Insights into cotranslational protein
folding and protein quality control systems on ribosomes
Steffen Preißler
2011
Insights into cotranslational protein
folding and protein quality control systems on ribosomes
Dissertation
zur Erlangung des akademischen Grades
des Doktors der Naturwissenschaften (Dr. rer. nat.) an der Universität Konstanz
Fachbereich Biologie vorgelegt von
Steffen Preißler
geboren in Heidelberg
Tag der mündlichen Prüfung: 08.12.2011
Referenten: Prof. Dr. Elke Deuerling
Prof. Dr. Christof Hauck
Prof. Dr. Martin Scheffner
Table of contents
1. Summary 1
1.1. Summary (english version) 1
1.2. Zusammenfassung (deutsche Version) 5
2. Introduction 9
2.1. Protein homeostasis 9
2.2. Protein synthesis by ribosomes 10
2.2.1. Ribosomes: Architecture and function 10
2.2.2. Nascent polypeptide chains in the ribosomal exit tunnel 12
2.2.3. SecM-mediated ribosome stalling 13
2.2.4. The ribosomal tunnel exit site 15
2.2.5. Processes coupled with protein synthesis 16
2.2.6. Processing and covalent modification of nascent polypeptides 16
2.2.7. Transport of newly synthesized proteins 18
2.2.8. Folding of newly synthesized proteins 20
2.3. De novo protein folding 20
2.3.1. Principles of protein folding 20
2.3.2. De novo protein folding in the cell 21
2.3.3. Models for de novo protein folding 22
2.4. Cellular strategies to support protein folding 24
2.4.1. Molecular chaperones 24
2.4.2. The Hsp70 chaperone machinery 26
2.4.3. Ribosome-associated chaperones in pro- and eukaryotes 27
2.4.4. Trigger Factor (TF) 29
2.4.5. The nascent polypeptide-associated complex (NAC) 32 2.4.6. The ribosome-associated chaperone triad from Saccharomyces cerevisiae 34
2.5. Protein misfolding and aggregation 37
2.5.1. Protein aggregation 37
2.5.2. Clearance of protein aggregates 39
2.6. Protein quality control on ribosomes 40
2.6.1. The Ccr4-Not complex 41
3. Aims of this work 43
4. Results and discussion 46
4.1. Cotranslational structure acquisition of nascent polypeptides monitored
by NMR spectroscopy. 46
4.1.1. Objective 46
4.1.2. Experimental strategy 47
4.1.3. The SH3 domain as a model nascent polypeptide 48
4.1.4. SH3 constructs used for NMR analysis 49
4.1.5. Expression, metabolic labeling, and purification of arrested RNCs 50 4.1.6. Expression, metabolic labeling, and purification of ribosome-free samples 52
4.2.1. Objective 55
4.2.2. Experimental strategy 55
4.2.3. Summary of the experimental data 56
4.3. SecA interacts with ribosomes in order to facilitate posttranslational
translocation in bacteria. 59
4.3.1. Objective 59
4.3.2. Summary of the experimental data 60
4.4. Components of the Ccr4-Not complex associate with polyribosomes and play an important role in cellular protein homeostasis. 63
4.4.1. Objective 63
4.4.2. Not4 and Caf1 associate with polysomes in vivo 64 4.4.3. Components of the Ccr4-Not complex are important to maintain the cellular
protein homeostasis 66
4.4.4. Protein folding is impaired in not4Δ cells 69
4.5. Structural analysis of the ribosome-associated complex (RAC) reveals
an unusual Hsp70/Hsp40 interaction. 71
4.5.1. Objective 71
4.5.2. Summary of the experimental data 72
4.6. A dual function for chaperones SSB-RAC and the NAC nascent
polypeptide-associated complex on ribosomes. 75
4.6.1. Objective 75
4.6.2. Summary of the experimental data 76
4.7. Directed PCR-free engineering of highly repetitive DNA sequences. 79
4.7.1. Objective 79
4.7.2. Summary of the experimental data 80
5. Outlook 84
6. Abbreviations 87
7. Literature 89
7.1. References 89
7.2. Publications and manuscripts from this thesis 110
8. Acknowledgments (Danksagung) 111
9. Appendix 113
Summary
1. Summary
1.1. Summary (english version)
Upon their synthesis by ribosomes, proteins have to fold into unique three-dimensional structures to become biologically active. Understanding the mechanisms by which newly synthesized proteins acquire and maintain their shapes under cellular conditions is therefore particularly important. In addition, many nascent polypeptides become modified, targeted to membranes, or marked for degradation when their synthesis was defective. For this, all cells contain specialized factors that directly act on nascent polypeptide chains. Although not much is known about their functional interplay, these factors are thought to establish a cotranslational quality control network for newly made proteins. This work focused on investigating the principles of de novo protein folding and the cellular strategies to support the fidelity of this process in both, pro- and eukaryotic model organisms. The following results were obtained:
I. Analysis of cotranslational protein folding and ribosome-associated factors in Escherichia coli
(A) This work revealed for the first time the cotranslational folding pathway of a model nascent polypeptide at the atomic level. In a collaborative effort, NMR spectroscopy was used to monitor the conformation of the SH3 domain from α-spectrin at sequential stages during its synthesis. The translation of SH3 was therefore site-specifically arrested on ribosomes in Escherichia coli cells to generate 15N,13C-labeled nascent polypeptides. To provide snapshots of the translation process, nascent chains were designed to either expose the entire SH3 domain or C-terminally truncated versions thereof. The data showed that nascent SH3 remains unstructured during elongation but adopts a native-like conformation as soon as the entire sequence information is available outside the ribosome. In addition, the ribosome neither imposes conformational constraints nor forms significant contacts with the unfolded nascent SH3 domain. Thus, SH3 folds on ribosomes in a domainwise manner without populating folding intermediates.
Eichmann C.*, Preissler S.*, Riek R., Deuerling E. (2010) PNAS; *shared first authorship, equal contribution
(B) The molecular interaction between the bacterial ribosome-associated chaperone Trigger Factor (TF) and nascent polypeptides was investigated using a site-specific crosslinking approach. For this, radioactively labeled nascent chains were produced in vitro and subjected to binding experiments with TF. The contribution of this work was to clone DNA templates encoding model nascent chains and to generate a suitable in vitro transcription/translation system, which was derived from Escherichia coli cells, to study the chaperone activity of TF on ribosomes. The analysis revealed that ribosome-bound TF can accommodate nascent chains of different lengths and folding states in its interior to protect them against aggregation or premature degradation. This may explain how TF is able to assist cotranslational folding of a broad spectrum of nascent proteins.
Merz F., Boehringer D., Schaffitzel C., Preissler S., Hoffmann A., Maier T., Rutkowska A., Lozza J., Ban N., Bukau B., Deuerling E. (2008) EMBO J.
(C) It has been suggested that SecA-mediated translocation of secretory proteins across the cytoplasmic membrane of Escherichia coli cells occurs posttranslationally. This work contributed the key finding that SecA associates directly with ribosomes in vitro. Together with other results, a new model was proposed, according to which secretory proteins are recognized already cotranslationally by ribosome-bound SecA to direct them efficiently to the posttranslational translocation pathway.
Huber D., Rajagopalan N., Preissler S., Rocco M. A., Merz F., Kramer G., Bukau B. (2011) Molecular Cell
II. Analysis of the ribosome-associated protein quality control system of Saccharomyces cerevisiae
(A) The E3 ubiquitin-protein ligase Not4 is a component of the conserved eukaryotic Ccr4- Not complex and has been suggested to target stalled nascent polypeptides for degradation.
