Characterization of the ribosome-associated complex RAC from S. cerevisiae

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Characterization of the

ribosome-associated complex RAC from S. cerevisiae

Dissertation

zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.)

an der Universität Konstanz

Fachbereich Biologie

vorgelegt von

Janina Horst

Tag der mündlichen Prüfung: 25.03.2011

1. Referent/in: Prof. Dr. Elke Deuerling 2. Referent/in: Prof. Dr. Andreas Marx

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Summary

Zuotin and Ssz from yeast are members of the conserved Hsp40 and Hsp70 family of molecular chaperones, respectively, and are thought to be involved in the folding of newly synthesized proteins. Hsp70s use an ATP-controlled cycle for substrate binding and release which is regulated by their Hsp40 co-chaperones. However, in contrast to canonical Hsp70/40 members, Zuotin and Ssz form an unusually stable heterodimer termed ribosome-associated complex (RAC), which is bound to the ribosome. Thus RAC acts as a co-chaperone for another ribosome-bound Hsp70, Ssb. The aim of this thesis was to elucidate the unusual pairing of RAC and the influence of ATP on the complex by investigating the conformational changes and dynamics in solution. These conformational studies were performed using amide hydrogen exchange (HX) combined with high resolution mass spectrometry (MS). Additional analyses were conducted using mutational analyses, fluorescence measurements and electron paramagnetic resonance (EPR) measurements.

HX-MS experiments with recombinant, purifiedS. cerevisiae Ssz, revealed specific ATP-induced conformational changes in Ssz individually and in complex. Upon ATP binding the nucleotide binding domain (NBD) was stabilized, resulting in a more compact structure. However, in contrast to canonical Hsp70s no influence of ATP binding on the substrate binding domain (SBD) was observed. Complex formation did not influence the nucleotide binding domain, but decreased the conformational dynamics within the SBD, indicating the engagement of the SBD in complex formation.

Biochemical binding analysis of the individual domains NBD and SBD, respectively, revealed an interaction of the NBD of Ssz with the N-terminal domain of its complex partner Zuo. This indicates an involvement of both domains (NBD and SBD of Ssz) in complex formation of RAC.

HX-MS experiments with recombinant, purified S. cerevisiae Zuo, revealed an ex- tremely flexible region at the beginning of the N-terminal region of Zuo (aa 1 - 79).

Upon complex formation this structure was conformationally stabilized, leaving only

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a short linker-like-region (aa 53 - 79) unstructured. Deletion of the highly flexible N- terminus of Zuo abolished stable association with Sszin vitroand caused a phenotype resembling the loss of Ssz’s function in vivo. Thus, the C-terminal domain of Ssz, the N-terminal extension of Zuo and their mutual stabilization are the major structural determinants for RAC assembly.

Moreover, HX-MS experiments revealed dynamic changes in the J-domain of Zuo upon complex formation that might be crucial for RAC’s co-chaperone function. Fur- ther EPR analysis using cysteine mutants of Zuo indicated that the conformational changes are not restricted to the interacting HPD motif itself but rather to the whole J-domain.

The results obtained in this work allowed to develop a model presenting a novel mechanism for converting Zuo and Ssz chaperones into a functionally active heterodimer.

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Zusammenfassung

Zuo und Ssz aus der Hefe gehören zu den hochkonservierten Familien der moleku- laren Hsp40 bzw. Hsp70 Chaperone. Es wird vermutet, dass Zuo and Ssz an der Faltung von neu synthetisierten Proteinen beteiligt sind. Hsp70 Chaperone nutzen einen ATP kontrollierten Zyklus für die Substratbindung und -freigabe, welcher von ihren Hsp40 Co-Chaperonen reguliert wird. Im Gegensatz zu typischen Hsp70/40 Mitgliedern bilden Ssz und Zuo ein ungewöhnlich, sehr stabiles Heterodimer, genannt Ribosome-Associated-Complex (RAC), welcher in der Lage ist an das Ribosom zu binden. Dort agiert RAC als Co-Chaperon für ein anderes ribosomgebundenes Hsp70 Chaperon, Ssb. Ziel dieser Arbeit war es, durch Untersuchung von Konformationsän- derungen und der Dynamik der Wechselwirkungen von Zuo und Ssz aufzuklären, wie diese ungewöhnliche Paarung von RAC zustande kommt und welchen Einfluss ATP auf den Komplex hat. Diese Studien wurden mit Hilfe von Amid Hydrogen Austausch (HX) Experimenten in Verbindung mit hochauflösender Massenspektrometrie (MS) durchgeführt. Anschliessende Untersuchungen wurden mit Hilfe von Mutationsanaly- sen, Fluoreszenzmessungen und Elektronenspinresonanz (ESR) Messungen ausgeführt.

Durch HX-MS Experimente mit rekombinant gereinigtem S.cerevisiae Ssz konnten spezifische ATP induzierte Konformationsänderungen in Ssz alleine wie auch im Kom- plex mit Zuo (RAC) gezeigt werden: Die Nukleotidebindedomäne (NBD) wird durch ATP-Bindung stabilisiert, was sich in einer kompakteren Struktur zeigt. Einen Ein- fluss der ATP-Bindung auf die Substratbindedomäne (SBD), wie sie typischer Weise bei klassischen Hsp70 Chaperonen zu finden ist, war jedoch nicht nachweisbar. Die Komplexbildung zeigte nur geringen Einfluss auf die NBD von Ssz, führte jedoch zu einer stark verringerten konformativen Dynamik innerhalb der Substratbindedomäne, was auf eine Beteiligung dieser Domäne an der Komplexbildung hindeutet. Biochemis- che Bindeanalysen der individuellen Domänen NBD und SBD von Ssz deuten darauf hin, dass auch die NBD mit der N-terminale Domäne von Zuo interagiert. Somit scheinen beide Domänen (NBD und SBD von Ssz) an der Komplexbildung von RAC

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beteiligt zu sein.

HX-MS Experimente mit rekombinant gereinigtem S.cerevisiae Zuo ergaben eine extrem flexible Region am Beginn der N-terminalen Domäne von Zuo (aa 1 - 79).

Die Bildung des Komplexes führte zu einer Stabilisierung der Konformation in dieser Struktur, lediglich ein kleiner, Linker ähnlicher Bereich (aa 53 - 79) blieb unstruk- turiert. Die Deletion dieses hochflexiblen N-terminus von Zuo verhinderte eine stabile Interaktion mit Ssz in vitro und erzeugt einen Phänotypen in vivo der dem eines funktionslosen Ssz ähnelte. Zusammenfassend sind die SBD von Ssz, die N-terminale Region von Zuo und deren gegenseitige Stabilisierung die grundlegenden strukturellen Faktoren für die Assemblierung von RAC.

