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Constant-pH Molecular Dynamics and Applications
KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics. (Hess)
2024 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Although it has been long known that pH plays an important role in biology, it was only in the 20th century that people first realized that pH can greatly affect the structure and function of proteins on a molecular level. A commonly encountered problem when studying the effect of pH in proteins is that experimental techniques such as X-ray crystallography and cryogenic electron microscopy often lack the resolution required to determine the protonation states of titratable residues. Even with neutron diffraction methods, which can theoretically resolve protons, the electrostatic environment in a crystal or grid does not necessarily correspond to the cellular environment. Where experimental methods fall short, in silico approaches such as molecular dynamics (MD) simulations can provide additional insight. However, pH is not typically captured dynamically in classical MD simulations. Rather, the protonation states of titratable sites such as aspartic and glutamic acid are usually set at the start of the simulation and remain constant throughout the run. Despite significant efforts in theMDfield to model protonation more realistically, a comprehensive and efficient constant-pH implementation in GROMACS has so far been lacking.

The aim of this work was to implement a continuous constant-pH method in GROMACS, to improve the ease of use of such methodology for the GROMACS user community, and to apply such methodology to study pH-gated ion channels. To this end, the work presented in this thesis can be divided into three parts. First, in papers I and II, I discuss the implementation of a λ-dynamics-based constant-pH algorithm in GROMACS, including several important methodological aspects. Next, in paper III, I present phbuilder, a Python-based simulation builder tool that automates the often complicated, tedious, and error-prone set up process of constant-pH simulations in GROMACS. Finally, in papers IV and V, I investigate how the constant-pH method can be used to better understand the pH gating mechanism in the bacterial GLIC and sTeLIC ligand-gated ion channels.

The work presented in this thesis has contributed to both the methodological and application sides in the field of MD. I have helped to develop and implement a λ-dynamics based constant-pH method in GROMACS, and I have made such methodology available to the broader GROMACS user community by providing a tool that automates its setup process. On the application side, I have demonstrated the feasibility of the constant-pH implementation for studying large and complex protein systems, such as ion channels, and furthered our understanding of pH-mediated gating in GLIC and sTeLIC.

Abstract [sv]

Även om det länge har varit känt att pH spelar en viktig roll inom biologin, var det först under 1900-talet som människor insåg att pH kan ha stor påverkan på vilken struktur och funktion proteiner har på molekylär nivå. Ett vanligt problem vid studier av pHs effekt på proteiner är att experimentella tekniker, såsom röntgenkristallografi och kryoelektronmikroskopi, ofta saknar den upplösning som krävs för att bestämma protoneringsstadierna hos titrerbara aminosyror. Även med neutronspridningsmetoder, som teoretiskt sett har tillräcklig upplösning för att detektera enskilda protoner, motsvarar inte den elektrostatiska miljön i en kristall eller gitter nödvändigtvis den cellulära miljön. Där experimentella metoder inte räcker till kan in silico-tekniker som molekyldynamik (MD)-simuleringar ge ytterligare insikter. Dock fångas pH normalt sett inte dynamiskt in i klassiska MD-simulationer. Istället sätts protoneringsstadierna för titrerbara platser som asparagin- och glutaminsyra vanligtvis i början av simulationen och förblir konstanta under hela simulationen. Trots betydande ansträngningar inom MD-området för att modellera protonering mer realistiskt har en heltäckande och effektiv konstant-pH-implementering i GROMACS hittills saknats.

Syftet med detta arbete var att implementera en kontinuerlig konstant-pH-metod i GROMACS, att förbättra användarvänligheten för sådan metodik för GROMACS-användare och att tillämpa en sådan metodik för att studera pH-styrda jonkanaler. Utifrån detta kan arbetet som presenteras i denna avhandling delas in i tre delar. Först diskuterar jag i artikel I och II implementeringen av en λ-dynamikbaserad konstant-pH-algoritm i GROMACS, samt flera viktiga metodologiska aspekter. Därefter presenterar jag i artikel III phbuilder, ett Python-baserat verktyg för simuleringsgenerering som automatiserar den ofta komplicerade, omständliga och felbenägna processen att konfigurera konstant-pH-simulationer i GROMACS. Slutligen undersöker jag i artikel IV och V hur konstant-pH-metoden kan användas för att bättre förstå pH-styrningsmekanismen hos de bakteriella jonkanalerna GLIC och sTeLIC.

