IMPROvER: the Integral Membrane Protein Stability Selector

Harborne, Steven P. D.; Strauss, Jannik; Boakes, Jessica C.; Wright, Danielle L.; Henderson, J. Gordon; Boivineau, Jacques; Jaakola, Veli-Pekka; Goldman, Adrian

doi:10.1038/s41598-020-71744-x

“…This might explain why despite the lower sequence similarity, we observe similar interactions with anionic lipids in the distal area. The distal lipid interaction sites may play a role in mPPase stability, as the region was identified during a case study of the IMPROvER (integral membrane protein stability selector) pipeline for stabilising mutations [ 43 ]. Further, three of the Cp- PPase residues identified by IMPROvER were found to interact with anionic lipids in our study (F 1.53 , R 3.67 and R 7.62 ).…”

Section: Discussionmentioning

confidence: 99%

mPPases create a conserved anionic membrane fingerprint as identified via multi-scale simulations

Holmes

¹

,

Goldman

²

,

Kalli

³

2022

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Membrane-integral pyrophosphatases (mPPases) are membrane-bound enzymes responsible for hydrolysing inorganic pyrophosphate and translocating a cation across the membrane. Their function is essential for the infectivity of clinically relevant protozoan parasites and plant maturation. Recent developments have indicated that their mechanism is more complicated than previously thought and that the membrane environment may be important for their function. In this work, we use multiscale molecular dynamics simulations to demonstrate for the first time that mPPases form specific anionic lipid interactions at 4 sites at the distal and interfacial regions of the protein. These interactions are conserved in simulations of the mPPases from Thermotoga maritima, Vigna radiata and Clostridium leptum and characterised by interactions with positive residues on helices 1, 2, 3 and 4 for the distal site, or 9, 10, 13 and 14 for the interfacial site. Due to the importance of these helices in protein stability and function, these lipid interactions may play a crucial role in the mPPase mechanism and enable future structural and functional studies.

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“…This might explain why despite the lower sequence similarity, we observe similar interactions with anionic lipids in the distal area. The distal lipid interaction sites may play a role in mPPase stability, as the region was identified during a case study of the IMPROvER (integral membrane protein stability selector) pipeline for stabilising mutations (Harborne et al, 2020). Further, three of the Cp -PPase residues identified by IMPROvER were found to interact with anionic lipids in our study (F 1.53 , R 3.67 and R 7.62 ).…”

Section: Discussionmentioning

confidence: 99%

mPPases create a conserved anionic membrane fingerprint as identified via multi-scale simulations

Holmes

¹

,

Goldman

²

,

Kalli

³

2022

Preprint

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Membrane-integral pyrophosphatases (mPPases) are membrane-bound enzymes responsible for hydrolysing inorganic pyrophosphate and translocating a cation across the membrane. Their function is essential for the infectivity of clinically relevant protozoan parasites and plant maturation. Recent developments have indicated that their mechanism is more complicated than previously thought and that the membrane environment may be important for their function. In this work, we use multiscale molecular dynamics simulations to demonstrate for the first time that mPPases form specific anionic lipid interactions at 4 sites at the distal and interfacial regions of the protein. These interactions are conserved in simulations of the mPPases from Thermotoga maritima, Vigna radiata and Clostridium leptum and characterised by interactions with positive residues on helices 1, 2, 3 and 4 for the distal site, or 9, 10, 13 and 14 for the interfacial site. Due to the importance of these helices in protein stability and function, these lipid interactions may play a crucial role in the mPPase mechanism and enable future structural and functional studies.Author summaryIn this work we have been able to demonstrate conservation of lipid-interaction sites on proteins from distinct species that deviated from their evolutionary common ancestors a long time ago, as in the case of the membrane-integral pyrophosphatases from a thermophilic bacteria species and a plant species studied here. This retention of a common lipid interaction profile or “fingerprint” and our ability to predict this for other proteins in this family may indicate that they are more integral to protein function than previously thought. By identifying lipid interactions that may act to stabilise the protein structure, these properties could be exploited to gain protein structures, and the interfacial site’s potential involvement in inter-subunit communication may be useful for further investigation of the catalytic cycle of this clinically relevant membrane protein family.

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“…Expression of hENT1 variants identified by IMPROvER ( Harborne et al, 2020 ) was achieved using the Bac-to-Bac® (Invitrogen) expression system in Sf9 cells. Expression cultures were set up using fresh mid-log phase Sf9 cells, diluted to a density of approximately 1.0 x 10 6 cells/mL in 15 ml pre-warmed Insect-XPRESS™ Protein-free Insect Cell Medium (Lonza).…”

Section: Methodsmentioning

confidence: 99%

“…However, to understand the mechanism fully, it is important to stabilise full-length transporter in different states. We have used the program IMPROvER for rational design of stabilising mutations in integral membrane proteins including hENT1 ( Harborne et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

Novel variants provide differential stabilisation of human equilibrative nucleoside transporter 1 states

Boakes

¹

,

Harborne²,

Ngo

³

et al. 2022

Front. Mol. Biosci.

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Human equilibrative nucleoside transporters represent a major pharmaceutical target for cardiac, cancer and viral therapies. Understanding the molecular basis for transport is crucial for the development of improved therapeutics through structure-based drug design. ENTs have been proposed to utilise an alternating access mechanism of action, similar to that of the major facilitator superfamily. However, ENTs lack functionally-essential features of that superfamily, suggesting that they may use a different transport mechanism. Understanding the molecular basis of their transport requires insight into diverse conformational states. Differences between intermediate states may be discrete and mediated by subtle gating interactions, such as salt bridges. We identified four variants of human equilibrative nucleoside transporter isoform 1 (hENT1) at the large intracellular loop (ICL6) and transmembrane helix 7 (TM7) that stabilise the apo-state (∆Tm 0.7–1.5°C). Furthermore, we showed that variants K263A (ICL6) and I282V (TM7) specifically stabilise the inhibitor-bound state of hENT1 (∆∆Tm 5.0 ± 1.7°C and 3.0 ± 1.8°C), supporting the role of ICL6 in hENT1 gating. Finally, we showed that, in comparison with wild type, variant T336A is destabilised by nitrobenzylthioinosine (∆∆Tm -4.7 ± 1.1°C) and binds it seven times worse. This residue may help determine inhibitor and substrate sensitivity. Residue K263 is not present in the solved structures, highlighting the need for further structural data that include the loop regions.

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IMPROvER: the Integral Membrane Protein Stability Selector

Cited by 10 publications

References 79 publications

mPPases create a conserved anionic membrane fingerprint as identified via multi-scale simulations

mPPases create a conserved anionic membrane fingerprint as identified via multi-scale simulations

mPPases create a conserved anionic membrane fingerprint as identified via multi-scale simulations

Novel variants provide differential stabilisation of human equilibrative nucleoside transporter 1 states

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