To investigate whether Not4 plays a role in the ribosome-bound protein quality control system of Saccharomyces cerevisiae, the interaction of Not4 with ribosomes was analyzed.
The results showed that Not4 and Caf1, another subunit of the complex, associate with polyribosomes in vivo. Ribosome-association of both factors, however, was dependent on the presence of nascent polypeptides, suggesting that Not4, or the entire Ccr4-Not complex, senses the presence of nascent peptides and thus may function in cotranslational protein quality control. Moreover, deletion of the NOT4 gene caused temperature-dependent aggregation of a broad range of different protein species and the constitutive upregulation of
Summary
heat-shock responsive reporters, indicating folding stress. These data functionally connect the Ccr4-Not complex to the cellular protein homeostasis network.
Preissler S., Koch M., Scior A., Deuerling E.; to be submitted
(B) In yeast, the stable heterodimeric ribosome-associated complex (RAC) is composed of the Hsp70 and Hsp40 chaperones Ssz and Zuotin, respectively. RAC acts as cochaperone for the Hsp70 chaperone Ssb on ribosomes.
(i) To investigate the functional interplay between the Ssb-RAC system and the nascent polypeptide-associated complex (NAC), another ribosome-anchored complex, genetic and biochemical approaches were applied. Deletion of the genes encoding Ssb resulted in the accumulation of protein aggregates consisting predominantly of ribosomal proteins and ribosome biogenesis factors. Additionally, the levels of ribosomal particles and actively translating ribosomes were reduced in these cells. These defects were aggravated in nacΔssbΔ cells, suggesting that both, Ssb and NAC, play a role in the regulation of ribosome biogenesis. The present work contributed the quantification of ribosome levels to this study, as well as experiments showing that ribosomal proteins are specific components of the aggregates formed in ssbΔ and nacΔssbΔ cells.
Kolplin A., Preissler S., Ilina Y., Koch M., Scior A., Erhardt M., Deuerling E. (2010) JCB
(ii) To analyze the architecture of the RAC complex, which consists of Ssz and Zuotin, pulldown experiments were performed showing that the N-terminal region of Zuotin is sufficient to form a stable interaction with Ssz in vivo. This result complements the findings obtained by amide hydrogen exchange experiments and mutational analyses. Together, the data suggest that the mutual stabilization of the highly flexible N-terminus of Zuotin and the C-terminal domain of Ssz constitutes the molecular basis for RAC complex formation.
Fiaux J., Horst J., Scior A., Preissler S., Koplin A., Bukau B., Deuerling E. (2010) JBC
III. Development of a method to study protein folding and aggregation
(A) To investigate protein aggregation of disease-related Poly-Q (poly-glutamine) proteins, a new method was developed for generating constructs containing repetitive sequences.
Cloning of repetitive DNA sequences using standard PCR-based methods is challenging due
was designed to assemble highly repetitive nucleotide sequences. By this approach, DNA templates were generated to produce proteins containing defined stretches of consecutive glutamine residues in bacteria. With these proteins an improved assay was established to study the aggregation of Poly-Q polypeptides in vitro.
Scior A.*, Preissler S.*, Koch M., Deuerling E. (2011) BMC Biotechnology; *shared first authorship, equal contribution
Summary
1.2. Zusammenfassung (deutsche Version)
Nach ihrer Synthese durch Ribosomen müssen Proteine in ihre native Struktur falten um biologisch aktiv zu werden. Der Mechanismus, durch welchen neu synthetisierte Proteine falten und wie sie in der Lage sind unter zellulären Bedingungen ihre Struktur zu wahren, ist eine wichtige Frage der Molekularbiologie. Zudem werden viele naszierende Polypeptide modifiziert, an Membranen transportiert oder wenn Fehler während der Synthese auftreten für Abbau markiert. Um diese vielfältigen Aufgaben bewerkstelligen zu können, besitzen alle Zellen spezialisierte Faktoren, die direkt mit naszierenden Polypeptidketten interagieren.
Obwohl bislang wenig über ihr funktionelles Zusammenspiel bekannt ist, wird angenommen dass diese Faktoren ein kotranslationales Qualitätskontrollsystem für neusynthetisierte Proteinen darstellen. In dieser Arbeit wurden die Prinzipien der de novo Proteinfaltung, sowie die zellulären Strategien von Pro- und Eukaryonten, welche den effizienten und korrekten Ablauf dieses Prozesses gewährleisten untersucht. Die folgenden Resultate wurden erzielt:
I. Analyse kotranslationaler Proteinfaltung und ribosomen-assoziierter Faktoren in Escherichia coli
(A) Diese Arbeit beschreibt erstmalig den kotranslationalen Faltungsweg eines naszierenden Modellproteins auf atomarer Ebene. In einer Kollaboration wurde die Konformation der SH3- Domäne des Proteins α-Spektrin während aufeinanderfolgenden Stufen ihrer Synthese mittels NMR-Spektroskopie untersucht. Dazu wurde die Translation von SH3 an definierten Aminosäurepositionen an Ribosomen in Escherichia coli Zellen arretiert um 15N, 13C- markierte naszierende Polypeptide herzustellen. Um verschiedene Momentaufnahmen des Translationsprozesses zu erhalten wurden naszierende Ketten erzeugt, welche entweder die gesamte SH3-Domäne oder C-terminal verkürzte Versionen exponieren. Die Daten zeigten, dass SH3 während der Elongation unstrukturiert bleibt, aber in die native Struktur faltet sobald die gesamte Sequenz außerhalb des Ribosoms zur Verfügung steht. Desweitern konnte gezeigt werden, dass das Ribosom weder die Konformation der entfalteten naszierenden SH3-Domäne einschränkt, noch signifikante Interaktionen mit ihr eingeht. Die SH3-Domäne faltet daher domänenweise an Ribosomen ohne Faltungsintermediate zu bilden.
Eichmann C.*, Preissler S.*, Riek R., Deuerling E. (2010) PNAS; *geteilte Erstautorenschaft
(B) Mittels einer positions-spezifischen chemischen Quervernetzungsstrategie wurde die molekulare Interaktion des bakteriellen ribosomen-assoziierten Chaperons Trigger Factor (TF) mit naszierenden Polypeptiden untersucht. Dazu wurden radioaktiv markierte naszierende Polypeptidketten in vitro hergestellt und für Bindungsexperimente mit TF eingesetzt. Diese Arbeit trug dazu bei DNS-Template, welche naszierende Modellketten kodieren, zu klonieren. Außerdem wurde ein geeignetes, auf Escherichia coli Zellen basierendes, in vitro Transkriptions-/Translationssystem generiert, um die Chaperonaktivität von TF an Ribosomen zu studieren. Die Untersuchung zeigte, dass ribosomen-gebundener TF naszierende Polypeptidketten unterschiedlicher Längen und Faltungszuständen in seinem Inneren aufnehmen kann, um diese vor Aggregation oder vorzeitigem Abbau zu schützen. Dies könnte erklären wie TF in der Lage ist die kotranslationale Faltung eines breiten Spektrums an naszierenden Polypeptiden zu unterstützen.
Merz F., Boehringer D., Schaffitzel C., Preissler S., Hoffmann A., Maier T., Rutkowska A., Lozza J., Ban N., Bukau B., Deuerling E. (2008) EMBO J.
(C) Es wurde angenommen, dass SecA-abhängige Translokation sekretorischer Proteine über die Zytoplasmamembran von Escherichia coli Zellen posttranslational erfolgt. Ein wichtiger Befund dieser Arbeit ist, dass SecA in vitro selbst in der Lage ist an Ribosomen zu binden. Zusammen mit weiteren Daten konnte ein neues Modell vorgeschlagen werden.