Desweiteren zeigten die HX-MS Experimente eine dynamische Veränderung im Bere- ich der J-Domäne von Zuo, welche durch die Komplexbildung hervorgerufen wird und eine wichtige Rolle für die Co-Chaperon Funktion von RAC spielen könnte. Darauf aufbauende EPR Messungen mit Cysteinmutanten von Zuo wiesen darauf hin, dass die Konformationsänderungen sich wahrscheinlich nicht direkt auf das HPD-Motif selbst auswirken, sondern eher die gesamte J-Domäne betreffen.

Die Ergebnisse dieser Arbeit führten zu Entwicklung eines Modells, welches einen neuartigen Mechanismus präsentiert, wie die Chaperone Zuo and Ssz zu einem funk- tionellen Heterodimer assembliert werden.

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1. Introduction

The conversion of genetic information into functional proteins is a fundamental process in all living cells. Ribosomes are essential in this process as they translate the mRNA into the primary amino acid sequence. Subsequently the newly synthesized polypeptide folds into its native three dimensional structure to constitute a functional protein.

Molecular chaperones (folding helpers) are crucial for many proteins in this final step.

1.1. Ribosomes

Ribosomes are large structures found in all living cells. They are the working machinery for protein synthesis. A ribosome is composed of two subunits, that associate to form a functional machinery. Both subunits consist of large ribosomal RNAs (rRNA) and many ribosomal proteins (RPs) (Tab 1.1). Ribosomes from all kingdoms of life are functionally conserved, but differ in size, composition and the ratio of protein to RNA. In particular, functional domains show a high level of sequence conservation.

During evolution the eukaryotic ribosomes have increased in complexity by additional ribosomal proteins and extra rRNA segments, leading to a gain in molecular weight (Tab 1.1). Around 30 % of the total cell mass in bacteria and eukaryotes are ribosomes (Kramer et al., 2009).

Table 1.1.: RNA and protein composition of ribosomes.

Prokaryotic ribosome Eukaryotic ribosome

70S (2.4 MDa) 80S (4 MDa)

small subunit large subunit small subunit large subunit

(30S) (50S) (40S) (60S)

rRNA 16S 5S, 23S 18S 5S, 5.8S, 28S

RPs 21 (S1-S21) 31 (L1-L31) 33 (S1-S33) 50 (L1-L50)

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During the last few years a breakthrough in understanding the mechanism of protein synthesis was achieved (Ramakrishnan, 2002) by solving a number of ribosomal crystal structures at atomic resolution . First the high-resolution structure of an archaean 50S subunit (Ban et al., 2000) was solved, followed by the 30S structure ofT. thermophilus (Schluenzen et al., 2000; Wimberly et al., 2000). A few years later the atomic structure of a complete 70S ribosome in different functional states was solved (Schuwirth et al., 2005; Selmer et al., 2006; Korostelev et al., 2006; Berk et al., 2006), followed by ribosome structures with bound ligands (Blaha et al., 2009; Schmeing et al., 2009; Gao et al., 2009). Very recently a 5.5 Å model based on cryoEM and single-particle recon- struction (Armache et al., 2010) was published followed by a 4.15 Å resolution from crystal structure (Ben-Shem et al., 2010), providing insight into eukaryote-specific features of protein synthesis. Ribosomal translation can be divided into three phases:

initiation, elongation and termination, with each step being accompanied by a large number of specific translation factors. Bacterial ribosomes synthesize proteins with rates of about 20 aa (amino acids) per second, whereas eukaryotes translate at a speed of 5-9 aa per second only.

1.2. Protein Folding

Protein synthesis at the ribosome results in a linear amino acid chain. To properly fulfill their function inside the cell, proteins need to adopt a defined three dimensional structure. The amino acid sequence (or primary structure) of the polypeptide includes all the information necessary for the protein to assume the proper secondary and tertiary structure. This has been demonstrated by the classical protein refolding experiments of Anfinsen (Anfinsen, 1973). However, folding processes inside the cell are highly sensitive and error prone and the newly synthesized proteins constantly face the risk to undergo wrong or non-productive interactions. Since proteins are synthesized in a vectorial manner that proceeds from the N-terminal to the C-terminal part of the polypeptide chain, the N-terminal part of the polypeptide chain already faces the crowded environment of the cytosol, while the C-terminal part is still being synthesized by the ribosome. The exposure of hydrophobic side chains, especially the close proximity of the same type of nascent chain displaying the same unfolded features in the non-folded intermediate states, would render them prone to intermolecular aggregation (Frydman, 2001). Moreover, the molecular crowding of macromolecules

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1.3. Molecular Chaperones inside the cell can significantly accelerate nonproductive inter- and intramolecular in-

teractions (Minton, 2005; Ellis and Hartl, 1999; Zimmerman and Minton, 1993).

To protect the new polypeptide chain emerging from the ribosome against aggregation and misfolding events, molecular chaperones are present directly at the ribosomal exit tunnel, until translation is completed and full information for the native folding is available. (Netzer and Hartl, 1998; Hartl and Hayer-Hartl, 2002). However, about one third of newly synthesized proteins must be translocated across membranes to fulfill their functions. These proteins are targeted as nascent or loosely folded polypeptides to their translocation machinery, and only after reaching their intended destination they fold to their native states. Targeting is achieved primarily through the co-translational action of the trans-acting factor SRP (Driessen et al., 2001; Walter and Johnson, 1994), and through molecular chaperones like Hsp70 (Schatz and Dobberstein, 1996).

Along with molecular chaperones, nature has evolved folding helper enzymes, so called "folding catalysts". Slow steps in protein folding are catalyzed by two classes of enzymes, namely the cis/trans conformational interconversion of certain peptide bonds of the polypeptide backbone by peptidyl-prolyl cis/trans-isomerases (PPIases) and thiol/disulfide exchange processes characterized by reshuffling of covalent bonds by the protein disulfide isomerases (PDIs). By acceleration of slow refolding kinetics due to polypeptide restructuring, folding helper enzymes avoid the accumulation of reaction intermediates that are prone to form non-native protein conformations (Schiene and Fischer, 2000).

