Structural and functional characterisation of the fungal plasma membrane proton pump, Pma1, and related P-Type ATPases
<p>P-type ATPases are a large family of ubiquitous ion pumps that carry out fundamental biological processes, ranging from generating membrane potentials to muscle contraction.</p> <p>Part I of this thesis focuses on the fungal plasma membrane proton pump, Pma1. Pma1 is responsibl...
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| Other Authors: | |
| Format: | Thesis |
| Language: | English |
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2022
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| _version_ | 1797108908840452096 |
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| author | Geurts, MMG |
| author2 | Bublitz, M |
| author_facet | Bublitz, M Geurts, MMG |
| author_sort | Geurts, MMG |
| collection | OXFORD |
| description | <p>P-type ATPases are a large family of ubiquitous ion pumps that carry out fundamental biological processes, ranging from generating membrane potentials to muscle contraction.</p>
<p>Part I of this thesis focuses on the fungal plasma membrane proton pump, Pma1. Pma1 is responsible for generating the proton-motive force that drives nutrient import into the cell via secondary active transporters. Uniquely, Pma1 forms ring shaped hexamers in the plasma membrane and is regulated through C- and N-terminal extensions. Due to its critical role in the cell, it poses as a target for novel antifungals against invasive mycoses. To date, the structure of Pma1 from Neurospora crassa has been determined in an autoinhibited state, and the structure from Saccharomyces cerevisiae has been determined in both an activated and autoinhibited state. However, whilst the structures have elucidated the molecular basis for function and regulation, they did not provide an understanding of the role of the surrounding lipids. In this thesis, N. crassa Pma1 was studied in a native-like membrane using coarse-grained molecular dynamic (MD) simulations. The results explain Pma1’s dependency on anionic lipids such as phosphatidylserine and phosphatidylinositol ceramide but also reveal a further understanding of how Pma1 cooperatively interacts with the membrane: it induces a membrane deformation that could play a critical role in the mechanism and energetics of proton pumping. In addition to computational studies, the expression and purification of Pma1 was optimised, leading to inhibitory assays and preliminary cryo electron microscopy. Finally, first steps toward a recombinant expression system for purification of N. crassa Pma1 from yeast have been made.</p>
<p>Part II of this thesis addresses the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). SERCA is the most widely studied and well-characterised member of the family, forming the basis of understanding other P-Type ATPases. Whilst being well characterised, the central residue, Glu340 and its mutant, E340A, have not been understood until now. Previous functional studies of the mutant showed that the mutation impacts the Ca2+-binding kinetics, but a E340A crystal structure did not explain this effect. By studying the wild-type, E340A and an in silico E340A structure of SERCA in a lipid membrane using all-atom MD, we found that the mutant has altered dynamics around both the Ca2+- and the ATP-binding sites. Altogether, the work allowed us to propose that E340A stabilises a more substrate-occluded state in SERCA. Conversely, the conserved Glu340 ensures a highly dynamic and coupled mechanism of action by aiding inter-domain communication between the head piece and transmembrane domain of SERCA.</p> |
| first_indexed | 2024-03-07T07:34:37Z |
| format | Thesis |
| id | oxford-uuid:3b9f20b3-5a6f-456c-a489-a5d6a1128d55 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T07:34:37Z |
| publishDate | 2022 |
| record_format | dspace |
| spelling | oxford-uuid:3b9f20b3-5a6f-456c-a489-a5d6a1128d552023-02-23T12:19:49ZStructural and functional characterisation of the fungal plasma membrane proton pump, Pma1, and related P-Type ATPasesThesishttp://purl.org/coar/resource_type/c_db06uuid:3b9f20b3-5a6f-456c-a489-a5d6a1128d55Molecular dynamicsBiochemistryMembrane proteinsStructural biologyEnglishHyrax Deposit2022Geurts, MMGBublitz, MSansom, MSauer, D<p>P-type ATPases are a large family of ubiquitous ion pumps that carry out fundamental biological processes, ranging from generating membrane potentials to muscle contraction.</p> <p>Part I of this thesis focuses on the fungal plasma membrane proton pump, Pma1. Pma1 is responsible for generating the proton-motive force that drives nutrient import into the cell via secondary active transporters. Uniquely, Pma1 forms ring shaped hexamers in the plasma membrane and is regulated through C- and N-terminal extensions. Due to its critical role in the cell, it poses as a target for novel antifungals against invasive mycoses. To date, the structure of Pma1 from Neurospora crassa has been determined in an autoinhibited state, and the structure from Saccharomyces cerevisiae has been determined in both an activated and autoinhibited state. However, whilst the structures have elucidated the molecular basis for function and regulation, they did not provide an understanding of the role of the surrounding lipids. In this thesis, N. crassa Pma1 was studied in a native-like membrane using coarse-grained molecular dynamic (MD) simulations. The results explain Pma1’s dependency on anionic lipids such as phosphatidylserine and phosphatidylinositol ceramide but also reveal a further understanding of how Pma1 cooperatively interacts with the membrane: it induces a membrane deformation that could play a critical role in the mechanism and energetics of proton pumping. In addition to computational studies, the expression and purification of Pma1 was optimised, leading to inhibitory assays and preliminary cryo electron microscopy. Finally, first steps toward a recombinant expression system for purification of N. crassa Pma1 from yeast have been made.</p> <p>Part II of this thesis addresses the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). SERCA is the most widely studied and well-characterised member of the family, forming the basis of understanding other P-Type ATPases. Whilst being well characterised, the central residue, Glu340 and its mutant, E340A, have not been understood until now. Previous functional studies of the mutant showed that the mutation impacts the Ca2+-binding kinetics, but a E340A crystal structure did not explain this effect. By studying the wild-type, E340A and an in silico E340A structure of SERCA in a lipid membrane using all-atom MD, we found that the mutant has altered dynamics around both the Ca2+- and the ATP-binding sites. Altogether, the work allowed us to propose that E340A stabilises a more substrate-occluded state in SERCA. Conversely, the conserved Glu340 ensures a highly dynamic and coupled mechanism of action by aiding inter-domain communication between the head piece and transmembrane domain of SERCA.</p> |
| spellingShingle | Molecular dynamics Biochemistry Membrane proteins Structural biology Geurts, MMG Structural and functional characterisation of the fungal plasma membrane proton pump, Pma1, and related P-Type ATPases |
| title | Structural and functional characterisation of the fungal plasma membrane proton pump, Pma1, and related P-Type ATPases |
| title_full | Structural and functional characterisation of the fungal plasma membrane proton pump, Pma1, and related P-Type ATPases |
| title_fullStr | Structural and functional characterisation of the fungal plasma membrane proton pump, Pma1, and related P-Type ATPases |
| title_full_unstemmed | Structural and functional characterisation of the fungal plasma membrane proton pump, Pma1, and related P-Type ATPases |
| title_short | Structural and functional characterisation of the fungal plasma membrane proton pump, Pma1, and related P-Type ATPases |
| title_sort | structural and functional characterisation of the fungal plasma membrane proton pump pma1 and related p type atpases |
| topic | Molecular dynamics Biochemistry Membrane proteins Structural biology |
| work_keys_str_mv | AT geurtsmmg structuralandfunctionalcharacterisationofthefungalplasmamembraneprotonpumppma1andrelatedptypeatpases |