Danach werden sekretorische Proteine schon kotranslational durch SecA an Ribosomen erkannt, was dazu beiträgt diese effizient dem posttranslationalen Translokationsweg zuzuführen.
Huber D., Rajagopalan N., Preissler S., Rocco M. A., Merz F., Kramer G., Bukau B. (2011) Molecular Cell
II. Analyse des kotranslationalen Proteinqualitätskontrollsystems von Saccharomyces cerevisiae
(A) Es wurde beschrieben, dass die E3 Ubiquitin-Protein Ligase Not4, eine Komponente des Ccr4-Not Komplexes, am Abbau arretierter naszierender Polypeptidketten beteiligt ist. Um zu untersuchen ob Not4 tatsächlich eine Rolle im Proteinqualitätskontrollsystem an Ribosomen von Saccharomyces cerevisiae spielt, wurde die Interaktion von Not4 mit Ribosomen analysiert. Die Ergebnisse zeigten, dass Not4 und Caf1, eine weitere Untereinheit des Komplexes, mit Polyribosomen assoziieren. Die Ribosomenbindung beider Faktoren war von der Anwesenheit naszierender Polypeptidketten abhängig. Dies deutet
Summary
Dieser Befund unterstützt die Vermutung, dass der Komplex eine Funktion in der kotranslationalen Proteinqualitätskontrolle haben könnte. Des Weiteren hatte die Deletion des NOT4 Gens die temperaturabhängige Aggregation einer großen Bandbreite an Proteinen sowie eine konstitutive Hochregulierung von Hitzeschockreportern zur Folge. Dies deutet darauf hin, dass in diesen Zellen Proteinfaltungsstress vorherrscht. Die Daten stellen somit einen funktionalen Zusammenhang zwischen dem Ccr4-Not Komplex und dem zellulären Proteinhomeostasenetzwerk her.
Preissler S., Koch M., Scior A., Deuerling E.; fertiges Manuskript
(B) In Hefen besteht der stabile heterodimere ribosome-associated complex (RAC) aus den Hsp70 und Hsp40 Chaperonen Ssz und Zuotin und fungiert als Co-Chaperon für das Hsp70 Ssb.
(i) Mittels genetischer und biochemischer Methoden wurde das funktionelle Zusammenspiel zwischen dem Ssb-RAC System und dem nascent polypeptide-associated complex (NAC), einem weiteren ribosomen-gebundenen Komplex, untersucht. Die Deletion der Gene, welche für Ssb kodieren, führte zur Akkumulation von Proteinaggregaten, welche hauptsächlich aus ribosomalen Proteinen und Ribosomenbiogenesefaktoren bestanden. Zudem war der Gehalt an ribosomalen Partikeln und aktiv translatierenden Ribosomen in diesen Zellen reduziert.
Diese Defekte waren jeweils in nacΔssbΔ Zellen verstärkt, was darauf hindeutet, dass Ssb und NAC eine Rolle in der Regulation der Ribosomenbiogenese spielen. In dieser Arbeit wurde der Gehalt der Ribosomen quantifiziert und Experimente durchgeführt die zeigten, dass ribosomale Proteine spezifische Komponenten der Aggregate aus ssbΔ und nacΔssbΔ Zellen sind.
Kolplin A., Preissler S., Ilina Y., Koch M., Scior A., Erhardt M., Deuerling E. (2010) JCB
(ii) Um den Aufbau des RAC Komplexes zu analysieren, welcher aus Ssz und Zuotin besteht, wurden Pulldown-Experimente durchgeführt die zeigten, dass der N-terminale Bereich von Zuotin ausreicht um in vivo eine stabile Interaktion mit Ssz einzugehen. Dieses Resultat ergänzt Ergebnisse aus Amid-Wasserstoffaustausch Versuchen und Mutationsanalysen. Gemeinsam zeigen die Daten, dass die gegenseitige Stabilisierung des flexiblen N-Terminus von Zuotin und der C-terminalen Domäne von Ssz die molekulare Basis für die Bildung des RAC Komplexes darstellt.
III. Entwicklung einer Methode zur Untersuchung von Proteinfaltung und -aggregation
(A) Um die Aggregation von pathogenen Poly-Q (Polyglutamin) Proteinen zu untersuchen, wurde eine neue Methode für die Herstellung von Konstrukten mit repetitiven Sequenzen entwickelt. Aufgrund des Fehlens spezifischer Hybridisierungsstellen für Primer ist die Klonierung von repetitiven DNS-Sequenzen mittels Standard-Klonierungsverfahren schwierig. Deshalb wurde eine PCR-freie Klonierungsstartegie entwickelt, die es ermöglicht hochrepetitive Nukleotidsequenzen lückenlos zusammenzufügen. Mit dieser Methode war es möglich DNS-Template zu generieren, die genutzt werden konnten um Proteine in Bakterien herzustellen, die definierte Bereiche aufeinanderfolgender Glutamine enthielten. Diese Proteine konnten darüberhinaus genutzt werden um ein verbessertes Versuchsprotokoll zu etablieren, welches es erlaubt die Aggregation von Poly-Q Proteinen in vitro zu analysieren.
Scior A.*, Preissler S.*, Koch M., Deuerling E. (2011) BMC Biotechnology; *geteilte Erstautorenschaft
Introduction
2. Introduction
2.1. Protein homeostasis
The term protein homeostasis, or proteostasis, refers to the regulation of the concentration, conformation, binding interactions, and location of polypeptides in the cell to keep the proteome in a balanced state (Balch et al, 2008). Therefore, a variety of cellular pathways constitute an integrated network controlling all aspects of a protein’s life cycle, including protein biosynthesis, trafficking, and degradation (Figure 1).
Figure 1: Pathways of the cellular proteostasis network. Arrows represent the individual pathways involved in proteostasis. All illustrated steps underlie cellular regulation and can be adjusted according to the respective physiological conditions.
The so-called proteostasis network has two major tasks. First, it maintains the cellular pool of
Transcription
Translation
Aggregate Transport to
destination Degradation or
deposition
Unfolding Chaperone- &
enzyme-assisted folding
Misfolding
Chaperone- mediated unfolding
Aggregation Active disaggregation Targeting &
translocation factors Unfolded polypeptide
Folded protein Misfolded protein
conditions and makes it robust against a multitude of stresses. As it will be described in the following chapters, their chemical nature makes polypeptides susceptible to misfolding and aggregation. Therefore, a proper balance between quality control mechanisms is essential for protein homeostasis. These mechanisms either facilitate correct protein folding and refolding of misfolded proteins, or eliminate the irreversibly damaged ones (Powers et al, 2009). Since the individual proteostasis pathways are thoroughly interwoven, impairment of any of them challenges the whole network. Prolonged perturbations of the proteostasis network can evoke numerous metabolic, oncological, and cardiovascuolar diseases (Douglas
& Dillin, 2010). Especially neurodegenerative disorders correlate with protein misfolding and the accumulation of aggregated polypeptides (Douglas & Dillin, 2010). Besides environmental stresses and mutations that affect protein stability, changes in proteostasis regulation during aging are assumed to be the main causes of pathogenic defects in protein homeostasis.
2.2. Protein synthesis by ribosomes
The amino acid sequence of each protein is encoded on the genomic DNA. Upon transcription into messenger RNA (mRNA) the genetic information is delivered to ribosomes where it is converted into linear amino acid sequences. In the past decade structural and mechanistic studies provided detailed insights into the translation process.