1.3. Molecular Chaperones

Most molecular chaperones are constantly present in the cell under normal conditions to assist folding and prevent aggregation during de novo protein synthesis. Alongside their function inde novo protein folding, chaperones are also involved in other cellular processes such as protein- targeting, degradation and signal transduction (Ellis and Hart, 2000). Under heat shock conditions, the level of many chaperones is elevated, leading to the term "heat-shock-protein" (Hsp) (Ellis, 1987), and they are involved in the prevention of aggregation of misfolded proteins as well as refolding. Hsps are present in all living organisms, and exist in several evolutionary conserved families, which are named according to their molecular weight; e.g. Hsp110, Hsp70, Hsp40 or the small Hsps.

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Molecular chaperones can be divided into two functional groups: holdases and foldases. Holdases bind the folding intermediates and prevent their aggregation but they do not support the folding of the substrate protein (Beissinger and Buchner, 1998). They are usually ATP independent proteins and include the group of small heat shock proteins (sHsp). Foldases, such as members of the Hsp70 family (Mayer et al., 2001) have been shown to actively support the folding of substrate proteins to their native state (Martin and Hartl, 1997). Foldases do not provide structural information nor do they become part of the folded product (Ellis and Hartl, 1999). They facilitate the folding process for the substrate protein to fold correctly on its own.

Foldases are usually ATP dependent and their binding affinity is often regulated by protein co-factors.

Chaperones can be further distinguished based on their cellular localization. Mem- bers of one class of chaperones perform their function as soluble proteins in the cytosol.

The second class comprises the ribosome-associated chaperones, which are found to interact with the nascent chain early during ongoing protein synthesis, indicating its specialization in de novo folding. The existence of ribosome-associated chaperones is a evolutionary highly conserved principle in eukaryotes and prokaryotes, although the types of chaperones differ completely.

1.4. Ribosome-associated chaperones

The best characterized ribosome-associated chaperone is the bacterial trigger factor (TF), which is found in prokaryotes and chloroplasts only. Recently it was shown that TFs interaction with ribosomes and nascent chains is modulated by the nature of the nascent chain itself in a dynamic reaction cycle (Kaiser et al., 2006; Raine et al., 2006;

Rutkowska et al., 2008). Eukaryotes lack TF, obviously it did not meet the demands of eukaryotic protein folding and was not maintained during evolution. Instead, a completely different set of proteins has evolved that shows no structural homology to TF. So far, two different ribosome-associated systems are found, which are assumed to act co-translationally in protein folding: A Hsp70/40 based chaperone machine and the nascent polypeptide-associated complex (NAC).

The yeast Saccharomyces cerevisiae contains both chaperone systems. The heterodimeric nascent polypeptide-associated complex (NAC) consists of α-NAC and a β- NAC subunit, which anchors the complex to the ribosome. The second chaperone

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1.5. The Hsp70/40 Chaperone System system is an Hsp70/40 chaperone system formed by two Hsp70 members Ssz and Ssb

and one Hsp40 member Zuotin (Zuo). Zuo and Ssz form a complex termed ribosome- associated complex (RAC), which is the subject of this study. Recently a functional connection between both chaperone systems NAC and RAC could be shown (Koplin et al., 2010), suggesting a parallel or overlapping pathway in de novo protein folding.

1.5. The Hsp70/40 Chaperone System

1.5.1. The Hsp70 family

Ssz as well as Ssb belong to the class of the 70 kDa heat shock protein (Hsp70) chaperones. Hsp70s are present in most compartments of eukaryotic cells, in eubacteria and in many archaea. They are the central part of a ubiquitous chaperone system and play an important role in different cellular processes (Mayer et al., 2001; Mayer and Bukau, 2005) such as the folding of newly synthesized or misfolded proteins as well as the prevention or reversion of protein aggregation (Deuerling et al., 1999; Goloubinoff et al., 1999; Mogk et al., 1999; Teter et al., 1999). Most quality control functions of Hsp70s require co-chaperones or chaperones of other families and play an important role in stress situations like heat-shock or harsh environmental conditions. All these functions of Hps70 chaperones are driven by their basic property to interact with short hydrophobic peptide sequences in an ATP dependent fashion. Under normal conditions Hsp70s take over housekeeping functions such as disassembly of protein complexes e.g. clathrin coats (Chappell et al., 1987; Ungewickell, 1985), import of proteins into the lumen of the endoplasmic reticulum (ER) (Lyman and Schekman, 1997; Matlack et al., 1999) and the mitochondrial matrix (Voisine et al., 1999), regulation of the heat-shock response and the control of regulatory proteins in cooperation with the Hsp90 system. In E.coli the main Hsp70 chaperone is DnaK which is constitutively expressed at a high level even under normal conditions. Under heat-shock conditions the expression is even higher, upregulated by the heat-shock transcription factor σ³². Higher eukaryotes contain two Hsp70 isoforms, the constitutively expressed Hsc70s and the stress induced Hsp70s which are under the control of the heat-shock response.

Most organisms contain multiple members of the Hsp70 family, some of which co- exist in the same cellular compartment. The yeast Saccharomyces cerevisiae contains four canonical Hsp70s and three fungal-specific ribosome-associated and specialized

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Hsp70s. Several studies demonstrated functional specificity among the Hsp70 isoforms, indicating significance of the requirement for multiple Hsp70s (Kabani and Martineau, 2008).

1.5.1.1. Structure of Hsp70 Chaperones

Hsp70 chaperones are structurally highly conserved and consist of two functional domains: a ~44 kDa N-terminal nucleotide binding domain (NBD) and a substrate binding domain (SBD) at the C-terminus of about ~25 kDa; the two domains are connected by a short hydrophobic linker (Bukau and Horwich, 1998) (Fig 1.1a). In the case of DnaK, an almost full length structure (aa 1 - 605) has been gained recently by the combination of NMR-RDC & X-ray analysis (Bertelsen et al., 2009) (Fig 1.1b).

Figure 1.1.: Domain Architecture and Structure of Hsp70 -(a)Domain organization of DnaK with nucleotide binding domain (NBD) and substrate binding domain (SBD) (b) Secondary structure representation of E.coli DnaK (PDB code 2KHO). In gray, secondary structure representation of SBD (393-605) and in blue, secondary structure representation of NBD (1-366) with lobes I and II, formed by subdomains A and B, respectively.