Arbetet som presenteras i denna avhandling har bidragit till både metodologiska och tillämpade aspekter inom MD-området. Jag har hjälpt till att utveckla och implementera en λ-dynamikbaserad konstant-pH-metod i GROMACS, och jag har gjort en sådan metodik tillgänglig för den bredare GROMACS-användargruppen genom att tillhandahålla ett verktyg som automatiserar dess installationsprocess. På den tillämpade sidan har jag visat genomförbarheten av konstant-pH-implementeringen för att studera stora och komplexa proteinsystem, såsom jonkanaler, och vidareutvecklat vår förståelse för pH-medierad gating hos GLIC och sTeLIC.

Abstract [nl]

Hoewel het al lange tijd bekend is dat pH een belangrijke rol speelt in de biologie, realiseerde men zich pas in de 20e eeuw voor het eerst dat pH een grote invloed kan hebben op de structuur en functie van eiwitten op moleculair niveau. Een veelvoorkomend probleem bij het bestuderen van het effect van pH op eiwitten is dat experimentele technieken zoals röntgenkristallografie en cryogene elektronenmicroscopie vaak niet de benodigde resolutie bieden om de protonatietoestanden van titreerbare aminozuren te bepalen. Zelfs met neutrondiffractiemethoden, die in theorie protonen kunnen onderscheiden, komt de elektrostatische omgeving in een kristal of rooster niet noodzakelijk overeen met de celomgeving. Waar experimentele methoden tekortschieten, kunnen molecular dynamics (MD) simulaties aanvullend inzicht bieden. pH wordt echter doorgaans niet dynamisch vastgelegd in traditionele MD-simulaties. In plaats daarvan worden de protonatietoestanden van titreerbare aminozuren meestal aan het begin van de simulatie ingesteld en blijven constant tijdens de run. Ondanks aanzienlijke inspanningen in het MD-veld om protonatietoestanden realistischer te modelleren, ontbrak tot nu toe een moderne en efficiënte constant-pH-implementatie in GROMACS.

Het doel van dit proefschrift was om een continue constant-pH-methode te implementeren in GROMACS, om de gebruiksvriendelijkheid voor de GROMACS gebruikersgemeenschap te verbeteren, en om deze methodologie toe te passen om pH-gemoduleerde ionkanalen te bestuderen. Eerst bespreek ik in papers I en II de implementatie van een λ-dynamica-gebaseerd constant-pH-algoritme in GROMACS, inclusief verschillende belangrijke methodologische aspecten. Vervolgens presenteer ik in paper III phbuilder, een op Python gebaseerde system builder tool die het vaak ingewikkelde, tijdrovende en foutgevoelige instelproces van constant-pH-simulaties in GROMACS automatiseert. Ten slotte onderzoek ik in papers IV en V hoe de constant-pH-methode kan worden gebruikt om de pH-modulatie in de bacteriële GLIC- en sTeLIC-ligandgestuurde ionkanalen beter te begrijpen.

Het werk in dit proefschrift heeft bijgedragen aan zowel de methodologische als de toepassingsgerichte aspecten op het gebied van MD. Ik heb geholpen bij de ontwikkeling en implementatie van een λ-dynamica-gebaseerde constant-pH-methode in GROMACS, en ik heb deze methodologie beschikbaar gemaakt voor de GROMACS-gebruikersgemeenschap door een tool te ontwikkelen die het instelproces automatiseert. Aan de toepassingskant heb ik de haalbaarheid van de constant-pH-implementatie aangetoond voor het bestuderen van grote en complexe eiwitsystemen zoals ionkanalen, en ons begrip van de pH-gating in GLIC en sTeLIC vergroot.

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2024. , p. 151
Series
TRITA-SCI-FOU ; 2024:21
National Category
Biophysics
Research subject
Biological Physics
Identifiers
URN: urn:nbn:se:kth:diva-346447ISBN: 978-91-8040-908-7 (print)OAI: oai:DiVA.org:kth-346447DiVA, id: diva2:1857880
Public defence
2024-05-31, F3 (Flodis), Lindstedtsvägen 26, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

QC 2024-05-16

Available from: 2024-05-16 Created: 2024-05-14 Last updated: 2024-05-21Bibliographically approved
List of papers
1. Scalable Constant pH Molecular Dynamics in GROMACS
Open this publication in new window or tab >>Scalable Constant pH Molecular Dynamics in GROMACS
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2022 (English)In: Journal of Chemical Theory and Computation, ISSN 1549-9618, E-ISSN 1549-9626, Vol. 18, no 10, p. 6148-6160Article in journal (Refereed) Published
Abstract [en]