2.2.1. Ribosomes: Architecture and function
Ribosomes are macromolecular machineries in the cytosol that catalyze the conversion of genetic information into the amino acid sequences of proteins. A single bacterial cell is estimated to contain up to 105 ribosomes, whereas eukaryotic cells can be equipped with several million ribosomes depending on the cell type and synthetic activity (Bashan &
Yonath, 2008; Wegrzyn & Deuerling, 2005). Prokaryotic ribosomes synthesize polypeptide chains at rates of about 15-20 amino acids per second. By comparison, translation in higher eukaryotes is typically slower (5-7 amino acids per second).
The basic mechanism by which ribosomes perform their biosynthetic function and their overall structural organization are conserved in all kingdoms of life. Ribosomes are composed of two different subunits, a small and a large one, which associate into functional complexes. Each subunit consists of ribosomal ribonucleic acid (rRNA) and ribosomal
Introduction
(MW ~2,500 kDa) consist of 3 rRNAs (5S, 23S, 16S) and 55 proteins, the eukaryotic 80S ribosomes (MW ~4,200 kDa) contain 4 rRNA molecules (S5, 28S, 5.8S, 18S) and approximately 82 proteins (Bashan & Yonath, 2008; Wilson & Nierhaus, 2005).
It was for a long time challenging to obtain insights into the mechanism by which ribosomes synthesize polypeptides. A series of recent x-ray crystallography studies provided structural details at high resolution of the ribonucleoprotein architecture of ribosomes. Structures of the 50S ribosomal subunit from the archaeon Haloarcula marismortui (Ban et al, 2000) and the mesophilic Gram-positive bacterium Deinococcus radiodurans (Harms et al, 2001) as well as the 30S subunit structures of the bacterium Thermus thermophilus (Harms et al, 2001;
Wimberly et al, 2000) paved the way for modeling the entire 70S ribosome from Thermus thermophilus (Yusupov et al, 2001). Meanwhile crystal structures of the 70S ribosome from Escherichia coli (Yusupov et al, 2001), the eukaryotic 40S subunit from Tetrahymena thermophila (Yusupov et al, 2001), and the 80S ribosome from Saccharomyces cerevisiae (Ben-Shem et al, 2010) are available.
Figure 2: Structure of the bacterial 70S ribosome. A) Surface representation of the E. coli 70S ribosome. The small subunit is illustrated in light gray (rRNA of 30S) and yellow spheres (30S ribosomal proteins), whereas the large subunit is shown in dark gray (rRNA of 50S) and blue spheres (50S ribosomal proteins). The figure was generated using PyMOL (composite of PDB entries 2AVY and 2AW4). B) Architecture of the translating 70S ribosome based on Cryo-EM reconstitution. The PTC is located at the interface between the small (light yellow) and the large (blue) subunit and contains A- (red), P- (green), and E-site (brown) tRNAs. Half of the 50S subunit is cut away to show a cross-section of the polypeptide tunnel. A stretch of mRNA (magenta) and the nascent polypeptide chain (yellow) were modeled into the structure. Taken from (Mitra & Frank, 2006).
The structural data provided important insights into the catalytic mechanism of translation.
A B
(Nissen et al, 2000). In contrast, the ribosomal proteins are found predominantly at the surface (Figure 2). Although the ribosomal subunits play different functional roles, they cooperate in polypeptide synthesis. The small subunit binds to mRNA, provides the decoding center, and controls fidelity of translation. The large subunit harbors the peptidyltransferase center (PTC) where peptide bonds are formed and provides the exit tunnel through which the growing nascent polypeptide chain leaves the ribosome into the cytosol.
The entire translation process depends on additional non-ribosomal factors and can be subdivided into three different stages: initiation, elongation, and termination. During initiation the small and the large ribosomal subunits join together on mRNA to form catalytically active ribosomes. Each assembled ribosome contains three tRNA binding sites (A (aminoacyl), P (peptidyl), and E (exit)). tRNAs are the molecules that transport amino acids and decode the genetic information. The first aminoacylated tRNA binds to the start codon of the mRNA at the P-site. The next aminoacyl-tRNA enters the ribosome and attaches to the second codon at the A-site. While the peptide bond is formed between the amino acids in the PTC, the A- site tRNA is translocated to the P-site. Subsequently the deacylated tRNA leaves the ribosome though the E-site. At each elongation cycle both ribosomal subunits undergo dynamic conformational changes to translocate the bound tRNAs and mRNA by a single codon. At the end of an open reading frame, termination factors recognize the stop codon and catalyze the release of the newly synthesized polypeptide from the peptidyl-tRNA.
2.2.2. Nascent polypeptide chains in the ribosomal exit tunnel
Peptide bond formation takes place in the active site of the ribosome at the interface between the two subunits. As translation proceeds, the growing polypeptide chain leaves the ribosome through a long tunnel in the large ribosomal subunit, which connects the PTC with the cytosol (Figure 2B). The nascent-peptide exit tunnel is approximately 80-100 Å in length and its diameter varies between 10 and 20 Å (Nissen et al, 2000). Because the tunnel must allow the passage of all types of cellular polypeptide chains independent of their size, charge, and hydrophobicity, it must be non-sticky and promiscuous. Consistent with this notion, structural data indicate that the tunnel is primarily built of rRNA; its walls are lined by hydrated polar groups and lack extended hydrophobic patches (Nissen et al, 2000). Due to its dimensions, the exit tunnel is expected to accommodate extended peptides of 30 amino acids or up to 60 amino acids in α-helical conformation (Malkin & Rich, 1967; Picking et al, 1992; Voss et al, 2006). Since the average diameter of the tunnel is only about 15 Å, protein folding inside the ribosome is assumed to be limited to α-helix formation (Nissen et al, 2000).
Indeed, several studies provided evidence that folding of nascent polypeptides can start
Introduction
monitored near the PTC by measuring the distance between two residues within a nascent chain by the FRET technique (Woolhead et al, 2004). As the ribosomal tunnel is not uniform throughout its length, Deutsch and colleagues proposed distinct folding zones for nascent chains inside the tunnel, which allow the formation of compact secondary structures, such as α-helices (Lu & Deutsch, 2005a; Lu & Deutsch, 2005b; Tu & Deutsch, 2010). Even small tertiary structures (e.g. β-hairpins) were detected within the distal region of the tunnel close to its exit port (Kosolapov & Deutsch, 2009). In addition, single-particle cryo-EM reconstitutions of translation-arrested ribosomes demonstrated that nascent chains adopt distinct conformations inside the tunnel and interact with ribosomal components (Bhushan et al, 2010; Seidelt et al, 2009).
The ribosome exit tunnel was for a long time thought to be a passive conduit for growing nascent polypeptide chains. However, recent studies provided evidence that it plays a more active role in regulating translation and early protein folding events. In particular, a number of leader peptides were identified that regulate translation of a downstream open reading frame by modulating ribosomal activity. These peptides specifically interact with components of the ribosomal tunnel during their synthesis to induce ribosome stalling on mRNA, e.g. in response to the availability of an effector molecule (Hood et al, 2009; Lovett & Rogers, 1996;
Mankin, 2006; Tenson & Ehrenberg, 2002). Biochemical data indicate that the tunnel carries an overall electronegative potential (Lu et al, 2007). Accordingly, the translation of peptides containing elongated stretches of consecutive positively charged amino acids (e.g. poly- lysine or poly-arginine) is inefficient and induces transient elongation arrest (pausing) due to electrostatic interactions between the nascent chain and the ribosomal tunnel (Lu & Deutsch, 2008). This suggests that the charge distribution within nascent chains has an impact on translation rates.
