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«Structure of green type Rubisco activase from Nicotiana tabacum Mathias Michael Stotz München 2012 Dissertation zur Erlangung des Doktorgrades an ...»

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Structure of green type Rubisco

activase from Nicotiana tabacum

Mathias Michael Stotz

München 2012

Dissertation zur Erlangung des Doktorgrades

an der Fakultät Chemie und Pharmazie

der Ludwig–Maximilians–Universität


Structure of green type Rubisco

activase from Nicotiana tabacum

vorgelegt von

Mathias Michael Stotz

aus Tübingen, Deutschland

München, 2012


Diese Dissertation wurde im Sinne von §7 Absatz 1 der Promotionsordnung vom 28. November 2011

von Herrn Prof. F. Ulrich Hartl betreut.

Eidesstattliche Versicherung Diese Dissertation wurde selbständig, ohne unerlaubte Hilfsmittel erarbeitet.

München, den 20.04.2012 Dissertation eingereicht am: 20.04.2012 Erstgutachter: Prof. F. Ulrich Hartl Zweitgutachter: Prof. Patrick Cramer Tag der mündlichen Prüfung: 22.05.2012 Rule I "We are to admit no more causes of natural things, than such as are both true and sufficient to explain their appearances.

To this purpose the philosophers say, that Nature does nothing in vain, and more is in vain, when less will serve; for Nature is pleased with simplicity, and affects not the pomp of superfluous causes."

Sir Isaac Newton, The Mathematical Principles of Natural Philosophy Acknowledgement I want to thank Prof. Dr. F. Ulrich Hartl and Dr. Manajit Hayer-Hartl for the opportunity to conduct my PhD research in their Department at the Max PLanck Institute of Biochemistry (MPIB). This work has benefited greatly from their intellectual and scientific guidance.

I also want to thank Dr. Andreas Bracher and Dr. Oliver Müller-Cajar. I have learned a lot from both of them and the contrast between Andreas calm and critical suggestions and Olivers inspiring enthusiasm for science (and equally inspiring discussions) is what made working on this project so truly enjoyable.

I am grateful for valuable discussions with Thomas Hauser, Dr. Amanda Windhof, Dr. Paulo Durao, Dr.

Candace Tsai, Dr. Cuimin Liu, Zhuo Li and Leonie Mönkemeyer. Because of them the Rubisco office was and is a place for open-minded scientific exchange and discussions.

I am thankful for the discussions with my thesis advisory board members Prof. Dr. Jürgen Soll and Prof. Dr.

Elena Conti and for the support of the IMPRS organisation team Dr. Hans Joerg Schaeffer, Dr. Ingrid Wolf and Maximiliane Reif.

I especially want to thank all collaborators within and outside of the Department. Dr. Petra Wendler and Susanne Cyniawski for performing negative stain electron microscopy on Activase, Dr. Cyril Boulegue for mass spectrometry on crystallised Rca fragments, Reinhard Mentele for Edman sequencing, Karina Valer and the MPIB Crystallization Facility for support with initial screening of crystallisation conditions and the staff of the MPI Sequencing Facility. Thank you to Evelyn Frey-Royston, Silke Leuze-Bütün and Andrea Obermayr-Rauter for their administrative support as well as Emmanuel Burghardt, Bernd Grampp, Romy Lange, Nadine Wischnewski, Verena Marcus, Elisabeth Schreil, Albert Ries, Andreas Scaia, Tobias Wauer and Leonhard Popilka for their technical support. I also want to thank all members of the Department of Cellular Biochemistry at the MPI for their support.

I want to thank the members of my PhD comittee Prof. Dr. Patrick Cramer, Prof. Dr. Jürgen Soll, Dr. Petra Wendler, Prof. Dr. Ulrike Gaul and Prof. Dr. Elena Conti for critical evaluation of this thesis.

I am grateful for the continuous support of my parents Regine and Hans Martin Stotz during my studies.

Last but certainly not least I want to express my thankfulness to Anne Dueck for her continuous support and love.

