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Characterization of ATP-dependent protein dynamics under native-like conditions
Umeå University, Faculty of Science and Technology, Department of Chemistry.ORCID iD: 0000-0002-8795-6309
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins are biological macromolecules capable of accelerating biochemical reactions. To accomplish this, proteins undergo changes in their molecular structure. Advances in structural biology have resulted in ever-increasing numbers of high-resolution protein structures. However, the majority of transient intermediate states will not amendable with traditional structural determination methods. Therefore, understanding how protein structural changes are correlated with the biological function necessitates development of methods that characterize the reaction in the native environment. P-type ATPase membrane transporters and the adenylate kinase (AK) are two ATP-dependent proteins that undergo extensive conformational change in their reaction cycles. While P-type ATPases maintain concentration gradients of ions across the cellular membranes, AK regulates cellular energy homeostasis by catalyzing interconversion of nucleotides. Resolving P-type ATPase and AK temporal and spatial structural dynamics is crucial to understand how these proteins are triggered by ATP for functionality. 

To pave way for time-resolved X-ray characterization of ATP-dependent conformational changes, it was necessary to identify optimal conditions for triggering protein reactions. Therefore, time-dependent Fourier-Transform Infra-Red (FTIR) spectroscopy of a recombinant Zn2+-transporting ATPase was used to optimize activation by photolysis of caged ATP. These conditions were then used to track structural dynamics of the Ca2+-transporting sarcoplasmic reticulum ATPase (SERCA) in skeletal muscle native membranes. Fast single-cycle dynamics were registered with the formation of an intermediate state at 1.5 ms followed by steady-state accumulation at 13 ms. The molecular dynamic (MD)-based structural refinement procedure showed that the 13-ms transient intermediate represented an ADP-sensitive, phosphorylated Ca2+-bound E1 state (Ca2E1P), with a domain arrangement that has so far eluded structural characterization.

MD simulations of the identified SERCA transient intermediates further finetuned their positions in the reaction cycle. The 1.5-ms state was assigned to an ATP-bound state prior to phosphorylation, while the 13-ms state was stable in its Ca2E1P conformation. Because the simulations were performed in multicomponent lipid bilayers mimicking the native membrane, specific state-dependent lipid interactions were also identified. 

Finally, the wider applicability of the time-resolved X-ray method to study ATP-dependent protein dynamics was demonstrated by tracking AK structural dynamics. A transient intermediate at 5 ms was identified that showed closing of the ATP-binding domain prior to the NMP-binding domain, in the presence of both ATP and AMP substrates. This study provided conclusive experimental proof of the relative ordering of domain closure that had been predicted by several computational studies.In summary, the work presented in this thesis has contributed to developing the time-resolved X-ray method to study the structural dynamics of ATP-dependent proteins.

Abstract [sv]

Populärvetenskaplig sammanfattning

Proteiner är biologiska makromolekyler vars arbetsuppgifter i cellen möjliggör liv. Föratt kunna utföra sina tillordnade funktioner måste proteinets struktur i många fallförändras. Denna inneboende dynamik är inkodad i proteinets aminosyrasekvens ochhar optimerats genom evolutionsprocessen. I många fall finns det molekyler som kanstarta igång ett visst proteins funktion. En sådan molekyl är adenosintrifosfat (ATP)som är en organisk förening som tillhandahåller energi till ATP-beroende proteiner.Ett exempel är P-typs ATPaser som är proteiner insprängda i cellens membran somtransporterar olika joner. Denna transport möjliggörs av proteinets strukturellaförändringar som sätts igång av energi tillförd av ATP molekylen. Även proteinet somreglerar tillgången av ATP i cellen genomgår stora konformationsförändringar. Dettaprotein är adenylatkinas (AK) och katalyserar interkonversion av nukleotider. För attförstå hur dessa proteiner kan startas igång av ATP molekylen är det därför viktigt attbestämma hur deras strukturer förändras över tid till följd av tillgång på ATP.