At approximately 20-35 Å away from the PTC the exit tunnel is restricted by extensions of the ribosomal proteins L4 and L22 (L4 and L17 in eukaryotes, Figure 3A) (Nissen et al, 2000;
Wilson & Beckmann, 2011). The region between the PTC and the constriction point seems to be most crucial for functional interactions between regulatory nascent peptides and the ribosome. Mankin and coworkers demonstrated that specific nascent polypeptides can communicate with the A-site of the PTC to restrict its ability to catalyze peptide bond formation with a particular subset of amino acids, and thereby cause translation arrest (Ramu et al, 2011).
2.2.3. SecM-mediated ribosome stalling
The SecM (secretion monitor) peptide from E. coli is one of the best-studied cis-acting
(170 aa) contains a C-terminal arrest sequence, which is required for the regulation of SecA synthesis. SecA is a bacterial motor ATPase that drives posttranslational translocation of secretory polypeptides through the SecYEG protein-conducting channel (PCC) in the plasma membrane (Murakami et al, 2004). During translation of SecM, when the entire arrest sequence (F150xxxxWIxxxxGIRAGP166) is inside the exit tunnel, elongation is stalled at Pro166 (Figure 3A) (Nakatogawa & Ito, 2002). In the stalled complex the peptidyl-tRNA (SecM-tRNAGly) is located at the P-site and the Pro-tRNAPro at the A-site of the ribosome (16713584). Analysis of elongation arrest suppressor mutants showed that in this position W155 and I156 of SecM interact with the 23S rRNA (bases A2058 and A749-753) and a β- hairpin loop of the ribosomal protein L22 at the narrowest constriction of the exit tunnel (Nakatogawa & Ito, 2002). A recent cryo-EM reconstitution of SecM-stalled RNCs mapped the critical interactions between SecM and the ribosomal tunnel (Bhushan et al, 2011). They also report a shift of the tRNA-nascent chain ester linkage of the SecM-tRNA away from A- site tRNA that is likely to impair the activity of the PTC, thus providing a structural explanation of SecM-mediated elongation arrest.
SecM contains a N-terminal translocation signal, which is exposed outside the ribosome in the arrested state and mediates targeting for secretion. At the membrane, the SecA-PCC physically pulls the SecM polypeptide, whereby the translational arrest is released and SecM is exported into the periplasm (Butkus et al, 2003). This reversible ribosome stalling mechanism of SecM provides a feedback system for the regulation of the cellular SecA levels (Figure 3B). The secM gene is located upstream of secA in the same transcription unit (operon). The intergenic region on the secM-secA bicistronic mRNA forms a stem-loop structure that sequesters the Shine-Dalgarno sequence (RBS: ribosome-binding site) for secA translation (McNicholas et al, 1997). In the paused state, the stalled ribosome is positioned in a way that resolves the stem-loop structure on the secM-secA mRNA and thereby allows translation of SecA by another ribosome. However, when the levels of SecA are high, the nascent SecM polypeptide is efficiently exported whereby translation arrest is released. This leads to re-formation of the stem-loop and inhibition SecA synthesis (Nakatogawa & Ito, 2004; Nakatogawa et al, 2004). Besides its regulatory function in bacteria, the SecM arrest sequence became an excellent tool to stall translation of any desired polypeptide in vivo (Evans et al, 2005; Schaffitzel & Ban, 2007). Therefore, SecM was used in this study to produce ribosome-nascent chain complexes (RNCs) for structural investigations.
Introduction
Figure 3: SecM-mediated ribosome stalling in E. coli. A) SecM polypeptide in the exit tunnel of an arrested ribosome. G165 is the last amino acid incorporated into the nascent SecM chain. The residues I156 and W155 are critical for stalling and locate close to the tunnel constriction point formed by protrusions of the ribosomal proteins L4 (magenta), L22 (blue), and rRNA. The N-terminal translocation signal (green) is exposed outside the ribosome. The ribosomal protein L23 is shown in orange. Based on (Nakatogawa & Ito, 2001; Nakatogawa & Ito, 2002). B) SecM regulates the synthesis of SecA in vivo. Under secretion-defective conditions (upper panel) ribosomes become efficiently arrested on the secM-ORF (blue) allowing the translation of the secA-ORF (red) by another ribosome. Under normal conditions (lower panel) translation arrest is released by cotranslational translocation of the SecM polypeptide. As a consequence, a stem-loop structure on the secM-secA mRNA can form to occlude the RBS of the secA-ORF, thereby inhibiting SecA synthesis (red arrow). Based on (Butkus et al, 2003; McNicholas et al, 1997; Nakatogawa & Ito, 2004; Nakatogawa et al, 2004).
2.2.4. The ribosomal tunnel exit site
A characteristic feature of the ribosomal tunnel is that it widens up towards its exit site at the distal end (Figure 2B) (Ban et al, 2000; Lu & Deutsch, 2005a). The tunnel exit site is surrounded by a set of surface-exposed ribosomal proteins, which are embedded in RNA (Figure 4). Four of these proteins (L22, L23, L24, and L29) are ubiquitously conserved, whereas the additional ones are part of a kingdom-specific repertoire of ribosomal proteins.
The ribosomal exit site is of particular importance, as it forms the interface between polypeptide biosynthesis inside the ribosome and downstream events in the cytosol required for protein maturation. Such processes involve dozens of additional cellular factors, many of which have been shown to associate with ribosomes in close proximity to the polypeptide exit tunnel (Giglione et al, 2009). Therefore, a major function of the ribosomal tunnel exit site is to provide a binding platform for ribosome-associated factors and to coordinate their access to the nascent polypeptide chains.
L22 L23
I156 L4 W155 G165
secM RBS secA
RBS
secM secA
5‘
5‘
3‘
3‘
A B
mRNA
SecM
Figure 4: The ribosomal polypeptide tunnel exit. View on the tunnel exit sites of E. coli (A) and S. cerevisiae (B) ribosomes. Only proteins directly adjacent to the tunnel exit are illustrated by different colors. Homologous ribosomal proteins are represented in the same color. rRNA is shown in gray. The tunnel exit is indicated with T.
The figures were generated using PyMOL. A) The tunnel exit of the E. coli ribosome. Ribosomal proteins L23 (red), L22 (orange), L24 (magenta), and L29 (green) are shown. (PDB 2AW4) B) The tunnel exit site of the S.
cerevisiae ribosome. Besides the ubiquitously conserved ribosomal proteins Rpl25 (L23 homolog, red), Rpl17 (L22 homolog, orange), Rpl26 (L24 homolog, magenta), and Rpl35 (L29 homolog, green) additional proteins (Rpl31 (cyan), Rpl19 (blue), and Rpl39 (yellow)) surround the tunnel exit. (PDB 3O58).
2.2.5. Processes coupled with protein synthesis
In order to become biologically active, newly synthesized proteins are subjected to downstream maturation processes such as folding, covalent modification and/or transport to membranes. Many of these processes are initiated cotranslationally and involve factors that bind directly to ribosomes (Figure 5).
2.2.6. Processing and covalent modification of nascent polypeptides
Many polypeptides are subjected to chemical modifications, some of which occur during their synthesis (Figure 5). Nascent chains translated from mRNA start with a methionine residue, as most open reading frames begin with the universal start codon AUG. Therefore, N- terminal methionine excision is the first essential proteolytic event to occur for a large number of polypeptides in all organisms. Methionine aminopeptidases (MAPs) catalyze the removal of the N-terminal methionine residue from a specific subset of nascent polypeptides.