The nucleotide binding domain of Hps70s has a similar fold as monomeric actin, con-

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1.5. The Hsp70/40 Chaperone System sisting of two structural lobes forming a V-like shaped domain with a deep nucleotide-

binding cleft. Each lobe can be further divided into two subdomains IA, IB and IIA, IIB (Fig 1.1b). The binding pocket for the nucleotide and the required Mg²⁺ and K⁺ ions is formed at the bottom of the cleft by all four subdomains and the cross- connecting helices, contacting the adenosine nucleotide with two α- and β-binding loops (Flaherty et al., 1990). By X-ray structure analysis of the ATPase domains of bovine, human and bacterial Hsp70 only subtle conformational changes were found in response to nucleotide binding or hydrolysis (Flaherty et al., 1994; Harrison et al., 1997; Sondermann et al., 2001). In contrast, NMR data revealed a high flexibility in the NBD with an opening and closing motion of the nucleotide binding cleft (Zhang and Zuiderweg, 2004). Amide-Hydrogen-Exchange experiments on full length DnaK supports the findings by NMR analysis, revealing a significant flexibility in all four subdomains of the NBD relative to each other (Rist et al., 2006). The peptide binding domain of DnaK is divided into a β-sandwich subdomain which consists of two four-stranded α-sheets with four loops protruding upwards (two inner and two outer loops) and an α-helical extension that functions as a lid for substrate enclosure (Zhu et al., 1996). Binding of peptides occurs in between the loops L1,2 and L3,4 by forming a direct hydrogen bond with the peptide substrate backbone (Bertelsen et al., 1999;

Morshauser et al., 1999; Pellecchia et al., 2000; Stevens et al., 2003). Helix B constitutes a lid which closes the cavity through a salt bridge and two hydrogen bonds to the outer loops L3,4 and L5,6. The function of the subdomain formed by the distal part of Helix B and the 3 additional helices C, D, and E is still unknown.

1.5.1.2. Hsp70 functional cycle

To assist folding Hsp70 chaperones undergo repeated cycles of substrate binding and release between two nucleotide states (Bukau and Horwich, 1998; Hartl and Hayer- Hartl, 2002). During these cycles Hsp70 alternates between two different structural states, depending on the phosphorylation state of the nucleotide bound (Fig 1.2). In the ATP bound state, the lid over the peptide-binding cleft is in an open conformation:

The affinity for substrates is low, but the association and dissociation rates of the substrate are high (low-affinity state). In contrast, in the ADP bound form, the binding pocket is closed, resembling a high-affinity state for substrates, but has low association and dissociation rates.

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Figure 1.2.: Functional ATPase cycle of Hsp70 The cycle involves the high-substrate- affinity ADP state and the low-affinity ATP state. ATP hydrolysis is catalyzed by the syner- gistic action of J-domain-proteins of the Hsp40 family and the substrate. Nucleotide-exchange factors (NEF) accelerate nucleotide release and the subsequent ATP binding leads to substrate release.

However, the basal or substrate stimulated ATP hydrolysis rate is too low to be of physiological relevance (Flynn et al., 1989; Jordan and McMacken, 1995; McCarty et al., 1995). Therefore, J-domain proteins and nucleotide exchange factors (NEFs) regulate the cycling of Hsp70. Upon substrate binding to Hsp70 in the ATP-bound state, interaction with JDP stimulates ATP hydrolyzes of Hsp70, facilitating a tight peptide capture (Mayer et al., 2000). Substrate release from Hsp70 requires exchange of the bound ADP to ATP which is catalyzed by NEFs (Harrison et al., 1997). Rebinding of ATP resets Hsp70 to its initial low substrate affinity status, completing the reaction cycle.

1.5.2. Hsp40 co-chaperone family

The J-domain (Hsp40) protein family, is much larger and more heterogeneous than the Hsp70 family (Laufen et al., 1999; Sahi and Craig, 2007). Zuotin the second component of the ribosome-associated complex (RAC) belongs to this family. All Hsp40s use their

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1.5. The Hsp70/40 Chaperone System J-domain to stimulate the ATPase activity of their Hsp70 partners. The J-domains

consist of about 65 amino acids, forming an elongated domain composed of helical bundles with an absolutely conserved Hsp70 interacting motif His-Pro-Asp (HPD) exposed on a loop at one end (Qian et al., 1996; Cheetham and Caplan, 1998; Hennessy et al., 2005; Qiu et al., 2006).

Hsp40 proteins first act as a chaperone by binding unfolded peptides and forwarding them to their Hsp70 partner. Concurrently they stimulate the ATP activity of the Hsp70s, resulting in a more than 100-fold stimulated ATP hydrolysis compared to basal rate (Laufen et al., 1999). Despite the highly conserved J-domain, Hsp40s differ in their domain organization and localization within the cell (Walsh et al., 2004). Depending on their domain structure Hsp40s are organized in 3 different types (Fig 1.3).

Figure 1.3.: Structural classification of J-proteins Representation of type I, II and III J proteins from yeast aligned according to the N-terminus of the mature protein. The gray boxes represent each polypeptide and show the scale of the J domain (J, pink), glycine-rich region (G, yellow) and zinc-finger domain (Zn-finger, green). CTD, carboxy-terminal domain

Type I Hsp40 proteins have a compact helical J-domain that is linked by a glycine- rich region to a zinc-finger domain followed by a C-terminal domain. Type II proteins have a J domain linked by a glycine-rich region to a C-terminal domain, whereas type III proteins have the J-domain only. Type I and type II proteins are functionally similar. Both bind to non-native substrates for presentation to their Hsp70 partners and are found in the cytosol. In contrast, type III proteins are found at diverse intracellular sites such as the ER, mitochondria or attached to ribosomes. None of them has been shown to bind non-native polypeptides. Therefore, functionality as chaperone is unlikely. Zuotin fromS. cerevisiaeis a ribosome bound Hsp40, belonging to the type III J-domain proteins.

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1.6. The ribosome-associated chaperone triad in S.

cerevisiae

Together, Zuo and Ssz form the ribosome-associated complex (RAC) (Gautschi et al., 2001). Homologs of RAC are also found in mammals, indicating its conservation in the eukaryotic world (Otto et al., 2005; Hundley et al., 2005). In S. cerevisiae RAC together with the ribosome-attached Hsp70 Ssb forms a functional chaperone triad (Gautschi et al., 2002), while in mammals the cytosolic Hsp70 seems to complete this tripartite system. Ssb is encoded by two genes (ssb1, ssb2), which are more than 99%

identical (hereafter referred to as Ssb).

Figure 1.4.: The ribosome-associated chaperone triad inS. cerevisiaeconsists of Ssz (purple) and Zuotin (orange) that form the ribosome-associated complex (RAC). Only the Hsp40 Zuotin and the Hsp70 Ssb (blue) bind directly to the ribosome. Only Ssb can interact with the nascent polypeptide.