–  –  –

XII List of abbreviations ∆................... Deletion (w/v)................ Weight/volume E. coli............... Escherichia coli N. tabacum.......... Nicotiana tabacum AAA(+)............. ATPases associated with various cellular activities ADP................ Adenosine 5’-diphosphate AMP-PNP........... Adenosine 5’-(β,γ-imido)triphosphate APS................ Ammoniumpersulfate ATP................. Adenosine 5’-triphosphate ATPγS.............. Adenosine 5’-γ-thiotriphosphate CA1P............... 2-carboxy-arabinitol 1-phosphate CABP............... Carboxy-arabinitol-1,5-bisphosphate CBB................ Calvin Benson Bassham CCM]............... carbon concentrating mechanism CD................. Circular dichroism CIP................. Calf intestinal phosphatase dA.................. Desoxyadenosine DNA................ Deoxyribonucleic acid DSS................ Disuccinimidyl suberate dT.................. Desoxythymidine DTT................ Dithiothreitol ECL................ Enhanced chemoluminescence EDTA............... Ethylenediaminetetraacetic acid EM................. Electron microscopy HEPES.............. N-(2-hydroxyethyl)piperacin-N’-2-ethanesulfonic acid

XIIIList of abbreviations

His6 Ub.............. Hexa-histidine tagged ubiquitin HRP................ Horseradish peroxidase Hsp................. Heat shock protein IMAC............... Immobilised Metal Ion affinity chromatography IPTG................ Isopropyl β-D-thiogalactopyranoside ITC................. Isothermal titration calorimetry LB.................. Lysogenic broth media MW................ Molecular weight NAD+ / NADH...... Nicotinamide adenine dinucleotide oxidized / reduced NADP / NADPH..... Nicotinamide adenine dinucleotide phosphate oxidized / reduced Ni-NTA............. Nickel-Nitrilotriacetic acid NtRca............... Nicotiana tabacum Rubisco activase OD................. Optical densitiy PMSF............... Phenylmethylsulfonylfluoride RbcL................ Rubisco large subunit RbcS................ Rubisco small subunit Rca................. Rubisco activase RT.................. Room temperature Rubisco............. Ribulose 1,5-bisphosphate carboxylase/oxygenase RuBP............... Ribulose 1,5-bisphosphate SDS-PAGE.......... Sodiumdodecylsulfate polyacrylamid gelelectrophoresis SEC................ Size exclusion chromatography SeMet............... Selenomethionine SIRAS.............. Single isomorphous replacement with anomalous scattering TEMED............. N,N,N’,N’-tetramethylethylenediamine Tris................. Tris(hydroxymethyl)aminomethane

XIV1 Summary

Ribulose 1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) is a key enzyme of photosynthesis, it catalyses the carboxylation of ribulose 1,5-bisphosphate (RuBP), which is the main pathway for the fixation of atmospheric, inorganic CO2 into organic biomass. Despite its central role for plant metabolism, the enzyme suffers from several shortcomings that necessitate its abundance in nature. Compared to other metabolic enzymes the catalytic rate of plant Rubisco is rather slow (~2-3 s−1 ). Furthermore, a prominent side reaction is the oxygenation of the substrate RuBP. Importantly, Rubisco is also prone to inhibition under physiological conditions.

A lysine residue in the active site pocket of Rubisco must be carbamylated and bind a Mg2+ ion as cofactor for the enzyme to become catalytically active. Binding of RuBP to uncarbamylated Rubisco, or of 2-carboxy-arabinitol 1-phosphate (CA1P) to the carbamylated enzyme at night, results in the trapping of the sugar phosphate and inhibition of the enzyme. Inhibition of Rubisco provides a mechanism for the regulation of Rubisco activity in response to varying environmental conditions. The inhibited Rubisco complexes are stable and cannot reactivate spontaneously under physiological conditions. In plants, inhibited Rubisco must be reactivated in an ATP dependent manner by the protein Rubisco activase (Rca) an ATPase associated with various cellular activities (AAA+).

Rca catalyses the ATP-dependent remodeling of Rubisco and facilitates the release of the bound sugar phosphate but is not actively involved in carbamylation of the active site. Since Rca is essential for plant growth and required for maintaining Rubisco activation in vivo, it has been dubbed the "catalytic chaperone" of Rubisco. Interestingly, Rca forms polydisperse, heterogeneous oligomers in solution which vary in size between 50 kDa and 600 kDa. Additionally, Rca is heat labile and it has been proposed that it is a limiting factor for the photosynthetic potential of plants under moderate heat stress, which might become increasingly problematic as the Earth’s temperature increases due to climate change. However, in the absence of a structure, the oligomeric state of the active enzyme as well as a mechanism for its Rubisco activation function remained elusive.