MAPs perform their function early during protein synthesis, as soon as the first N-terminal residues of the nascent chains become exposed outside the ribosome, suggesting a physical interaction with the translation machinery (Ball & Kaesberg, 1973). This assumption is supported by data showing that yeast MAPs indeed associate with the large ribosomal
A B
E. coli S. cerevisiae
L23 L29 L22 L24
Rpl25 Rpl39 Rpl19
Rpl31
Rpl17
Rpl35 Rpl26
T T
Introduction
In eubacteria, a special initiator tRNA, charged with formylmethionine (tRNAifMet), binds to the start codon during translation initiation. Afterwards the formyl group is removed from nascent proteins by the action of the enzyme peptide deformylase (PDF) - a step required for the subsequent excision of the N-terminal methionine (Kramer et al, 2009). In E. coli, PDF associates with the ribosome via a C-terminal helix that binds to a groove between the ribosomal proteins L22 and L32, and thus orients its active site towards the ribosomal tunnel for nascent chain interaction (Bingel-Erlenmeyer et al, 2008).
Figure 5: Processes coupled to translation. Factors, which directly bind to translating ribosomes, interact with nascent polypeptide chains and initiate downstream protein maturation processes, such as covalent modifications, transport, or folding. Deformylation occurs only in prokaryotes whereas myristoylation is restricted to some eukaryotic and viral proteins (*).
N-terminal acetylation is one of the most common protein modifications, which occur cotranslationally to a vast majority of eukaryotic proteins (Polevoda & Sherman, 2000); yet its relevance in vivo is largely unclear. A recent study suggested that N-terminal acetylation is part of a protein quality control system and creates a specific degradation signal (AcN-degron) for the ubiquitin proteasome system (Hwang et al, 2010). N-acetyltrasferases (NATs) are non-essential hetero-trimeric complexes, which catalyze the acetyl group transfer from acetyl-CoA to the α-amino group of the N-terminal residue of a substrate polypeptide (Meinnel & Giglione, 2008). Cells are usually equipped with several NATs and each is required for the acetylation of different groups of proteins (Polevoda & Sherman, 2000). It
Targeting factors
Enzymes Chaperones
Transport Translocation Membrane insertion
Secretion Modification
Methionine cleavage Deformylation*
N-acetylation Myristoylation*
Folding Folding Complex formation
Ligand binding Translation
Nascent chain
termini (Palmiter et al, 1978). In line with this observation, the yeast N-acetyltrasferase NatA binds quantitatively to ribosomes and could be chemically cross linked to nascent polypeptides, indicating its localization close to the ribosomal tunnel exit (Gautschi et al, 2003). Meanwhile also other yeast NATs were shown to associate with ribosomes. Pulldown experiments identified the ribosomal proteins Rpl25p and Rpl35p as potential binding sites for NatA on the large subunit (Polevoda et al, 2008). Finally, myristoylation of N-terminal glycine residues of a few eukaryotic proteins by N-Myristoyltransferases (NMTs) has been suggested to occur cotranslationally (Deichaite et al, 1988; Martin et al, 2011; Wilcox et al, 1987).
2.2.7. Transport of newly synthesized proteins
Many proteins are transported into subcellular compartments, are inserted into membranes, or become secreted. About 30% of the genes code for membrane proteins (Wallin & von Heijne, 1998). Therefore, cells require efficient targeting and transport systems to deliver newly synthesized proteins to their correct destination. The signal recognition particle (SRP) is a ubiquitous targeting factor that delivers substrate proteins cotranslationally to the Sec translocon on the plasma membrane of bacteria or the endoplasmatic reticulum (ER) of eukaryotic cells (Luirink & Sinning, 2004). In all organisms, SRP binds to ribosomes via the conserved ribosomal protein L23, which is located at the tunnel exit (Halic et al, 2004; Halic et al, 2006; Schaffitzel et al, 2006). In this position SRP scans nascent polypeptide chains and interacts with signal sequences as soon as they emerge from the ribosome. Upon substrate recognition, SRP is targeted together with the ribosome to the membrane where it interacts with the SRP-receptor. This interaction facilitates the transfer of the ribosome to the Sec-PCC where the nascent polypeptide is inserted into the membrane or translocated into the lumen of the ER (Cross et al, 2009). Whereas in eukaryotic cells most proteins are targeted via SRP, membrane targeting in bacteria is much more versatile.
E. coli cells contain two major pathways to target proteins to the cytoplasmic membrane.
Most proteins of the cytoplasmic membrane are targeted cotranslationally by SRP as ribosome-bound nascent chains (Luirink & Sinning, 2004; Rapoport, 2007). In contrast, the majority of secretory proteins, including periplasmic and outer-membrane proteins, are targeted via a posttranslational pathway, which depends on the cytosolic chaperone SecB and the motor ATPase SecA (Driessen & Nouwen, 2008; Rapoport, 2007). Both pathways converge at the heterotrimeric SecYEG protein-conducting channel (Sec translocon) through which the polypeptides must pass in an unfolded conformation (Van den Berg et al, 2004).
Introduction
• SRP-dependent cotranslational protein targeting in E. coli
In bacteria, ribosome-bound SRP detects highly hydrophobic sequences, such as transmembrane segments, on nascent polypeptides as soon as they become exposed to the cytosol. Recognition of these signals leads to the stable association of SRP with the RNCs and initiates targeting to the membrane, where SRP interacts with its receptor FtsY to transfer of the RNCs to the Sec translocon (Driessen & Nouwen, 2008). Coupling protein synthesis to translocation provides thereby the energy for the directed passage of the nascent chains through the translocon.
• SecA/B-dependent posttranslational protein targeting in E. coli
The majority of secretory proteins, including periplasmic and outer-membrane proteins, are targeted via a late- or posttranslational pathway (Driessen & Nouwen, 2008; Rapoport, 2007) (Figure 17). Such secretory proteins are synthesized as precursors (preproteins) in the cytosol with an N-terminal signal sequence, which is cleaved upon translocation into the periplasm by membrane-anchored signal peptidases (SPase) (Paetzel et al, 2002). The secretion-dedicated cytosolic chaperone SecB forms a homotetramer (Xu et al, 2000) and is thought to keep secretory proteins in an unfolded and translocation-competent state. At the membrane, SecA, an ATP-dependent motor protein, pushes the substrates through the Sec translocon (Hartl et al, 1990). SecA forms a homodimeric complex in which the two protomers are arranged in an antiparallel orientation (Hunt et al, 2002; Papanikolau et al, 2007; Sharma et al, 2003; Zimmer et al, 2006). Each protomer contains a motor domain, which hydrolyzes ATP and thereby converts chemical energy in mechanical force to drive protein translocation. SecA localizes to the cytosol as well as to the cytoplasmic membrane (Cabelli et al, 1991) via interactions with anionic phospholipids (Lill et al, 1990) or the Sec translocon (Hartl et al, 1990). Based on the available data, a model for posttranslational targeting of secretory proteins was postulated. Thereby, SecB binds newly synthesized substrates in an unfolded conformation and targets them to the membrane, where it interacts with SecYEG-bound SecA (Fekkes et al, 1998; Hartl et al, 1990). The high-affinity interaction between SecB and SecA facilitates substrate transfer to the translocon and SecA mediates stepwise protein translocation through repeated cycles of ATP hydrolysis (Economou &
Wickner, 1994; Schiebel et al, 1991; van der Wolk et al, 1997).