Molecular dynamics (MD) computer simulations are used routinely to compute atomistic trajectories of complex systems. Systems are simulated in various ensembles, depending on the experimental conditions one aims to mimic. While constant energy, temperature, volume, and pressure are rather straightforward to model, pH, which is an equally important parameter in experiments, is more difficult to account for in simulations. Although a constant pH algorithm based on the λ-dynamics approach by Brooks and co-workers [Kong, X.; Brooks III, C. L. J. Chem. Phys.1996, 105, 2414–2423] was implemented in a fork of the GROMACS molecular dynamics program, uptake has been rather limited, presumably due to the poor scaling of that code with respect to the number of titratable sites. To overcome this limitation, we implemented an alternative scheme for interpolating the Hamiltonians of the protonation states that makes the constant pH molecular dynamics simulations almost as fast as a normal MD simulation with GROMACS. In addition, we implemented a simpler scheme, called multisite representation, for modeling side chains with multiple titratable sites, such as imidazole rings. This scheme, which is based on constraining the sum of the λ-coordinates, not only reduces the complexity associated with parametrizing the intramolecular interactions between the sites but also is easily extendable to other molecules with multiple titratable sites. With the combination of a more efficient interpolation scheme and multisite representation of titratable groups, we anticipate a rapid uptake of constant pH molecular dynamics simulations within the GROMACS user community.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2022
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-346446 (URN)10.1021/acs.jctc.2c00516 (DOI)000861595500001 ()36128977 (PubMedID)2-s2.0-85138860798 (Scopus ID)
Funder
Swedish Research Council, 2019-04477European Commission, H2020-INFRAEDI-02-2018-823830Academy of Finland, 311031Academy of Finland, 332743
Note

QC 20240515

Available from: 2024-05-14 Created: 2024-05-14 Last updated: 2024-05-15Bibliographically approved
2. Best Practices in Constant pH MD Simulations: Accuracy and Sampling
Open this publication in new window or tab >>Best Practices in Constant pH MD Simulations: Accuracy and Sampling
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2022 (English)In: Journal of Chemical Theory and Computation, ISSN 1549-9618, E-ISSN 1549-9626, Vol. 18, no 10, p. 6148-6160Article in journal (Refereed) Published
Abstract [en]

Various approaches have been proposed to include the effect of pH in molecular dynamics (MD) simulations. Among these, the A-dynamics approach proposed by Brooks and co-workers [Kong, X.; Brooks III, C. L. J. Chem. Phys. 1996, 105, 2414-2423] can be performed with little computational overhead and hfor each typeence be used to routinely perform MD simulations at microsecond time scales, as shown in the accompanying paper [Aho, N. et al. J. Chem. Theory Comput. 2022, DOI: 10.1021 /acs.jctc.2c00516]. At such time scales, however, the accuracy of the molecular mechanics force field and the parametrization becomes critical. Here, we address these issues and provide the community with guidelines on how to set up and perform long time scale constant pH MD simulations. We found that barriers associated with the torsions of side chains in the CHARMM36m force field are too high for reaching convergence in constant pH MD simulations on microsecond time scales. To avoid the high computational cost of extending the sampling, we propose small modifications to the force field to selectively reduce the torsional barriers. We demonstrate that with such modifications we obtain converged distributions of both protonation and torsional degrees of freedom and hence consistent pK(a) estimates, while the sampling of the overall configurational space accessible to proteins is unaffected as compared to normal MD simulations. We also show that the results of constant pH MD depend on the accuracy of the correction potentials. While these potentials are typically obtained by fitting a low-order polynomial to calculated free energy profiles, we find that higher order fits are essential to provide accurate and consistent results. By resolving problems in accuracy and sampling, the work described in this and the accompanying paper paves the way to the widespread application of constant pH MD beyond pK(a) prediction.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2022
National Category
Theoretical Chemistry
Identifiers
urn:nbn:se:kth:diva-322879 (URN)10.1021/acs.jctc.2c00517 (DOI)000897361300001 ()36107791 (PubMedID)2-s2.0-85138973066 (Scopus ID)
Note

QC 20230126

Available from: 2023-01-26 Created: 2023-01-26 Last updated: 2024-06-20Bibliographically approved
3. phbuilder: A Tool for Efficiently Setting up Constant pH Molecular Dynamics Simulations in GROMACS
Open this publication in new window or tab >>phbuilder: A Tool for Efficiently Setting up Constant pH Molecular Dynamics Simulations in GROMACS
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2024 (English)In: Journal of Chemical Information and Modeling, ISSN 1549-9596, E-ISSN 1549-960X, Vol. 64, no 3, p. 567-574Article in journal (Refereed) Published
Abstract [en]