Yeast strains with a deletion of either one or all of its chaperone triad members display three phenotypes: salt sensitivity, cold sensitivity, and hypersensitivity to amino- glycosides such as paromomycin that block ribosomal protein biosynthesis and impair translational fidelity (Gautschi et al., 2001; Yan et al., 1998; Hundley et al., 2002). Al- though the specific function of the ribosome-associated triad in co-translational protein folding is still unknown, it is likely that the triad functions as a chaperone system, since all components are members of classical chaperone families.

In support of the likely role of the triad as a chaperone, the prokaryotic ribosome bound chaperone TF was shown to be able to substitute in vivo the function of the chaperone triad when expressed in a RAC deletion strain ofS. cerevisiae(Rauch et al.,

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1.6. The ribosome-associated chaperone triad in S. cerevisiae 2005; Ito, 2005). TF’s ability to rescue these cells is dependent upon its ability to bind

to ribosomes.

The chaperone triad has also been shown to bind to the ribosome. Both, Zuo and Ssb directly bind to the ribosome, whereas Ssz seems to be only indirectly attached via its stable interaction with Zuo (Pfund et al., 1998; Gautschi et al., 2001; Yan et al., 1998). Binding of RAC to the ribosome could be shown in close proximity of Rpl31 at the exit of the polypeptide tunnel in yeast (Peisker et al., 2008). However, stable Zuo ribosome binding is thought to be predominantly achieved via interaction with rRNA (Yan et al., 1998; Peisker et al., 2008). Furthermore, the chaperone triad can be crosslinked to a large variety of nascent chains, indicating its role in de novo protein folding (Pfund et al., 1998; Hundley et al., 2002). In these experiments, only Ssb contacts the nascent chain, but the cross-linking efficiency depends on the presence of RAC.

Recently, Ssb has been linked to the co-translational folding of ribosomal proteins and biogenesis factors, indicating a potential client repertoire for the ribosome- associated chaperone triad (Koplin et al., 2010). Furthermore, Ssb is assumed to mod- ulate production and assembly of ribosomal components, thereby adjusting ribosome production and proteins synthesis with the folding capacity of ribosome-associated chaperones. Additionally, Zuo too has been linked to the ribosome maturation (Al- banèse et al., 2010), indicating the involvement of the whole chaperone triad RAC/Ssb in the biogenesis of ribosomes.

Some very interesting features have been found to render the RAC chaperone system quite unique: Namely, Ssz and Zuo do not show a typical Hsp70/40 behavior as demonstrated previously for e.g. the bacterial DnaK/DnaJ chaperone system. Instead Zuo and Ssz form a highly stable complex. It is Ssz (Hsp70) that appears to regulate the activity of Zuotin (Hsp40) and it does so in a manner independent of Ssz’s ATPase activity. Zuotin thus requires the assistance of Ssz to efficiently stimulate the ATPase activity of Ssb (Hsp70) (Pfund et al., 2001; Huang et al., 2005).

1.6.1. Ssz

Although Ssz belongs to the Hsp70 family of chaperones, functionality and structure are changed compared to canonical Hsp70 to cope with its different demands in RAC.

Ssz’s ATPase binding domain is similar to this of canonical Hsp70s and was shown to

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bind ATP. However, the Ssz-ATP complex is rather unstable and no ATPase activity could be detected so far (Huang et al., 2005; Conz et al., 2007). A non ATP binding mutant version of Ssz (Ssz-LKA) fully complements the ssz∆ phenotype suggesting that ATP binding is not a prerequisite for function (Conz et al., 2007).

The substrate binding domain (SBD) of Ssz is shortened compared to canonical Hsp70s, missing the lid structure which closes the binding pocket through a salt bridge and two hydrogen bonds. Substrate binding within in the SBD could not be shown so far (Huang et al., 2005). Interestingly, Ssz lacking its peptide-binding domain fully complements ssz∆ mutants, but fails to stably interact with Zuo, indicating that a tight interaction between Ssz and Zuo is not strictly required in vivo (Huang et al., 2005; Conz et al., 2007). However, the combination of the C-terminal deletion and the inability of the N-terminal domain to bind ATP results in a loss of function phenotype in vivo (Conz et al., 2007).

Figure 1.5.: Schematic model of the domain structure of Ssz~58 kDa, 538 aa

1.6.2. Zuo

Zuo, as a type III J-protein, holds a highly conserved J-domain, but differs in its domain organization compared to other Hsp40 members. At the N-terminus a so called Zuotin-homology-region is a characteristic of all Zuo homologs, however, so far no functionality could be assigned. At the C-terminal region Zuo contains an internal, highly charged region localized between aa 284 - 360, which was found to be important for Zuo’s interaction with the ribosome as well as for its ability to interact with nucleic acids (Yan et al., 1998). A mutant version of Zuo lacking aa 282-331 was still able to form a complex with Ssz, but the complex was not bound to the ribosome (Peisker et al., 2008). However, a severe mutation, replacing a total of 15 aa to alanines in the region aa 296 - 305, showed only minor effects on Zuo-ribosome interaction (Peisker et al., 2008). Instead a chimera mutant, containing the J-domain from the cytosolic Hsp40 protein Ydj1 and a part of the charged region spanning aa 306-363, could be

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1.7. Analysis of Protein Dynamics shown to stably interact with the ribosome (Scior, 2008).

Figure 1.6.: Schematic model of the domain structure of Zuo~49 kDa, 433 aa

Although several studies support the function of RAC/Ssb as a chaperone machine, the actual role of this system in the process ofde novoprotein folding is still unresolved.

It is unknown how the chaperone system is targeted to the ribosome and how it acts on nascent polypeptides on the ribosomal surface. One aspect which is of decisive importance for the functionality of the triad is the mode of assembly of Ssz and Zuo into the RAC heterodimer, for proper interaction with Ssb. It also remains unclear whether the nucleotide ATP plays a role in this binding/association, and what are the particular dynamics of the protein-protein interaction of Zuo with Ssz.

1.7. Analysis of Protein Dynamics

To monitor conformational properties of proteins on a global level, a variety of methods are available such as far-UV circular dichroism (CD) (Pelton and McLean, 2000), tryptophan fluorescence, infrared spectroscopy (Barth, 2000) as well as small-angle X-ray scattering and cryo-electron microscopy (Koch et al., 2003).