2.2.8. Folding of newly synthesized proteins
Newly synthesized proteins that reside in the cytosol need to fold into their native structure to become functional. A network of molecular chaperones, including ones that directly interact with the translation machinery, aids early steps of folding. As this work focuses on the de novo folding of newly synthesized proteins, a more detailed introduction on the factors involved in this process will be given in the following chapters.
2.3. De novo protein folding
2.3.1. Principles of protein folding
Protein structures are based on non-covalent intramolecular interactions within the peptide backbone and between amino acid side chains. The most fundamental insights about protein folding came from early refolding studies (Anfinsen, 1973). Since then, it was shown in numerous in vitro experiments that denatured full-length proteins are able to refold spontaneously into their native state upon removal of the denaturing condition. These findings demonstrated that all the information required for a polypeptide to fold correctly into a specific three-dimensional structure is inherent in its linear amino acid sequence.
Nevertheless, a polypeptide can adopt a tremendous number of theoretical conformations during folding. Therefore, sampling all possible conformations to establish the correct ones would render the folding process impossible to occur on a biologically relevant time scale.
However, proteins typically fold within a few seconds or even microseconds (Buchner et al, 2011; Gruebele, 2005; Kubelka et al, 2004). This discrepancy between theory and experimental observation is described in the so-called Levinthal’s paradox, according to which a protein would never reach its native structure by exploring its entire conformational space (Zwanzig et al, 1992).
Another important discovery was that in most cases, folding is a sequential process rather than a one-step transition. Thereby, proteins fold in a funnel-shaped energy landscape along a pathway of defined intermediates until they reach their free energy minimum in the native state (Levy & Onuchic, 2006). In aqueous solutions water provides the main driving force for folding. In a first phase, a fast hydrophobic collapse of the unfolded peptide gives rise to compact intermediates, or molten globules, that contain native-like secondary structures (e.g.
α-helices and β-sheets) but lack well-packed side chains and a defined tertiary structure
Introduction
peripheral elements and are therefore especially prone to misfold or to aggregate. In this transition state most proteins have to overcome an energetic bottleneck situation upon which the multiple secondary structure elements condense against each other to form the native tertiary structure.
2.3.2. De novo protein folding in the cell
As mentioned above, most of our current knowledge about the driving forces of protein folding is based on the results of in vitro refolding studies. Such experiments are usually performed with denatured full-length proteins under diluted conditions, where the protein concentrations are low. Thus, the entire sequence information is instantaneously available when refolding is initiated. In contrast, protein folding in the cell is considerably different from in vitro refolding. One of the most critical aspects is that the cytosol is a crowded environment composed of organic molecules that reach concentrations between 300 and 400 g/l (Goodsell, 1991; Zimmerman & Trach, 1991). Molecular crowding has been shown to cause excluded volume effects that significantly increase the affinities between interacting proteins (Minton, 2005). Therefore, a multitude of cellular components can potentially interact with nascent polypeptides or partially folded intermediates and influence their folding pathway. In addition, de novo folding goes along with protein synthesis. Protein synthesis by ribosomes, however, is a strict vectorial process that proceeds from N- towards the C- terminus of a polypeptide chain. Since incomplete polypeptide chains cannot fold into their final structures, nascent polypeptides expose hydrophobic sites, which provide a contact surface for unproductive interactions. The probability for the formation of non-native contacts is even enhanced by the fact that translation is slow (~20-80 seconds for a protein of 400 amino acids) compared to average protein folding kinetics, which are usually on the microsecond to second timescale (Jackson, 1998; Zwanzig et al, 1992). This leads to the prolonged exposure of nascent polypeptides in the non-native state. During their synthesis, proteins are therefore especially prone to misfold and to aggregate. In order to prevent folding errors, nature has evolved several strategies, including folding catalysts and molecular chaperones, to keep newly synthesized proteins on the correct folding pathway (Komar, 2009).
2.3.3. Models for de novo protein folding
Different models for de novo protein folding have been proposed (Figure 6). As postulated by Baldwin, protein biosynthesis without folding is energetically unfavorable, because the conformational space and the energy of the growing polypeptide continuously increase with ongoing translation (Baldwin, 1999; Fedorov & Baldwin, 1997). As described above, there is meanwhile substantial experimental support that nascent polypeptides can fold cotranslationally, e.g. during their synthesis. Whereas the ribosomal tunnel only allows the formation of secondary structure elements, such as α-helices, compact three-dimensional folding is supposed to occur outside the ribosome. The compaction of nascent polypeptides on ribosomes was probed with several elegant approaches, including limited proteolysis, analysis of correct disulfide bridge formation, or by conformation-specific antibodies (Hamlin
& Zabin, 1972; Komar, 2009; Land et al, 2003; Netzer & Hartl, 1997). However, the enzymatic activity of full-length nascent chains provided the most solid evidence that proteins can adopt native conformations while being attached to ribosomes (Kudlicki et al, 1995;
Makeyev et al, 1996). Importantly, it was shown that for some proteins cotranslational folding occurs faster (Kolb et al, 2000) and with higher yields of correctly folded species, compared to in vitro refolding (Katranidis et al, 2009; Ugrinov & Clark, 2010). Although cotranslational folding takes place at least for a subset of newly synthesized proteins, the precise mechanism underlying this process and the atomic details of nascent polypeptides on ribosomes remain largely unexplored. Two different scenarios for cotranslational folding can be envisioned (Figure 6). One possibility is that secondary and tertiary structures begin to form gradually as soon as sequence information becomes exposed outside the ribosome. An alternative model suggests that tertiary structure formation could occur in a domainwise manner. In this case, the individual structural units (domains) of a protein may stay largely extended until enough sequence information is available outside the ribosome for productive folding (Zhang & Ignatova, 2011). Thereby, intramolecular interactions would be formed in a hierarchical condensation process, starting with secondary structures based on hydrogen bonds within the peptide backbone, followed by near-range side chain interactions that lead to compact folding of the domain. The long-distance interactions would be established later during synthesis or even posttranslational. This model is especially attractive for larger multi- domain proteins with complex architectures. In both cotranslational folding modes, however, non-native intrachain contacts may become trapped and thereby make folding inefficient and error-prone. It is therefore possible, that although folding may start during synthesis, some proteins acquire their native structures posttranslationally (Figure 6) (Jansens et al, 2002).
Introduction
Figure 6: Models for initial protein folding. In the posttranslational folding mode polypeptides stay unfolded during their synthesis and fold into their native conformation upon release from the ribosome. On the contrary, proteins could fold cotranslationally by forming intermediate structures as soon as sufficient sequence information becomes available outside the ribosome. An alternative model suggests that cotranslational folding of multi- domain proteins could occur in a domainwise manner. In this case, an individual domain remains unfolded until its entire sequence is exposed to allow productive structure formation. The subsequent domains may fold likewise.
Based on (Deuerling & Bukau, 2004).
Nevertheless, it is still challenging to study the details of cotranslational folding mechanisms due to the lack of adequate methods that can reliably detect transient folding intermediates during synthesis. In the present study we applied a new structural approach to visualize the dynamics and conformations of nascent polypeptides by nuclear magnetic resonance (NMR) spectroscopy. This allowed us to obtain detailed insights into their cotranslational structure acquisition pathway.
