Elucidation structurale des facteurs de transcription végétaux à domaines MADS

249 Pages·2016·20.35 MB·English

THÈSE
Pour obtenir le grade de
DOCTEUR DE LA COMMUNAUTÉ UNIVERSITÉ
GRENOBLE ALPES
Spécialité : Biologie Structurale et Nanobiologie
Arrêté ministériel : 7 août 2006

Présentée par
Sriharsha PURANIK

Thèse dirigée par Chloe ZUBIETA
codirigée par Gordon LEONARD

préparée au sein du Structural Biology Group,
European Synchrotron Radiation Facility
dans l'École Doctorale de Chimie et Sciences du Vivant

Elucidation structurale des
facteurs de transcription
végétaux à domaines MADS

Structural elucidation of plant MADS
domain transcription factors

Thèse soutenue publiquement le 30th May 2016,
devant le jury composé de :

Dr. Pierre Emmanuel MILHIET Rapporteur

Dr. Pradeep DAS Rapporteur

Dr. Darren HART Président

Dr. Pau BERNADO Examinateur

Table of Contents
Acknowledgements .............................................................................................................. 4
Abstract ................................................................................................................................ 6
Résumé en Français ............................................................................................................ 8
Popularized summary of thesis ........................................................................................ 10
I INTRODUCTION...................................................................................................... 12
I.1 Summary .................................................................................................................. 12
I.2 Plant evolution and classification ............................................................................ 13
I.2.1 Role of MADS TFs in plants ........................................................................... 14
I.2.2 Molecular mechanisms for flower development.............................................. 20
I.2.3 MADS TF classification and structure: ........................................................... 22
I.3 Objectives of the thesis............................................................................................. 26
II SEPALLATA3............................................................................................................ 28
II.1 Summary .............................................................................................................. 28
II.2 Introduction.......................................................................................................... 30
II.3 Results and Discussion ........................................................................................ 33
II.3.1 Construct design and protein purification ........................................................ 33
II.3.2 Biophysical characterisation and DNA-binding studies of SEP3(75-178) .......... 51
II.4 Conclusions .......................................................................................................... 69
II.5 Materials and methods ......................................................................................... 71
II.5.1 Construct design and purification .................................................................... 71
II.5.2 Biophysical characterisation and DNA binding studies .................................. 76
III AGAMOUS ................................................................................................................. 81
III.1 Summary .............................................................................................................. 81
III.2 Introduction.......................................................................................................... 82
III.3 Results and Discussion ........................................................................................ 84
2
III.3.1 Construct design and protein purification ........................................................ 84
III.3.2 Biophysical characterisation of AG(74-173) ........................................................ 95
III.4 Conclusions ........................................................................................................ 104
III.5 Materials and methods ....................................................................................... 105
III.5.1 Construct design and purification .................................................................. 105
III.5.2 Biophysical characterisation .......................................................................... 106
IV SHORT VEGETATIVE PHASE............................................................................ 109
IV.1 Summary ............................................................................................................ 109
IV.2 Introduction........................................................................................................ 111
IV.3 Results and Discussion ...................................................................................... 114
IV.3.1 Construct design and protein purification .................................................. 114
IV.3.2 Biophysical characterisation and DNA binding studies of SVP(1-240) ........ 131
IV.4 Conclusions ........................................................................................................ 161
IV.5 Materials and methods ....................................................................................... 162
IV.5.1 Construct design and purification .............................................................. 162
IV.5.2 Biophysical characterization and DNA binding studies ............................ 166
V Conclusion ................................................................................................................ 171
Bibliography .................................................................................................................... 173
VI Appendix I ................................................................................................................ 193
VI.1 Library generation and purification techniques ................................................ 193
VI.1.1 Multi-vector expression screen .................................................................. 193
VI.1.2 Insect cell expression system at EMBL-Grenoble ..................................... 203
VI.2 Biophysical techniques....................................................................................... 205
VI.2.1 Atomic force microscopy ........................................................................... 205
VI.2.2 Small Angle X-ray Scattering .................................................................... 208
VII Appendix II – Collection of articles .................................................................... 215