In order to determine detailed information about amino acid structures within proteins, X-ray crystallography is capable of determining resolution in the range of 1-2 Å(Moffat, 2001). However, this can only be achieved using highly ordered crystallized protein.

Since a long time, attempts were made to crystallize RAC, Zuo and Ssz, respectively, but so far no crystals could be obtained.

Since many years multi-dimensional nuclear magnetic resonance (NMR) spectroscopy is used to monitor protein dynamics in solution. This is possible under steady state conditions and on multiple time scales at the amino acid level (Dyson and Wright, 2004). However, NMR experiments have long been limited to small, soluble proteins up to 35 kDa. New technologies (high magnetic fields and cryoprobes) and new pulse sequences now allow to analyze proteins up to 100 kDa and possibly even very large structures such as ribosomes, depending on the system used and the biological question

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to answer (Henzler-Wildman and Kern, 2007).

Determination of global or local dynamics within a protein can also be done by hydrogen exchange (HX), which can be coupled to either mass spectrometry or NMR spectroscopy. It provides a powerful tool for analysis on a high temporal resolution, reaching from the millisecond range up to hours. A method to detect local flexibility of distinct atoms within molecules is electron paramagnetic resonance (EPR). Mass spectrometry, HX measurements and EPR were the methodology used in this work to investigate conformational changes within chaperone proteins. Details of mass spectrometry, HX measurements and EPR can be found in the appendix. A short introduction into HX-MS and the setup used in this study will be described in the following section.

1.7.1. Amide hydrogen exchange and mass spectrometry (HX-MS) Mass spectrometry has proven to be a valuable analytical tool to measure amide hydrogen exchange to monitor protein dynamics and conformational changes (Hoofnagle et al., 2003; Kaltashov and Eyles, 2002; Wales and Engen, 2006). A major advantage of mass spectrometry is that it separates coexisting conformational states of a protein by their differences in deuterium contents and hence molecular masses. This feature of mass spectrometry is unique as other techniques, such as e.g. NMR which is also used to analyze amide hydrogen exchange, only measure average behavior of different conformational states. Additional advantages of mass spectrometry are the lower amount of sample, required for one run, the unlimited mass range for larger proteins and the ability to monitor individual peptides and proteins in a mixture. Due to its coupling to HPLC, ESI-MS is the most commonly used ionization method in HX studies.

In a typical HX experiment, undeuterated proteins are labeled with deuterium by dilu- tion in a D₂O buffer. Incubating for different time intervals in D₂O and monitoring the isotope exchange as a function of exchange time provides information on the conformational dynamics of a protein under equilibrium conditions. In an established labeling protocol (Zhang and Smith, 1993) the exchange reaction is performed at physiological conditions for certain amounts of time and subsequently quenched by lowering the pH to 2.5 and the temperature to 0^◦C, which decreases HX rates at the peptide amide linkages by up to five orders of magnitude. The half-life for amide hydrogen exchange under these quench conditions is 30 - 120 min. This allows to analyze the sample by

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1.7. Analysis of Protein Dynamics reversed phase HPLC coupled to ESI-MS, causing minimal loss of deuterons on the

amides.

Figure 1.7.: Experimental setup of an amide hydrogen exchange experiment com- bined with mass spectrometryThe protein is equilibrated under physiological conditions and then diluted into D2O buffer for certain time intervals (tHX). After a quench at low pH and low temperature, the deuterated protein is injected into a HPLC for desalting and sub- jected to ESI-MS. Measurement of deuteron incorporation in full-length protein mass spectra results in information about global exchange kinetics. In addition, the deuterated protein can be digested during the HPLC step by pepsin and the deuteration kinetics of the peptic peptides (i.e. shifts of the isotopic cluster to higher m/z by deuteration time) provides information about slow and fast exchanging regions in the protein.

To determine the exchange behavior of a protein on a global level, deuteron incorporation of full-length mass can be determined. To obtain information of regions in the protein that exchange either slowly or rapidly, a proteolytic digest of the deuterated protein and MS analysis of the resulting peptides can be accomplished. The most commonly used acidic protease is pepsin. Pepsin provides peptides with an average length between 4-15 amino acids. A disadvantage of this procedure is a continuous loss of deuterium label during HPLC analysis in aqueous buffers. This effect called

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"back exchange" can be reduced by fast sample processing at low temperatures (Feng et al., 2006).

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2. Aims of the work

In all living cells, the correct folding of newly synthesized proteins requires the assistance of molecular chaperones. In Eukaryotes, a conserved tripartite Hsp70/40- chaperone system associates with ribosomes and nascent polypeptides and is assumed to control the early folding steps of newly synthesized proteins. This system consists of the ribosome-bound heterodimeric RAC (ribosome associated complex) which is formed by Ssz (a Hsp70 chaperone) and Zuotin (a Hsp40 chaperone). The third partner is another Hsp70 protein which can be either ribosome tethered as in yeast (called Ssb) or a soluble partner in the cytosol.

So far little is known about RAC and its function. As an Hsp70 chaperone Ssz contains an ATPase domain, but so far hydrolysis of ATP was not shown. Its C-terminal peptide binding domain (PBD) is shortened, missing a lid-like structure, which closes the binding pocket of canonical Hsp70s. Interestingly it forms an unusual, stable complex with Zuo. Zuo, as an Hsp40 chaperone, contains a J-domain to stimulate the ATPase activity of its partner Hsp70, which is not Ssz, but another Hsp70 chaperone, Ssb. In addition it has a highly charged region at the C-terminal part which is supposed to accomplish ribosome binding and an N-terminal extension of unknown function, which is conserved in all Zuo-homologs.

This work aimed to gain insight into the structural arrangement of RAC from the eukaryoteSaccharomyces cerevisiae.

To this end, the molecular basis for the unusual pairing of the Hsp70/40 complex RAC was investigated using hydrogen exchange (HX) experiments combined with high resolution mass spectrometry (MS). In particular, (I) the precise role of the nucleotide ATP and its influence on the conformation of Ssz was analyzed, (II) the conformational rearrangements of Zuo and Ssz during heterodimerization were investigated and (III) structural elements which are involved in complex formation were identified.

Mutational analysis based on biochemical andin vivomethods were done to further characterize functional structures. The focus of this part of the thesis was on the

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analyze of the N-terminal domain of Zuotin and its function in complex formation.

To study the behavior of the J-domain of Zuo with respect to its flexibility upon complex formation, electron paramagnetic resonance (EPR) spectroscopy was used.

