University of Groningen

The Role of Structural Dynamics in Protein Function and EvolvabilityMuthahari, Yusran

DOI:10.33612/diss.155494424

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Appendices

Supplementary InformationSamenvatting

SummaryAcknowledgement

| Supplementary Information and Files 158

Supplementary Information and Files

Appendices| 159

b. Sequence-based Phylogenetic Tree

| Supplementary Information and Files 160

c. Rigid Domains Only

Appendices| 161

d. Rigid Domains Only

| Supplementary Information and Files 162

e. C-tail Only

SFL1. Supplementary S1 – Full Phylogenetic Trees

Structure- and sequence-based phylogenetic trees of the 53 identified cherry-core proteins with

known structures. Summary of this tree is presented in Figure 2.1A and Figure S2.1B-D. Trees are

constructed after the entire polypeptide chain (A, B), the CC only (C) and the C-tail only (D).

Every protein is represented by its PDB ID code.

Appendices| 163

SFL2.A – Interaction Map of LTTRS’ Structure

| Supplementary Information and Files 164

SFL2.B - Interaction Map of CynR Models derived from LTTRS’ Structure

Appendices| 165

SFL2.C - Interaction Map of LTTRS’ Structure

| Supplementary Information and Files 166

SFL2.D - Interaction Map of CynR Models derived from LTTRS’ Structure

Appendices| 167

SFL2.E - Interaction Map of LTTRS’ Structure

| Supplementary Information and Files 168

SFL2.F - Interaction Map of CynR Models derived from LTTRS’ Structure

SFL2. Supplementary S2 –Interaction of CynR connecting loop/dimerization helix with the CC.

The cynR sequence was modelled after the known oligomeric high-resolution structures (see Figure

4.2). The contact interfaces between the dimerization helix and the CC derived from the structures

(A,C,E) or from the corresponding models (B,D,F) were analyzed in the Protein Interaction

calculator server, using standard server settings. The residues forming such interface belong to the

connecting-loop or the dimerization helix (indicated as loop and helix respectively) and elements

of the CC (indicated according to the Figure 2.1). The nature of the interactions, hydrophobic,

aromatic, Cation-Pi, hydrogen-bonds, Ionic are represented with blue, cyan, green, orange and red

color respectively.

Appendices| 169

| Supplementary Information and Files 170

SFL3. Supplementary S3– Structure based phylogenetic trees of Class A Protein

Results as obtained from the Dali server. The structure based phylogenetic tree of class A proteins

with known oligomeric structures (see Figure 4.2). Top part contains full length class A

transcription factors (HTH motif/dimerization helix and CC) while bottom part, only the CC.

Appendices| 171

Samenvatting

Tijdens de evolutie worden 'peptiden' omgezet in 'eiwitten' door de specifieke functies op

een geavanceerde manier aan dergelijke biopolymeren te delegeren. Van alle beschikbare

conformeren moet de natuur een subset ervan selecteren om elk eiwit specifieke substraten te laten

herkennen. Tijdens de korte evolutieperiode kan een dergelijke functionele specialisatie van een

peptide met een vaste lengte getrouw worden verklaard door de theorie van de ontwikkelbaarheid

van eiwitten. Om de evolutie van de niet-vaste lengte peptiden die op een lange termijn kunnen

voorkomen te rationaliseren, hebben we ons gefocust op het uitbreiden van de huidige

evolueerbaarheidstheorie door het introduceren van de proteïne modulariteit in dit proefschrift.

Om het concept van eiwitmodulariteit volledig te begrijpen, hebben we in Hoofdstuk 1

een overzicht gegeven met de samenvatting van twee fundamentele studies: (i) de multi-tier

eiwitdynamiek en (ii) de evolueerbaarheid van eiwitten. Het hoofdstuk bevat ook een korte uitleg

van het energetische trechtermodel, omdat het ons in staat stelt om de evolutie van de proteïne-

energetica en -structuren op basis van functionele specialisatie te veronderstellen. Door deze

algemeen aanvaarde concepten uit te werken, zijn we in staat een werkhypothese te vormen over

de rol van de eiwitmodules, zoals we in het volgende Hoofdstuk 2 hebben gepresenteerd.