För P-typs ATPaser har ett stort antal strukturer av olika intermediära tillståndbestämts främst genom röntgenkristallografi. Dock så kommer det finnas instabilatillstånd som aldrig kommer att kunna fångas av traditionellastrukturbestämningsmetoder. Utöver detta så är det möjligt att proteinet inte kangenomgå sina naturliga strukturförändringar i en proteinkristall. För ettmembranprotein är dessutom funktionen beroende av dess omgivande lipidmembran.För att bättre förstå proteinets funktion är det därför viktigt att försöka studera denaktuella processen under förhållanden så nära som möjligt till de fysiologiskaförhållandena som råder i cellen.

Denna avhandling syftade till att vidareutveckla en existerande tidsupplöströntgenspridningsteknik (TR-XSS) för att karakterisera ATP-beroendeproteindynamik i lösning. För att erhålla en tillräckligt stark signal i dessa experimentmåste en stor del av proteinerna i provet utföra sin funktion samtidigt. Detta kanuppnås genom laseraktivering. Vi använde laseraktivering av en burförening av inaktivATP för att starta transportreaktionen för en zinktransportör från den patogenabakterien Shigella sonnei. Genom att följa signalen från ATP hydrolys med infrarödspektroskopi hittades lämpliga betingelser för TR-XSS synkrotronexperiment.Förutom att bana vägen för tidsupplösta röntgenförsök är kinetiken ochstrukturdynamiken av intresse eftersom denna zinktransportör är vanligtförekommande hos patogena bakterier men inte hos människor och därför utgör ettpotentiellt målprotein för utveckling av nya antibiotika.

Vid utvecklingen av TR-XSS-metoden användes laseraktivering av en ATP burföreningför att studera en strukturellt väl-karakteriserad kalciumtransportör (SERCA) i dessnaturliga membranmiljö istället för i proteinkristaller. Experimenten identifierade ettintermediärt tillstånd vid 1.5 millisekunder och ackumuleringen av ett annat hastighetsbegränsande intermediärt tillstånd vid 13 millisekunder. Med hjälp avmolekyldynamikdatorsimuleringar (MD) bestämdes sedan strukturen av dethastighetsbegränsande tillståndet i en konformation som ännu inte hade observeratsmed traditionella strukturbestämningsmetoder. MD-simulering avstrukturmodellerna erhållna från TR-XSS-metoden i membranmodeller somimiterade den naturliga lipidsammansättningen finjusterade placeringen av debestämda tillstånden i SERCA-reaktionscykeln samt karakteriserade deras interaktionmed de omgivande lipiderna.

Den utvecklade TR-XSS-metoden användes sedan för att studera ATP-beroendeproteindynamik för AK. Resultaten visade experimentellt den relativa ordningen på destrukturella förändringar som proteinet genomgår som respons på ATP tillgång. Dettavisar på bredden hos den utvecklade röntgenmetoden. Dessutom finns flera olika typerav burföreningar tillgängliga, såsom metaboliter, joner, och signalsubstanser, vilketytterligare öppnar upp för möjliga målproteiner.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2020. , p. 76
Keywords [en]
proteins, membrane proteins, structural dynamics, ATPases, MD simulations, X-ray scattering, FTIR spectroscopy, ATP, TR-XSS, structural biology.
National Category
Biophysics
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-170649ISBN: 978-91-7855-302-0 (print)ISBN: 978-91-7855-303-7 (electronic)OAI: oai:DiVA.org:umu-170649DiVA, id: diva2:1429878
Public defence
2020-06-08, Lilla Hörsalen KBE301, KBC, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2020-05-18 Created: 2020-05-12 Last updated: 2020-05-18Bibliographically approved
List of papers
1. Probing the activity of a recombinant Zn2+-transporting P-type ATPase
Open this publication in new window or tab >>Probing the activity of a recombinant Zn2+-transporting P-type ATPase
2018 (English)In: Biopolymers, ISSN 0006-3525, E-ISSN 1097-0282, Vol. 109, no 2, article id e23087Article in journal (Refereed) Published
Abstract [en]