Posttranslational
N
N C
N C
Cotranslational
N C
Cotranslational domainwise
N N
N N
N
2.4. Cellular strategies to support protein folding
Under cellular conditions proteins are at a constant risk to misfold or to aggregate. Although proteins are especially susceptible to misfolding during their synthesis, most proteins repeatedly unfold at least partially throughout their lifetime. One reason is that the proteins of an organism have to be conformationally flexible in order to perform their biological function and thus, they were optimized during evolution to be only marginally stable at the respective growth temperature. As a consequence, the energy barriers between the native and non- native conformations are usually small (Jahn & Radford, 2005). Therefore, cells contain an arsenal of factors, such as folding catalysts and molecular chaperones, which assist protein folding in vivo. Importantly, these factors are neither present in the final structure nor provide steric information for folding. Instead, they are thought to act in kinetic partitioning between productive folding and aggregation, and thereby improve the yield of native protein (Hartl &
Hayer-Hartl, 2009).
Folding enzymes catalyze the rate-limiting steps of protein folding, in which covalent bonds are formed or remodeled. For example, disulfide bond formation is essential for the correct folding of many secreted proteins. Therefore, protein disulfide isomerases (PDIs) promote the rapid exchange between paired disulfides and promote the formation of disulfide cross- bridges that stabilize the native structure (Messens & Collet, 2006). Peptidyl-propyl isomerases (PPIases) are the second important class of folding enzymes. They catalyze the cis/trans isomerization of propyl peptide bonds (Xaa-Pro) N-terminal to proline residues and thereby accelerate protein folding (Schiene & Fischer, 2000; Shaw, 2002).
Whereas folding enzymes act predominantly on a subset of polypeptides, all cellular proteins are potential substrates of chaperones. Per definition, molecular chaperones are proteins that interact transiently with non-native polypeptides in stoichiometric ratios and thereby stabilize their client proteins or support correct folding (Hartl & Hayer-Hartl, 2009). Cells are equipped with different types of molecular chaperones that play important roles in a broad spectrum of cellular functions, including de novo protein folding or refolding of stress- denatured polypeptides. In addition, processes, such as intracellular protein transport, assembly of oligomeric complexes, and assistance in proteolytic degradation of terminally misfolded proteins, depend on the chaperone network (Hartl & Hayer-Hartl, 2009).
2.4.1. Molecular chaperones
Molecular chaperones were originally discovered as proteins involved in the cellular
Introduction
and environmental stress conditions (Richter et al, 2010). The heat shock response is a universal defense mechanism of pro- and eukaryotic cells against an imbalance of protein homeostasis due to enhanced protein unfolding. However, chaperones are the only class of Hsps that show significant conservation between distantly related species. Chaperones are present in the cytosol as well as in all subcellular compartments and can be subdivided into highly conserved families based on their approximate molecular weight: Hsp40s, Hsp60s (chaperonins), Hsp70s, Hsp90s, Hsp100s and small heat shock proteins (sHsps). Besides these canonical classes, cells contain additional molecular chaperones, like Hsp33 and Trigger Factor, which are non-ubiquitous (Jakob et al, 1999). Although many chaperones are upregulated when unfolded proteins accumulate, most of them are also present at basal levels under physiological conditions (Richter et al, 2010). This indicates a constant need of molecular chaperones e.g. to assist de novo folding or to refold spontaneously denatured proteins. Moreover, cells contain a subpopulation of chaperones, which are constitutively expressed (e.g. Hsc70s or Hsc90s) and fulfill various housekeeping functions. A general feature of most molecular chaperones is that they interact with a broad range of non-native proteins (Richter et al, 2010). Such unfolded species expose hydrophobic amino acids, which are normally shielded in the native structure. Therefore, molecular chaperones are supposed to recognize hydrophobic patches, defined sequence motifs, or structural elements in non- native polypeptides (Hartl & Hayer-Hartl, 2009). Although all chaperones can prevent aggregation of their substrate proteins, only members of the ATP-dependent Hsp families can actively resolve inappropriate interactions and stimulate refolding. Hence, chaperones are commonly classified according to their mode of action. The so-called “foldases” (like Hsp60s, Hsp70s, and Hsp90s) bind transiently to unfolded client proteins and thereby prevent aggregation or promote refolding to the native state. Binding is followed by a controlled release that comes along with a drop in affinity of the chaperone to its substrate.
Such changes between different affinity states require conformational rearrangements driven by regulated cycles of ATP binding and hydrolysis (e.g. Hsp70 cycle, Figure 7) (Mayer, 2010). However, the precise contribution of ATP-dependent chaperones to the folding process is still unknown. In the case of Hsp70, Goloubinoff and coworkers proposed that the energy from ATP-hydrolysis is used to unfold misfolded or pre-aggregated polypeptides into intermediates that, upon dissociation from the chaperone, spontaneously refold to the native state (Sharma et al, 2010). By contrast, “holdases”, like the sHsps or Hsp40s, bind independent of ATP to unfolded proteins (Richter et al, 2010). They are assumed to play an important role during the early phase of the heat shock response and interact rapidly with their clients to prevent aggregation. However, the maintenance of an intact proteome requires the cooperation between individual chaperone systems (Balch et al, 2008). For
of aggregated proteins (Haslberger et al, 2008; Weibezahn et al, 2005). sHsps also cooperate with foldases, such as Hsp70 and Hsp100, to refold their substrates (Richter et al, 2010). In addition to chaperones involved in refolding processes, cells contain a chaperone network for de novo folding of newly synthesized proteins (Wegrzyn & Deuerling, 2005), including specialized chaperones that associate directly with ribosomes in order to interact with nascent polypeptides.
2.4.2. The Hsp70 chaperone machinery
Hsp70s constitute a ubiquitous class of chaperones involved in diverse cellular functions, including folding of newly synthesized proteins and refolding of stress-denatured proteins. In addition, they facilitate transport processes across membranes and mediate protein-protein interactions (Bukau et al, 2006; Hartl & Hayer-Hartl, 2009; Kampinga & Craig, 2010). Despite their functional diversity, Hsp70 chaperones share the same molecular architecture and show a high degree of sequence identity (Kampinga & Craig, 2010). Hsp70 chaperones consist of a nucleotide-binding domain (∼40 kDa) at their N-terminus as well as a C-terminal peptide-binding domain (∼25 kDa) (Bertelsen et al, 2009). Both domains are connected via flexible linker. The interaction between Hsp70s and their clients is governed by their nucleotide status. In the ATP-bound state, the on and off rates for substrate binding are fast.
However, ATP hydrolysis induces a conformational change and stabilizes the interaction with the substrate (Vogel et al, 2006a; Vogel et al, 2006b). Since the spontaneous transition between the two nucleotide states is very slow, Hsp70s work always in concert with cofactors, including J-proteins (also known as Hsp40 co-chaperones) and nucleotide exchange factors (NEFs), both of which control the ATP-dependent client-binding and - release cycle (Figure 7) (Bukau et al, 2006): Hsp40s recruit substrates to their Hsp70 partner and stimulate its ATPase activity. This leads to tight substrate binding by the Hsp70. In contrast, NEFs reset the cycle by triggering the exchange of ADP against ATP and thereby promote substrate release.
Hsp40s are much more diverse than Hsp70s (Kampinga & Craig, 2010). However, they all contain a common feature, the J-domain (∼70 aa), which forms a compact helical coiled-coil composed of four α-helices and contributes to the interaction with the Hsp70 partner (Greene et al, 1998; Jiang et al, 2007; Pellecchia et al, 1996). The loop region between the second and third helix bears a highly conserved His, Pro, Asp tripeptide sequence (also called HPD- motif) that is essential for the stimulation of the Hsp70’s ATPase activity (D'Silva et al, 2003;
Kelley, 1998; Tsai & Douglas, 1996; Yan & Craig, 1999).