Hoofdstuk 2 geeft een generieke werkhypothese van dit proefschrift door de rol van de modules om

de functie van eiwitten tijdens de evolutie te specialiseren ter discussie te stellen. Om deze

hypothese te verifiëren, hebben we een grote groep eiwitten onderzocht die een gemeenschappelijke

'primordiale' structuur aannemen, namelijk de 'cherry-core' (CC). De CC geïdealiseerde consensus,

die is samengesteld uit twee continue Rossmann-fold Domeinen (D1 en D2) verbonden via twee

anti-parallelle β-sheet 'hinges' (βH1 en βH2), vertoont een opmerkelijke symmetrie die kan

bijdragen aan de conformationele variabiliteit en plasticiteit. Vervolgens hebben we voorgesteld dat

de geometrische plaatsing van de modules (d.w.z. C-terminaltoevoegingen) in de CC de meerlaagse

dynamiek op de CCP's mogelijk maakt.

Om de hypothese uit het vorige hoofdstuk te testen, hebben we grootschalige

domeinbewegingen onderzocht via single-molecule Förster Resonance Energy Transfer (smFRET)

en lokale structurele fluctuaties via Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS)

van de CCP's en hun derivaten. In Hoofdstuk 3 ontdekten we dat de modules van CCP's een

| Samenvatting 172

verschillende structurele dynamiek op meerdere niveaus aan de CC geven. Een dergelijke dynamiek

maakt de diversificatie van functie en ligandspecificiteit van de CCP's mogelijk. In dit hoofdstuk

hebben we ons gericht op het benadrukken van de rol van asymmetrische C-staart om tier-0

dynamiek te 'genereren' op SBD2 en MalE van respectievelijk Klasse B en G. Dergelijke eiwitten

zijn ABC-transporter-gerelateerde eiwitten, die twee verschillende conformaties vereisen om hun

verwante liganden te vangen (d.w.z. open in de apo- en gesloten in de holo-toestand). Door

specifieke interacties op de C-staart te verstoren, destabiliseren we de open apo-toestand en

vergroten we hun bindingsaffiniteit. Dergelijke resultaten bevestigen de rol van de modules van de

ABC-transporter-gerelateerde CCP's om de 'open' conformatie als de oorspronkelijke apo-toestand

te genereren.

De CC van de LysR Type Transcription Regulators (LTTR's) heeft geen extra C-staart-

module en werkt als het effector-bindende domein (EBD). We hebben aangetoond dat Tier-0-

dynamiek niet waarneembaar is op de EBD van CynR en mogelijk niet vereist is tijdens

effectorbinding om de quaternaire veranderingen te verspreiden. In Hoofdstuk 4 hebben we de

dynamiek van het lagere niveau die ten grondslag ligt aan de functie van CynR gevolgd door de

combinatie van structurele modellering en dynamica-studies (d.w.z. HDX-MS) te gebruiken. Hoewel

er geen significante structurele veranderingen zijn waargenomen op de CC die is gedetecteerd met

onze experimentele procedures, stelden we voor dat de accumulatieve dynamiek van de lage niveaus

mogelijk voldoende is om de quartaire veranderingen te initiëren die essentieel zijn voor het DNA-

(ont)buigen.

In Hoofdstuk 5 hebben we het effect van de modules op de structurele dynamiek van een

van de moderne CCP's bepaald, namelijk het Maltose Binding Protein (MalE). Zulke modules zijn

voornamelijk ingebed in de C-terminy en geïntegreerd in de geconserveerde structuur, meer als

vervangers van consensus helices / sheets, zoals beschreven in Hoofdstuk 2. We hebben de rol van

dergelijke modules geverifieerd met behulp van een reeks biofysische tools (bijv. SmFRET, HDX-

MS en Isothermal Titration Calorimetry) en MD-simulaties waarmee we het energetische landschap

van MalE konden reconstrueren. Door dergelijke modules te veranderen met behulp van

plaatsgerichte mutagenese, hebben we de synergetische rollen van de modules bevestigd, die

cruciaal zijn om MalE te personifiëren om te functioneren als een transportgerelateerd bindend

eiwit door (i) het ligand in te sluiten en (ii) een aparte apo-toestand te creëren.

Appendices| 173

We verwachtten dat de energetische argumenten tijdens de evolutie zouden kunnen

worden gezien als de belangrijkste selectiedruk. Om aan de energetische behoefte te voldoen, zou

de natuur modules kunnen introduceren in de hoog-evolueerbare eiwitkernen tijdens de lange

evolutieperiode, zoals we in dit proefschrift hebben gepresenteerd over het modulariteitsconcept.

Zodra de modules zijn 'gehecht', is evolutie op een peptide met vaste lengte voldoende om de

eiwitfuncties te optimaliseren. Het latere evolutieproces zou in een relatief kortere evolutieperiode

kunnen plaatsvinden, zoals vermeld in de ‘avant-garde’ evolueerbaarheidstheorie.

| Summary 174

Summary

During evolution 'peptides' are turned into 'proteins' by delegating the specific functions

to such biopolymers in a sophisticated manner. Out of all available conformers, nature has to select

a subset of them to allow each protein to recognize specific substrates. During the short

evolutionary period, such functional specialization of a fix-length peptide can be faithfully explained

by the protein evolvability theory. To rationalize the evolution of the non-fixed length peptides that

might occur upon a long-term period, we focused on expanding the current evolvability theory by

introducing the protein modularity notion in this thesis.