3
Acknowledgements
“Feeling gratitude and not expressing it, is like wrapping a gift and not giving it”
-William Arthur Ward
It is impossible for any work to come to a conclusion without adding a warm note of
thanks to all those special people who have been kind enough to encourage me throughout
and helped me in completing this dissertation.
Firstly, I would like to express my sincere gratitude to my advisor Dr. Chloe Zubieta. I
have been amazingly fortunate to have an advisor who gave me the freedom to explore on my
own and at the same time the guidance to recover when my steps faltered. I would like to
thank her for her continuous support during the thesis, for her patience, motivation, immense
personal attention and encouragement. I could not have imagined having a better advisor and
mentor for my Ph.D. I am also really thankful to my co-supervisor Dr. Gordon Leonard for
his stimulating suggestions, optimism, valuable inputs and constructive criticisms during the
project and especially during writing of this dissertation. I am also grateful to Dr. Montse
Soler-Lopez, for taking out time from her busy schedules and providing insightful comments
on my dissertation. Her critical questioning, remarks and suggestions proved to be a helpful
in shaping the dissertation. I am grateful to the members of thesis committee, Dr. Francois
Parcy, Dr. Renaud Dumas, Dr. Veronique Hugouvieux and Dr. Jean-Luc Pellequer for their
motivation and suggestions every year which helped to progress in the right direction.
I wish to express my gratefulness to all the members of the Structural Biology group in
ESRF for their unconditional support and help, especially, Samira Acajjaoui for her
enormous contributions to the project. I also take this opportunity to express my heartfelt
thanks to Dr. Martha Brennich, Dr. Adam Round and Dr. Luca Costa for being resourceful
tutors for the subjects which were mystery to me such as SAXS and AFM. Their doors were
always open for attending and answering all my naïve queries.
This acknowledgment is incomplete without thanking my friends in Grenoble, especially
Lahari and Vipin for the number of drink and dinner meetings which helped me to forget the
failed experiments in laboratory; and also Elise and Benedicte for the coffee discussions, for
being my French translators and for the memories which I will cherish forever.
Most importantly, none of this would have been possible without the love and patience of
my family. I am thankful to my parents Mrs. Medha and Mr. Vivek Puranik for their
unconditional love, encouragement and support throughout my PhD thesis work. I admire
4
them for all of their accomplishments, for their efforts and most importantly for teaching me
the value of dedication and hard work. I would like to express my heartfelt appreciation to
my wife, Dr. Sakshi Sood for having faith in me and accompanying me on this adventure, I
look forward to our next one. Finally, I would like to dedicate this thesis to my late
grandparents, Mrs. Pramila and Mr. Gopal Gokhale who played an important role in the
development of my identity and shaping the individual that I am today.
I would like to add a note of thanks to all the different laboratories I worked with during
this project, especially, the ESRF structural biology laboratory and beamlines (BioSAXS-
BM29, ID14-4, ID23-1) , surface science laboratory (Dr. Fabio Comin, ESRF), high
throughput protein technologies-ESPRIT (Dr. Darren Hart, IBS/ISBG, Grenoble), eukaryotic
expression facility (Dr. Imre Berger, EMBL, Grenoble), cell free expression facility (IBS)
and other platforms used as a part of ‘Partners in Structural Biology’ (PSB).

5
Abstract
Virtually all terrestrial habitats are dominated by angiosperms, or flowering plants. Their
success in colonizing new habitats and supplanting other species is due to the advent of a
complex reproductive structure – the flower. The flower unites the male and female organs
into one compact structure and encloses the seed. Flowering plants are not only the dominant
type of land plants, but also are the primary source of food and habitat for all animals,
including humans. In evolutionary terms, flowers are considered a recent development and
have been a subject of speculation from the time of Charles Darwin who termed the dominant
rise and diversification of flowering plants as “an abominable mystery”* due to the lack of a
smooth transition from non-flowering to flowering plants in the fossil record. With the
sequencing of multiple genomes from gymnosperms (non-flowering seed plants), basal
angiosperms and higher flowering plants, certain gene families have been identified which
play a central role in the development and evolution of the flower. My research focuses on
one such family of high-level regulators, the MADS transcription factor (TF) family. This TF
family helps to orchestrate flower development among other functions. As such, there is great
interest in understanding the molecular mechanisms of the MADS family and how these
proteins are able to control complex reproductive pathways.
This project integrates different biophysical techniques including x-ray crystallography,
small angle x-ray scattering (SAXS) and atomic force microscopy (AFM) to investigate
protein-protein and protein-DNA interactions of MADS TFs. No studies to date have
investigated the molecular mechanisms of MADS TFs using this integrated structural
approach.
One important hurdle in the study of the MADS TFs has been recombinant protein
expression and purification. In this project, recombinant purification protocols for several
full length MADS TFs were established, allowing the structural and biochemical
characterisation of the proteins. The crystal structure of the oligomerisation domain of the
MADS family protein SEPALLATA3 (SEP3) is presented and used as a template for
understanding the oligomerisation patterns of the larger family and the molecular basis for
protein-protein interactions. Investigation of solution structures, derived from SAXS studies,
of AGAMOUS (AG) and SHORT VEGETATIVE PHASE (SVP) along with biochemical
characterisation of their oligomerisation states are also presented.
*Letter from Charles Darwin to Joseph Dalton Hooker, written 22 July 1879 (Source: Cambridge
University Library DAR 95: 485 – 488) (Friedman, 2009b).
6
In order to study protein-DNA interactions, complementary methods were used. An
important putative property of the MADS TFs is their ability to change the structure of DNA
through the formation of DNA loops. MADS TFs are hypothesized to oligomerise and bind
DNA at two different sites, potentiating looping of DNA. Using AFM, the first direct
evidence of DNA looping by SEP3 is described. The DNA binding characteristics of SVP
were studied using electrophoretic mobility shift assay (EMSA), microscale thermophoresis
(MST) and AFM. Unlike SEP3, SVP is dimeric and thus exhibits different DNA-binding
patterns.
The data presented here provide an atomic and structural basis for MADS TF function.
Based on this work, we now are beginning to understand some of the oligomerisation and
DNA-binding specificity determinants. These studies demonstrate how the MADS TFs
oligomerise and the results show that we can disrupt oligomerisation and potentially DNA-
binding very specifically through the introduction of point mutations. Future work will
investigate the in vivo consequences of altered oligomerisation and how this affects different
developmental programs in plant reproduction and floral organ morphogenesis.