In the present study, the structure, the oligomeric state and the mechanism of Rubisco activase were

1 Summary

investigated. The crystal structure of an N- and C-terminally truncated Rca construct from Nicotiana tabacum was solved at a resolution of 2.95 Å by seleno-methionine single isomorphous replacement with an anomalous scatterer (SIRAS). The structure of the truncated subunit shows the classical AAA+ architecture consisting of a N-terminal nucleotide binding domain and a C-terminal 4-helix bundle. Whereas the nucleotide binding domain is folded in a non-classical Rossmann-fold typical for AAA+ proteins, the 4-helix bundle domain differs from the classical AAA+ fold topology. It features a small helical insertion, containing critical amino acid residues required for the recognition of the substrate Rubisco.

Guided by the structure key features of Rca from Nicotiana tabacum were targeted for mutation.

Specifically, amino acid changes were introduced in the putative Rca oligomer interface and in the putative pore loop residues involved in the Rubisco remodeling. Mutations in the putative subunit-subunit oligomerisation interface generally disrupted oligomer formation of Rca in the nucleotide free state, but also identified an arginine to alanine mutation that lead to formation of stable hexamers in the nucleotide bound state while preserving wildtype activity. Negative stain electron microscopy of this mutant lead to a low resolution model of Rca in the hexameric state, which presumably reflects the active oligomer of the enzyme. Additional density was observed on the "top" side of the hexamer, which presumably corresponds to the N-terminal domain absent in the crystallised constructs. In analogy to other AAA+ domains, it is hypothesized that the N-terminal domain is acting as a substrate adaptor to Rubisco, consistent with reports of deletions and mutations in this domain. Mutation of amino acids in the loops facing the central pore in the hexameric state of Rca partially retained ATPase activity but showed greatly diminished Rubisco activation activity, indicating a role for the pore loops in Rubisco activation.

The combined structural as well as biochemical data presented in this thesis provides a framework for the detailed mechanistic analysis of Rubisco activation in plants. The data suggests that the active state of Rca is a hexamer. Initial Rubisco recognition is mediated by helix H9 and the N-terminal domain and remodeling of Rubisco might involve partial threading of Rubisco through the central pore by the pore loops.

2 Introduction

Proteins are abundant biological macromolecules made up of amino acids and named after the Greek word "proteios" which means elementary. They are found ubiquitously in all domains of life and are essential for almost all biological functions. Proteins act as biological "machines" serving diverse roles, ranging from structure forming assemblies to signaling to catalytic activities. The function of a protein is determined by its structure, which ultimately is encoded in the sequence of the 20 proteinogenic amino acids forming the unbranched protein chains. The elucidation of protein structures is therefore a method to investigate the biological function and mechanism of a protein.

2.1 Protein folding

Since the structure of a protein defines its function, proper folding of the newly-synthesized, disordered chain into its native, three dimensional structure is essential. The information for the structure of the native state is encoded in the primary sequence of a protein, as demonstrated by Christian Anfinsen in the early 1960’s [1][2][3][4] through refolding experiments with Ribonuclease A. The main driving force of protein folding in solution is the burial of hydrophobic side chains in the interior of the protein and the entropy gain of solvent molecules (water) released during this process. Other stabilizing forces are salt bridges between opposite charges, hydrogen bonds and van der Waals contacts between atoms. The native state of a protein is therefore energetically lower than the unfolded state and usually represents the most stable state of the system [4]. However, since proteins usually require some degree of flexibility for their biological function, the native state is only 10-15 kcal/mol more stable than the unfolded state.

Thus the temperature range for protein stability is relatively narrow[5].

Since random sampling of all possible conformations to reach the native state requires a timescale orders of magnitudes longer than measured refolding times of proteins in vitro, a pathway on which proteins fold must exist [6]. This can be illustrated by the multidimensional energy landscape of protein folding, called the folding "funnel" in which the coordinates of a conformational state (usually expressed

2 Introduction

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