Constant pH molecular dynamics (MD) is a powerful technique that allows the protonation state of residues to change dynamically, thereby enabling the study of pH dependence in a manner that has not been possible before. Recently, a constant pH implementation was incorporated into the GROMACS MD package. Although this implementation provides good accuracy and performance, manual modification and the preparation of simulation input files are required, which can be complicated, tedious, and prone to errors. To simplify and automate the setup process, we present phbuilder, a tool that automatically prepares constant pH MD simulations for GROMACS by modifying the input structure and topology as well as generating the necessary parameter files. phbuilder can prepare constant pH simulations from both initial structures and existing simulation systems, and it also provides functionality for performing titrations and single-site parametrizations of new titratable group types. The tool is freely available at www.gitlab.com/gromacs-constantph. We anticipate that phbuilder will make constant pH simulations easier to set up, thereby making them more accessible to the GROMACS user community.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2024
National Category
Other Electrical Engineering, Electronic Engineering, Information Engineering
Identifiers
urn:nbn:se:kth:diva-343938 (URN)10.1021/acs.jcim.3c01313 (DOI)001161607500001 ()38215282 (PubMedID)2-s2.0-85182581195 (Scopus ID)
Note

QC 20240305

Available from: 2024-03-05 Created: 2024-03-05 Last updated: 2024-05-14Bibliographically approved
4. Constant-pH Molecular Dynamics Simulations of Closed and Open States of a Proton-gated Ion Channel
Open this publication in new window or tab >>Constant-pH Molecular Dynamics Simulations of Closed and Open States of a Proton-gated Ion Channel
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Although traditional molecular dynamics simulations successfully capture a variety of di erent molecular interactions, the protonation states of titratable residues are kept static. A recent constant-pH molecular dynamics implementation in the GROMACS package allows pH e ects to be captured dynamically, and promises to provide both the accuracy and computational performance required for studying pH-mediated conformational dynamics in large, complex systems containing hundreds of titratable residues. Here, we demonstrate the applicability of this constant-pH implementation by simulating the proton-gated ion channel GLIC at resting and activating pH, starting from closed and open structures. Our simulations identify residues E26 and E35 as especially pH-sensitive and reveal state-dependent pKa shifts at multiple residues, as well as side chain and domain rearrangements in line with the early stages of gating. Our results are consistent with several previous experimental  ndings, demonstrating the applicability of constant-pH simulations to elucidate pH-mediated activation mechanisms in multidomain membrane proteins, likely extensible to other complex systems.

National Category
Natural Sciences
Identifiers
urn:nbn:se:kth:diva-346443 (URN)10.1101/2023.11.30.569372 (DOI)
Note

QC 20240515

Available from: 2024-05-14 Created: 2024-05-14 Last updated: 2024-05-20Bibliographically approved
5. Molecular Dynamics of an Alkaline-gated Ion Channel Using Constant-pH Simulations
Open this publication in new window or tab >>Molecular Dynamics of an Alkaline-gated Ion Channel Using Constant-pH Simulations
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Ligand-gated ion channels are critical to electrochemical signal transduction in many organisms, yet the molecular details of their activation and modulation remain unclear. A recently reported bacterial membrane receptor, sTeLIC, constitutes a promising model system to study structure, function, and regulation in the pentameric ligand-gated ion channel family. Along with several of its mammalian homologs, sTeLIC is sensitive to extracellular pH, specifically activating under alkaline conditions. However, experimental structures of sTeLIC do not allow direct observation of protonation states or other pH-driven dynamics. Here, we employed a recent implementation of constant-pH molecular dynamics to simulate open and closed states of sTeLIC under both resting and activating conditions. Despite the acidic pKa of glutamate side chains in solution, pH- and state-dependent changes in protonation were particularly observed at residues E106 in the extracellular vestibule and E160 at the intersubunit interface. Rearrangements in these regions were associated with coordinated contraction of the extracellular domain and expansion of the transmembrane domain. Together, our results demonstrate the applicability of constant-pH simulations to support a testable mechanistic model for pH-gating, potentially extensible to a range of physiologically relevant systems.

National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-346444 (URN)
Note

QC 20240515

Available from: 2024-05-14 Created: 2024-05-14 Last updated: 2024-05-20Bibliographically approved

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