7
Résumé en Français

Virtuellement tous les habitats terrestres sont dominés par les angiospermes, ou plantes à
fleurs. Leur capacité à coloniser de nouveaux habitats et supplanter une autre espèce est dûe à
l'avènement d'une nouvelle structure reproductrice – la fleur. La fleur uni les organes mâles et
femelles dans une structure compacte et contient la graine. Les plantes à fleurs ne sont pas
seulement le type dominant des plantes terrestres, mais sont également la principale source de
nourriture et l'habitat de tous les animaux, y compris les humains. En termes d'évolution, les
fleurs sont considérées comme un développement récent. Elles ont fait l'objet de spéculations
depuis l'époque de Charles Darwin qui à nommé l’évolution dominante et la diversification
des plantes à fleurs comme «un abominable mystère» en raison de l'absence d'une transition
en douceur de la non-floraison vers la floraison des plantes dans le registre fossile. Avec le
séquençage de plusieurs génomes de gymnospermes (semences de plantes non-florales),
d’angiospermes basals et de plantes à fleurs supérieures, certaines familles de gènes jouant un
rôle central dans le développement et l'évolution de la fleur ont été identifiées. Notre
recherche se concentre sur une de ces familles de régulateurs de niveau supérieur
appelée « famille de facteur de transcription MADS » (TF). Cette famille de TF permet
d'orchestrer le développement des fleurs. Nous nous sommes intéressés à la compréhension
des mécanismes moléculaires de la famille des MADS et à la façon dont ces protéines sont
capables de contrôler les fonctions de reproduction complexes.
Ce projet intègre différentes techniques biophysiques comme la cristallographie aux
rayons X, la diffusion des rayons X aux petits angles (SAXS) et la microscopie à force
atomique (AFM) afin d’étudier les interactions protéine-protéine et protéine-ADN des FT
MADS. Aucune étude n’a, à ce jour, porté sur les mécanismes moléculaires des FT MADS en
utilisant cette approche structurale intégrée.
Un obstacle important dans l'étude des FT MADS a été l’expression des protéines
recombinantes et leur purification. Dans ce projet, les protocoles de purification de plusieurs
recombinants FT MADS entières ont été établis, permettant la caractérisation structurale et
biochimique des protéines dans leurs intégralités. La structure aux rayons X du domaine
d'oligomérisation de la protéine de la famille MADS, SEPALLATA3 (SEP3) est présenté et
utilisé comme modèle pour comprendre les motifs d'oligomérisation de la famille élargie et
les bases moléculaires des interactions protéine-protéine. Des solutions de structures
8
provenant d'études SAXS de AGAMOUS (AG) et de la phase végétative courte (SVP) sont
présentées et complétés par la caractérisation biochimique de leur état d'oligomérisation.
Afin d'étudier les interactions protéine-ADN, des procédés complémentaires ont été
utilisés. Une propriété importante des FT MADS est leur capacité à modifier la structure de
l'ADN grâce à la formation de boucles d'ADN. De manière hypothétique, les FT MADS
oligomérisent et fixent l'ADN sur deux sites différents, bouclant potentiellement l'ADN. En
utilisant l'AFM, la première preuve directe de la formation de boucle d'ADN par SEP3 est
obtenue. Les caractéristiques de liaison d'ADN de SVP ont été étudiées par analyse de
décalage de mobilité électrophorétique (EMSA), par thermophorèseà échelle microscopique
(MST) et par AFM. Contrairement au cas de SEP3, l’EMSA et l’AFM ont montrés que SVP
est un dimère et présente différents modes de liaison à l'ADN.
Ces données fournissent une base atomique et structurale de la fonction des FT MADS.
Sur la base de ce travail, nous commençons à comprendre l’oligomérisation et certaines
spécificités déterminantes de liaison à l'ADN. Ces études montrent comment les FT MADS
s’oligomérisent. De plus, les résultats montrent que nous pouvons très précisément perturber
cette oligomérisation et potentiellement la liaison à l'ADN grâce à l'introduction de mutations
ponctuelles. Les travaux à venir porteront sur les conséquences de l’altération de
l’oligomérisation in vivo et son effet sur les différents programmes de développement dans la
reproduction des plantes et la morphogenèse des organes floraux.
9