To fully comprehend the concept of protein modularity, we provided an overview

in Chapter 1 that includes the summary of two fundamental concepts: (i) the multi-tier protein

dynamics and (ii) the protein evolvability. The chapter also includes a short explanation of the

energetic funnel model, as it allows us to hypothesize the evolution of the protein energetics and

structures upon functional specialization. By elaborating on those widely-accepted concepts, we

are able to form a working hypothesis on the role of the protein modules, as we presented in the

subsequent Chapter 2.

Chapter 2 provides a generic working hypothesis of this thesis by questioning the modules'

role to specialize the function of proteins during evolution. To verify this hypothesis, we investigated

a large group of proteins adopting a common 'primordial' structure, i.e., the 'cherry-core' (CC).

The cherry-core idealized consensus, which is composed of two continuous Rossmann-fold topology

domains (D1 & D2) connected via two anti-parallel β-strand 'hinges' (βH1 & βH2), exhibits a

remarkable symmetry which may contribute to its conformational variability and plasticity. We

then proposed that the modules' geometrical placement (i.e., C-terminal additions) in the CC

enables the multi-tier dynamics on the CCPs.

To test the hypothesis stated in the previous chapter, we probed large-scale domain

motions via single-molecule Förster Resonance Energy Transfer (smFRET) and local structural

fluctuation via Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) of the CCPs and

their derivatives. In Chapter 3, we found that the modules of CCPs confer distinct multi-tier

structural dynamics to the CC. Such dynamics allow the diversification of function and ligand

specificity of the CCPs. In this chapter, we focused on highlighting the role of the asymmetric C-

Appendices| 175

tails to 'generate' tier-0 dynamics on SBD2 and MalE of Class B and G, respectively. Such proteins

are ABC transporter-related proteins, which require two distinct conformations to capture their

cognate ligands (i.e., open in the apo- and closed in the holo-state). By disrupting specific

interactions on the C-tail, we destabilize the open apostate and increase their binding affinity. Such

results confirm the role of the modules of the ABC transporter-related CCPs to generate the 'open'

conformation as the native apo-state.

The cherry-core of the LysR Type Transcription Regulators (LTTRs) is having no

additional C-tail module and acting as the effector binding domain (EBD). We showed that Tier-

0 dynamics are not observable on the EBD of CynR and may not be required during effector binding

to propagate the quaternary changes. In Chapter 4, we monitored the lower tier dynamics that

underlie the function of CynR by using the combination of structural modeling and dynamics studies

(i.e., HDX-MS). While there are no significant structural changes observed on the CC detected

with our experimental procedures, we proposed that the accumulative low tiers dynamics are

possibly sufficient to initiate the quaternary changes essential for the DNA-(un)bending.

In Chapter 5, we determined the modules' effect on the structural dynamics of one of the

modern CCPs, i.e., the Maltose Binding Protein (MalE). Such modules are embedded

predominantly at the C- terminy and integrated within the conserved structural more as the

substitutes of consensus helices/sheets, as detailed in Chapter 2. We verified the role of such

modules using an array of biophysical tools (i.e., smFRET, HDX-MS, and Isothermal Titration

Calorimetry) and MD simulations that allowed us to reconstruct the energetic landscape of MalE.

By altering such modules using site-directed mutagenesis, we confirmed the modules' synergistic

roles, which are critical to personify MalE to function as a transport-related binding protein by (i)

entrapping the ligand and (ii) creating a distinct apo-state.

We anticipated that the energetic arguments might be seen as the main selective pressure

during evolution. To fulfill the energetic requirement, nature might introduce modules into the

highly-evolvable protein cores during the long-period evolution, as we presented on the modularity

concept in this thesis. Once the modules are 'attached,' evolution on a fix-length peptide is

sufficient to optimize the protein functions. The later evolutionary process might take effect in a

relatively shorter evolutionary-period, as stated in the ‘avant-garde’ evolvability theory.

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Acknowledgement

| Acknowledgement 178

Acknowledgement

I would like to gratefully acknowledge the funding support that has been received through

The Indonesia Endownment Fund for Education (Lembaga Pengelola Dana Pendidikan - LPDP)

scholarschip scheme during the 4-years period of my PhD. I would like to thank also the Graduate

School of Science and Engineering of the University of Groningen, The Board of The Zernike

Institute and the Doctoral School of Biomedical Sciences of the Katholieke Universiteit Leuven for

establishing and financially support the Double Degree Program between the two universities.