In order to measure the kinetics of binding of RAC to the ribosome a technique based on fluorescently labeled cysteine mutants of Zuo was to be established. This method represents an alternative "in solution" assay as compared to surface dependent BiaCore measurements.

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3. Results & Discussion

3.1. Conformational studies of Ssz and Zuo by HX-MS

In order to analyze the conformational flexibility and stabilizing ability of the nucleotide ATP on the complex, native state amide hydrogen exchange (HX) technology was used in combination with mass spectrometry (MS) to map the solvent accessibility of the backbone amides in S. cerevisiae RAC in dependence of bound nucleotide.

All proteins used in this study were cloned into pSUMO vector and recombinantly expressed in E. coli. The protein purification was accomplished using Ni²⁺ affinity chromatography and ion exchange chromatography. The N-terminal HIS₆-SUMO-tag was cleaved by Ulp to gain authentic protein.

Data analysis of the complex mass spectra is challenging; all data sets have to be analyzed manually. By hand selections are subject to variations and can thus lead to slightly different results, depending on the person who analyzes the data and even when one particular person analyzes the data several times. For this thesis, data were partially revised and can thereby slightly differ from the figures already published.

However, there are only minor changes which do not influence the main conclusion of the data published.

3.1.1. Nucleotide dependent solvent accessibility changes in Ssz Purification of Ssz turned out to be demanding. Pure protein was obtained in small amounts only, as a result of continuous loss of protein due to aggregation, indicating a problem in stability during purification procedure. Although Ssz does not efficiently hydrolyze ATP (Conz et al., 2007), addition of ATP reduced the aggregation problems during purification of Ssz, suggesting a stabilizing role of the nucleotide. However, it was not possible to gain nucleotide-free Ssz by removing the nucleotides ATP and ADP (data not shown) after purification, as then the protein tended to rapidly aggregate.

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Therefore, no nucleotide-free Ssz was used in this study and whenever the term "-ATP"

is used, the protein contained residual ATP of unknown quantity from the purification.

To analyze overall kinetics of the HX reaction of Ssz, initially deuteron incorporation into wild type Ssz in the absence of additional nucleotides was measured. To this end Ssz was incubated for different time intervals (10 s - 2 h) in D₂O. The reaction was quenched by lowering the pH to 2.2 and shifting the temperature to 0^◦C, and subsequently analyzed on a HPLC electrospray ionization tandem mass spectrometry setup (HPLC-MS) (Rist et al., 2003, 2005b). Deuteron incorporation into full-length Ssz occurs at an unusually fast exchange kinetic. Approximately 73% of amide protons after 2 min and 84% after 30 min (Fig 3.1 a) were exchanged. In contrast, a well folded native protein typically shows a protected core of 40 to 50% of all amide protons after 1h incubation in D₂O (Rist et al., 2006). This indicates a highly dynamic and loosely folded protein conformation of Ssz, which also explains the pronounced aggregation phenomenon during purification.

To assess the effect of a nucleotide on the solvent accessibility of amide protons of Ssz, 0.6 mM ATP (concentration was chosen based on the limited reaction volume) was added to the reaction, incubated in D₂O and analyzed after different time intervals by the HPLC-MS setup (for details see section 1.7.1). The presence of ATP resulted in a strongly decreased deuteron incorporation, leaving 55% of amide protons unexchanged after 2min, rather comparable to that observed for canonical Hsp70 chaperones like DnaK (Fig 3.1 a)(Rist et al., 2006; Andreasson et al., 2008b). However, the nucleotide- induced stabilization vanished with longer incubation times in D₂O, implying that the nucleotide is released within minutes (Fig 3.1 a). Similar results were obtained using ADP as nucleotide (data not shown), suggesting a transient binding of nucleotides to the NBD of Ssz.

To localize regions in Ssz which exchange more slowly in the presence of nucleotide, the analysis of the HX reaction on the HPLC-MS setup was done including a column with immobilized pepsin. The acidic protease is active under quench conditions at 0^◦C and pH 2.2, and generates on-the-fly peptide fragments of Ssz. The average peptide size was around 15 residues and the overall sequence coverage with peptides that could be detected in almost every run was about 85% (Fig 3.1 b).

Secondary structure representation Ssz is shown color-coded according to the difference in deuteron incorporation between Ssz in the presence (D[Ssz+ATP]) and absence

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3.1. Conformational studies of Ssz and Zuo by HX-MS

Figure 3.1.: Conformational flexibility ofS. cerevisiae Ssz. (a)Time-dependent mass increase of full-length Ssz in the absence of nucleotide (open circles) and in the presence of ATP (filled circles). (b)Amino acid sequence of Ssz and peptic peptides used for the analysis (underlined). Sequence coverage was about 85%. (c)Secondary structure representation of Ssz (residues 1-533 modelled on bovine Hsc70 NBD 1HKM using Swiss-Model (Guex and Peitsch, 1997; Peitsch, 1996)). Peptides colored according to deuteron difference in % of D[Ssz+ATP]

- D[Ssz-ATP] as indicated on the left side after 10 s. The image was created in PyMOL (http://www.pymol.org). (d)Schematic model of the domain structure of Ssz.

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(D[Ssz-ATP]) of nucleotide after 10 s of HX (Fig 3.1 c). A detailed peptide pattern plot is attached in the appendix (Fig A.5).

In the presence of nucleotide the lobe II of the nucleotide binding domain (NBD) of Ssz gets strongly stabilized (Fig 3.1 c, 10 s), indicating a specific compaction of this part of the NBD. Only small effects were found within lobe I of the nucleotide binding domain. However, almost no effect on the C-terminal substrate binding domain (SBD) was found. This is in contrast to canonical Hsp70s where ATP binding results in an opening of the SBD, allowing binding to hydrophobic patches of substrates (Rist et al., 2006). Since so far no peptide binding within the SBD of Ssz was demonstrated (Huang et al., 2005), this further implicates a role for the C-terminus of Ssz that is different from that shown for canonical Hsp70s. These data show a specific influence of ATP on Ssz, mainly in the nucleotide binding domain.

3.1.2. Conformational flexibility of Zuotin

Purification of Zuo results in good amounts (25 mg) of pure protein, indicating a more stable protein compared to Ssz. However the purification has to be fast in order to circumvent degradation with degradation products appearing at a marginally smaller molecular weight. In order to analyze the conformational flexibility of Zuotin, HX-MS experiments as described for Ssz were performed. Deuterons incorporated into full length Zuotin to 64% after 2 min, indicating high structural dynamics of the protein.

