approach - to better understand why magnesium is ubiquitously involved in the catalysis of phosphate cleavage and transfer catalytic reactions. We will use our calculated structures and mechanism to develop and test novel inhibitor design strategies.

Planned Impact

Phosphate processing enzymes are essential in all biological processes, therefore our research is potentially relevant to most biological research fields that study processes related to phosphate cleavage and transfer. For example, research related to regulation and signal transduction by kinases, gene expression and replication by polymerases or topoisomerases, ATPase function in molecular motors such as ATP synthase, myosin, transporters, and other activated mechanical, structural or chemical processes occurring in the cells that requires the energy transfer from ATP hydrolysis.
The proposed project is driven by computational molecular modelling-based data and methods, and it is embedded in an inter-disciplinary research environment. Our research results on applications and developments of molecular modelling methods (e.g., sampling enhanced methods for rare events, statistical analysis of stochastic simulation results to obtain free energies and molecular kinetics) are beneficial for a wide range of research fields related to atomistic computational modelling in general. This includes biomolecular simulations, as well as material science modelling, biotechnology, and synthetic biology modeling.

Public Sector, Business, Industry
On long term, health-related public sectors will benefit from basic research on structure and function of phosphate processing enzymes. Our study may be inspirational to a large number of projects targeting phosphate-processing enzymes that are relevant to many diseases. Phosphate processing enzymes are validated targets of a large number of drugs used in current clinical practices treating a wide range of diseases. These include reverse transcriptase and integrase inhibitors used against HIV and hepatitis B, proton pump inhibitors used in gastric diseases, kinase and topoisomerase inhibitors used in chemotherapy to treat cancers.
In particular, BRAF inhibitor drugs present recent examples for targeted cancer therapy: (
Another example is related to basic research at the US National Cancer Institute, which supports Federally Funded Research and Development Center (FFRDC) devoted to biomedical research. Within these efforts, the "Ras initiative" is announced as a major initiative and research priority to "improving treatment, diagnosis, and prevention of the many human cancers driven by mutant RAS genes" by studies of mutant Ras and downstream signalling proteins (including BRAF): Both Ras (GTPases) and downstream kinases are examples of enzymes processing NTP hydrolysis and transfer, the subject of this proposal.
Our second example, Ddl is a validated target in combating antimicrobial resistance, and Ddl inhibitors are used as second line of treatment drugs against tuberculosis. For example, mutation in the chromosomal Ddl gene, encoding a cytoplasm enzyme is directly involved in glycopeptides resistance.
Our basic research results are also relevant for charities linked to these diseases e.g., Cancer Research UK. In addition to drug design, insights related to controlling enzyme activity is also relevant for biotechnology industries, e.g., businesses developing industrial enzymes such as Novozymes.

General Public, Education
The general public, high school and university students will benefit from new basic research developments in general, by public lectures in the UK and world-wide (e.g., via the Alchemy Today seminar series of the Eotvos University, where I've been recently invited as a speaker), or by the Open Days at King's. My lab also hosted 6 high school students to date in the past 2 years, who were introduced to on-going research in my lab via the In2Science and Nuffield Research Placement programs.


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Huggins D (2019) Biomolecular simulations: From dynamics and mechanisms to computational assays of biological activity in Wiley Interdisciplinary Reviews: Computational Molecular Science

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Jambrina PG (2016) Phosphorylation of RAF Kinase Dimers Drives Conformational Changes that Facilitate Transactivation. in Angewandte Chemie (International ed. in English)

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Sharma V (2017) Insights into functions of the H channel of cytochrome oxidase from atomistic molecular dynamics simulations. in Proceedings of the National Academy of Sciences of the United States of America

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Sánchez-Sanz G (2016) MST2-RASSF protein-protein interactions through SARAH domains. in Briefings in bioinformatics

Description We have discovered structural features of RAF dimers that are linked to transactivation and the paradoxical activation of RAF inhibitor melanoma drugs to wild type RAF. Based on our study, we were able to propose a combination of drugs that synergistically enhance their inhibitory effects on RAF dimers, furthermore, also show effectiveness for the Ras-mutated MAPK pathway. We also analysed structural features of NTP hydrolysis enzymes, and discovered a novel Arginine finger for dUTPases, which is the first pyrophosphatase enzyme that has an essential Arginine finger.
Exploitation Route Drug discovery for melanoma and various other diseases where phosphate catalysis is essential.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

Description BioNet - Dynamical Redesign of Biomolecular Networks
Amount £1,184,999 (GBP)
Funding ID 757850 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 02/2018 
End 01/2023
Description Collaboration with Beata Vertessy 
Organisation Budapest University of Technology and Economics
Department Department of Biotechnology and Food Sciences
Country Hungary 
Sector Academic/University 
PI Contribution We initiated a collaborative project to studey the role of conserved arginine residues in the dUTPase enzyme. We first performed a PDB-side structural analysis to compare arginine residues in NTP cleaving enzymes. Subsequently, we carried out QM/MM and MD calculations to establish the function of the key arginine finger residue in dUTPases.
Collaborator Contribution In this collaboration the experimental group led by Beata Vertessy performed X-Ray crystallography experiments togeter with biochemical experiments to use this in our joint project.
Impact Nagy et al, Journal of the American Chemical Society, 2016, DOI: 10.1021/jacs.6b09012 Multidisciplinary collaboration with experimental X-Ray crystallography and biochemistry group and our theoretical and computational biophysical chemistry group.
Start Year 2014
Description collaboration with Walter Kolch 
Organisation University College Dublin
Country Ireland 
Sector Academic/University 
PI Contribution Computational MD simulations to reveal structure and dynamics of RAF kinases.
Collaborator Contribution Prof. Kolch's group carried out in vitro and in cell biochemical experiments to reveal RAF kinase activity and validate key mutations suggested by computational results.
Impact Jambrina et al, Angewandte Chemie, 2016, DOI: 10.1002/anie.201509272 Sanchez-Sanz et al, PLOS Computational Biology, 2016, DOI: 10.1371/journal.pcbi.1005051 Interdisciplinary research with essential biochemical and biological experiments from the Kolch group (Systems Biology Ireland) and computational work from our group.
Start Year 2012
Description Markov modelling and free energy calculation workshop 2016 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact About 50 participants attended the Markov modelling workshop we organised at King's College London, some of which were international scientists from India, Europe or the US. Several participants were from pharmaceutical companies, such as Novartis, GSK, and UCB Pharma. The workshop initiated further collaborations and research visits with both academic groups and pharma companies.
Year(s) Of Engagement Activity 2016