I have been received the enormous support from people that wholeheartedly contributed

for the completion of this thesis and my PhD. Without their support, it would be impossible for

me to face the ups-and-downs of this challenging journey and finish my PhD.

Firstly, I would like to thank Dr. Giorgos Gouridis. I would like to say sorry to you as I

couldn’t mentioned your contributions and supports here one-by-one, as any other readers will end

up by reading a novel. During my PhD, we were evolving together not only as a scientist, but also

as a friend. We are ended up being a best friend - inside and outside the lab, and I hope you also

enjoyed our laughs and scientific fights. Thank you for investing your time and energy so much for

being my mentor and not only as my supervisor. I have never enjoying science better as you always

told me that science is similar as a game and It should be fun. I want to share the value that you

showed me as other people might also need to feel the same excitement while doing science not

only as a scientist but also as a human.

Secondly, I would like to thank Prof. Thorben Cordes for the having me as a PhD student

in your lab. I am grateful to have such a freedom to think and to collaborate. Thank you for

trusting me on getting my PhD and letting me grow with the best people in the field. I would like

to thank Prof. Anastassios ‘Tassos’ Economou and Dr. Spirydoula ‘Lily’ Karamanou for letting me

having a second home in Leuven. I enjoyed every time I spent in your lab with every member of

the group. The broad field and expertise that your lab provide helping me grow a lot. I would also

give my thank to Prof. Wouter Roos for your help and welcoming me in your group during my

PhD phase in Groningen.

Appendices| 179

For my Assessment Committee, especially Prof. Joost Schymkowitz and Prof. Frederic

Rousseau for your sharp and constructive comments of my thesis. I couldn’t thank you enough for

all your inputs as those comments significantly expand our perspectives regarding our results

included in this thesis. We never knew if our study would be applicable in a broader fields.

Special thanks to my mentor: Dr. Rukman Hertadi (Asssc.Prof.). I am so lucky to be

your student. Thanks for not limiting me but even challenging me to have a better scientific impact

than your study. Thanks for letting me grow and evolved – as a scientist and as a person. It must

be some kind of fluke (not anymore a matter of luck) for me to meet you and Dr. Gouridis that

have similar mentality, optimism, positiveness while doing science in my early phase of academic

carreer.

I would like to acknowledge my colleagues as without their support, I wouldn’t be able

to get my PhD. All the Single Molecule Biophysics (SMB) members. Kak Florence ‘Reren’ Husada,

thank you for welcoming and having me as a family in the SMB group. Without your help, I

wouldn’t be able to survive my PhD phase in Groningen. I can’t thank you enough for all you’ve

done. All the best for you, Kak. Semangat! Kostas Tassis, your molecular biology skills are

incredible. Thanks for teaching me your scientific protocol, experimental ‘tricks’, and social

methods on ‘how to bother people so they are willing to help and recognize us’. Marijn de Boer

for being an incredible scientist in the Lab and openly discuss tons of fruitful ideas with me and

Giorgos. Your views are always one-step ahead of everybody’s mind. Yichen Li, for being so calm

and warm office mate. All the best for you, Yichen. Jochem Smit, thanks for your help especially

in correcting my samenvatting of this thesis. Thanks also for Atieh, Monique, Jasper, and Jelle and

ITB-ZIAM student visited the group: M. Arizki and Yovin Sugijo. Thanks for the members of

MBP: Melissa, Sourav, Pedro, Yukun, Guus, and Yuzhen.

All the members of Lab Molecular Bacteriology- Rega Institute. Dr. ‘Eleftheriadis-

Nikolaos-Elef-Niko-…-‘ Nikolaos Eleftheriadis, thank you for being an excellent ‘collaborator.’ I will

miss your voice forever. Thanks for being such an excellent colleagues and a best friend (read:

family). Your energy and optimism is at another level, which everyone in the world should have.

Thank you for teaching me all the stuff that you are expert at and also the stuff that I told you

just 5 minutes before. I am always wondering how you can learn everything that fast. I really hope

we can collaborate in the future. Ruixue Xu, we knew only for 2 years but we became so click that

| Acknowledgement 180

we can even do and think almost anything in the lab. You’re incredible colleagues and person.