P‐type ATPase proteins maintain cellular homeostasis and uphold critical concentration gradients by ATP‐driven ion transport across biological membranes. Characterization of single‐cycle dynamics by time‐resolved X‐ray scattering techniques in solution could resolve structural intermediates not amendable to for example crystallization or cryo‐electron microscopy sample preparation. To pave way for such time‐resolved experiments, we used biochemical activity measurements, Attenuated Total Reflectance (ATR) and time‐dependent Fourier‐Transform Infra‐Red (FTIR) spectroscopy to identify optimal conditions for activating a Zn2+‐transporting Type‐I ATPase from Shigella sonnei (ssZntA) at high protein concentration using caged ATP. The highest total activity was observed at a protein concentration of 25 mg/mL, at 310 K, pH 7, and required the presence of 20% (v/v) glycerol as stabilizing agent. Neither the presence of caged ATP nor increasing lipid‐to‐protein ratio affected the hydrolysis activity significantly. This work also paves way for characterization of recombinant metal‐transporting (Type‐I) ATPase mutants with medical relevance.

Place, publisher, year, edition, pages
John Wiley & Sons, 2018
Keywords
FTIR spectroscopy, membrane protein, membrane transport, P-type ATPases
National Category
Biophysics
Research subject
Biochemistry
Identifiers
urn:nbn:se:umu:diva-170614 (URN)10.1002/bip.23087 (DOI)000428629000003 ()
Available from: 2020-05-12 Created: 2020-05-12 Last updated: 2020-05-14Bibliographically approved
2. Tracking Ca2+ ATPase intermediates in real time by x-ray solution scattering
Open this publication in new window or tab >>Tracking Ca2+ ATPase intermediates in real time by x-ray solution scattering
Show others...
2020 (English)In: Science Advances, E-ISSN 2375-2548, Vol. 6, no 12, article id eaaz0981Article in journal (Refereed) Published
Abstract [en]

Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) transporters regulate calcium signaling by active calcium ion reuptake to internal stores. Structural transitions associated with transport have been characterized by x-ray crystallography, but critical intermediates involved in the accessibility switch across the membrane are missing. We combined time-resolved x-ray solution scattering (TR-XSS) experiments and molecular dynamics (MD) simulations for real-time tracking of concerted SERCA reaction cycle dynamics in the native membrane. The equilibrium [Ca-2] E1 state before laser activation differed in the domain arrangement compared with crystal structures, and following laser-induced release of caged ATP, a 1.5-ms intermediate was formed that showed closure of the cytoplasmic domains typical of E1 states with bound Ca2+ and ATP. A subsequent 13-ms transient state showed a previously unresolved actuator (A) domain arrangement that exposed the ADP-binding site after phosphorylation. Hence, the obtained TR-XSS models determine the relative timing of so-far elusive domain rearrangements in a native environment.

Place, publisher, year, edition, pages
American Association for the Advancement of Science, 2020
National Category
Atom and Molecular Physics and Optics
Identifiers
urn:nbn:se:umu:diva-169875 (URN)10.1126/sciadv.aaz0981 (DOI)000521937000029 ()32219166 (PubMedID)
Available from: 2020-04-29 Created: 2020-04-29 Last updated: 2020-06-05Bibliographically approved
3. Stability and lipid-interaction in transient Ca2+ P-type ATPase (SERCA1a) intermediates
Open this publication in new window or tab >>Stability and lipid-interaction in transient Ca2+ P-type ATPase (SERCA1a) intermediates
(English)Manuscript (preprint) (Other (popular science, discussion, etc.))
Identifiers
urn:nbn:se:umu:diva-170713 (URN)
Available from: 2020-05-13 Created: 2020-05-13 Last updated: 2020-05-13
4. Tracking ATP binding conformational change in Adenylate kinase using time-resolved X-ray solution scattering
Open this publication in new window or tab >>Tracking ATP binding conformational change in Adenylate kinase using time-resolved X-ray solution scattering
(English)Manuscript (preprint) (Other academic)
National Category
Biophysics
Research subject
Biochemistry
Identifiers
urn:nbn:se:umu:diva-170647 (URN)
Available from: 2020-05-12 Created: 2020-05-12 Last updated: 2020-05-14

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Ravishankar, Harsha

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