Thanks for trusting me and also showing me the persistence. You’re evolving to become a biologist

in a very short time. Our friendship will not end here, it will last even more! Live happily my friend,

you deserved it. Bindu Srivinasu, for being such an open-minded person. The observer of the lab

who always brings warmness and happiness to all the members. The lab is ‘chaotic’ without your

presence. Mohamed Belal, for being a too nice person in the lab. Your sincerety will help you one

day, my friend. God bless you and your family. Athina Portaliou, for being an excellent teacher

and a warm friend. And other Lab members: Luit Barkalita, Srinath Krisnamurthy, Dries, Rengky,

Jos, Sonya, Guillaume, and Maria.

All the Indonesian community in Groningen. Kak Ali Syariati, Teh Liany Septiany, and

Cici Naureen: Nuhun untuk semuanya. Udah ngajarin semua hal baik tentang hidup dan

berkeluarga. Sehat dan sukses terus nya. Mas Salva Yurista udah ngasih semua petuah-petuah

hidup. Sukses terus mas, bahagia terus ya. Inget-inget kalau sudah jadi Menteri. Keluarga besar

Kolak (extended): Marina Ika & Mang Shiddiq – semangat PhD dan dota-nya! Caecil, Kak Dina,

Kak May – makasih pisan buat semuanya ya. Mas Ali Abdurahman, Teh Yosay, Fatih, Dedenya

Fatih – sukses dan bahagia terus ya. Bagus ‘Sugab’ Angga, Kang Fikri, Mas Bin-Kak Sofi, Pak

Hengky, Ika. Keluarga PL: Mas Kus-Mbak Fitri, Mas Zaenal-Mbak Ayu, Mas Kris-Mbak Ica,

Makasih udah semua kebaikan kalian dan selalu ngebantuin aku. Kang Azis-Teh Nna, Mas Bino-

Teh Susan, Pak-Bu Asmoro, Kang Irfan-Teh Liza, Kang Fajar-Teh Monik, Mas Adyhat-Mbak

Nuri, Mas Didik-Mbak Rosel, Mbak Nur, Mbak Ira, Mbak Tiur, Bu Ima, Mas Didin-Anis. And for

all the pople who welcome me during the first phase of my PhD: Rasyida, Keissha, Natasya Witto,

Mas Fean, Mbak Adel, Erlang, Enrico, Mas Guntur, Mas Shiddiq. And also KMP: Krystle and

Selva! Indonesian Day 2017-2018+PPIG Team: Mas Amak-Mbak Putri, Cancan, Mbak Era, Mas

Alfian, Yovita, Mas Bimo, Rachel, and many others – thanks for your kindness. And to the warmest

people: Bude Arie and (alm) Om Herman – makasih untuk semua kehanganatannya. The sandwich

student: Ila, Abed, Nelson, Fras, Romel! Kami sayang bude dan om. Tante Indah – Om Yon,

makasih tante Indah dan om Yon, bahagia selalu ya!

I would like to thank Ibu and Ayah for the endless support and duas: Alhamdulillah aa

sudah lulus, makasih doa dan keihklasannya selama ini. Semoga aa bisa lebih membahagiakan ibu

dan ayah. Hanifah Az Zahra for designing such a beautiful cover for this thesis and being a

wonderful old sister to Isfa Amalia. Thanks for Isfa Amalia for taking care Ayah and Ibu while your

Appendices| 181

old siblings are away from home all this time – Semangat kalian ya! Jalanin apa yang kalian suka

atau sukai apa yang kalian jalani. Keluarga Blitar: Bapak, Mamah, Edo: terima kasih untuk semua

kasih sayangnya. Sehat dan bahagia selalu ya. Thanks to Mbah Putri, the whole family of KBBR-

Jogja and keluarga Garut.

Thanks to the apples of my eyes, the jewels of my heart: Ibu Meissha Ayu Ardini and

Cantik Rinjani Faza Ayudya Yusran. I will work harder and smarter to give you love that both of

you deserved. I love you both to the moon! Terima kasih udah nemenin ayah ya, Ibu. Tetep jadi

wanita terbaik buat ayah dan Jani. Semoga ayah juga bisa nemenin kalian untuk raih cita-cita dan

mimpi-mimpi kita ya! Jani jangan lupa sayangin ibu terus ya. Be kind and courageous, Jani!

Semangat ya cantik!

And lastly: Allah SWT, Prophet Mohammad and his family and followers. Thank you for

giving me such wonderful life with incredible people around me. Alhamdulillah.

For the reader: I want to sincerely thank you for taking your time reading this thesis.

Investing those amount of time to read my thesis is not trivial and is such an honor for me. I would

be glad to hear any input, critic, comment, which will help us improve our study. Those

contributions of yours will probably not be included in this printed version of thesis, but those will

be carved in our minds and credited in our future work/manuscript. Please bear with us to expand

this study even more.