Addition of ATP had no effect, confirming that Zuotin is ATP independent (Fig 3.2 a)

To localize fast and slow exchanging regions within Zuotin, the analysis of the HX reaction on the HPLC-MS setup was performed including a column with immobilized pepsin. The average peptide size was around 15 residues and the overall sequence coverage with peptides that were detected in every run was about 70% (Fig 3.2 b). Due to the measurement conditions, highly charged peptides are unaccessible for analysis.

Therefore, information about the charged region, spanning residues 289 - 366, are missing. A representation of the domain architecture of Zuo is shown in Fig 3.2 d.

The deuteron incorporation in the N-terminal part of Zuotin is striking (residues 1- 79). Within 10 sec, the whole region is almost completely (average of 96%) exchanged, displaying a highly flexible and loosely folded structure. In contrast, the rest of the protein appears more folded, leaving 35% of amide protons unexchanged after 10 min

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3.1. Conformational studies of Ssz and Zuo by HX-MS

Figure 3.2.: Conformational flexibility of S. cerevisiae Zuotin. (a)Time-dependent mass increase of full-length Zuo in the absence of nucleotide (open circles) and in the presence of ATP (filled circles). (b) Amino acid sequence of Zuo and peptic peptides used for the analysis (underlined). Sequence coverage was about 70%. The charged region is marked in red. (c)Percentage of deuteron incorporation in Zuo after different incubation times (10 s to 30 min) in D₂O. The data were resolved to individual peptic peptides as indicated by the start and end residue numbers of the corresponding segments. (d)Schematic model of the domain structure of Zuo.

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(Fig 3.2 c).

From these primary data it can be suggested that both proteins, Zuotin and Ssz, when analyzed individually, are highly dynamic and partially unstructured. This is in contrast to other Hsp70 or Hsp40 members which are much more structured but not involved in the formation of a stable complex such as RAC.

3.2. HX-MS analysis of conformational alterations in RAC upon complex formation

In order to analyze complex formation, the exchange behaviors of the protein of interest measured individually and in complex were taken and compared. Differences regarding the time dependent deuteron incorporation will then reveal hints to conformational changes induced by complex formation or to the complex interfaces. For all final data the measurement in complex was always subtracted from the measurement of the individual protein. Thus stronger protection in the complex is indicated by a positive prefix (+) and deprotection within the complex by a negative prefix (-). To securely analyze complex induced changes only, all samples were measured in the presence of 0.6 mM ATP. Since ATP is bound only transiently to the N-terminal domain of Ssz, longer incubation times might still influence the nucleotide state of Ssz and therefore only short HX time measurements should be free of ATP dependent effects. The data analysis is therefore mainly focused on the short HX reaction times 10 s and 2 min.

3.2.1. Complex formation dependent solvent accessibility changes in Ssz

First the overall change in deuteron incorporation of Ssz when in complex with Zuo (RAC) was analyzed. Therefore, deuteron incorporation measurements with Ssz purified in complex with Zuotin (designated hereafter Ssz(RAC)) and in the presence of ATP to specifically report about the effect of Zuo binding were performed. Initial measurements analyzed on the HPLC-MS setup revealed a pronounced exchange protection of Ssz(RAC) (Fig 3.3 a). Ssz(RAC) incorporated 59 deuterons less than Ssz alone in a 2 min reaction. This protection persisted up to HX reaction times of 2 h, indicating that the complex is kinetically very stable.

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3.2. HX-MS analysis of conformational alterations in RAC upon complex formation

Figure 3.3.: Conformational alterations in Ssz upon complex formation. (a) Time- dependent mass increase in full-length Ssz in the presence (filled triangle) and absence (filled circle) of its complex partner Zuo. (b)HX properties of Ssz uncomplexed and bound to Zuo.

Mass spectra of representative peptic peptides of Ssz incubated in H₂0 (0%) or for 10 s in D2O buffer in the uncomplexed state (Ssz), in the complexed state (Ssz(RAC)), and in the reconstituted complex (Ssz(Zuo)). 100% designates a control spectrum for the same peptides obtained from fully deuterated RAC. The samples during HX contained 0.5 µM Ssz, or 0.8 µM RAC, or 0.3 µM Ssz and 0.6 µM Zuo, and 0.6 mM ATP. (c) Difference in deuteron incorporation between uncomplexed Ssz and Ssz(Zuo) (grey bars) or Ssz(RAC) (black bars) after 10 s of incubation in D2O buffer. The data were resolved to individual peptic peptides as indicated by the start and end residue numbers of the corresponding segments.

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3.2.2. Localization of complex formation-induced conformational alterations in Ssz

To localize the specific regions within Ssz that are affected by complex formation and thus are potentially involved to form the interface surface, the analysis of the HX reaction on the HPLC-MS setup was performed including a column with immobilized pepsin. Comparison of the peptide profile of Ssz and Ssz(RAC) in the presence of ATP revealed two categories of peptides: (I) peptides that incorporated less deuterons in the complex than in Ssz alone (HX protection), and (II) peptides that exhibited no changes between the two conditions. Examples of mass spectra of the analyzed peptides are shown in Fig 3.3 b, displaying either strong HX protection (e.g. 499 - 510) or no changes in their HX pattern (e.g. peptide 102 - 117). Particularly strong HX protection effects were found in regions 396 - 447 and 499 - 538, located in the SBD of Ssz (Fig 3.3 c). These regions localized to the predicted β-sheet subdomain and α-helical lid region of the SBD (Fig 3.1 c), respectively and are likely to include the major Zuo interaction surface. Additional protection was observed for peptides encompassing peptides 171 - 179 and 209 - 216 (Fig 3.3 c, black bars) of the ATPase domain of Ssz(RAC). Importantly, purified Ssz co-incubatedin vitro with Zuo, designated hereafter Ssz(Zuo), yielded results very similar to those obtained with purified RAC (Fig 3.3 c, gray bars). This confirms that purified Zuo and Ssz properly assem- ble into RAC, and that uncomplexed Ssz and Zuo can be taken asbona fide reference components.

3.2.3. Complex formation dependent solvent accessibility changes in Zuo

Deuteron incorporation measurements with Zuotin purified in complex with Ssz (designated hereafter Zuo(RAC)) revealed a small but significantly decreased deuteron incorporation in Zuo(RAC) at short HX times (Fig 3.4 a). The protection of 13 residues at 10 s HX in RAC decreased to 3 amide protons after 2 h, indicating that primarily fast exchanging regions within Zuo are affected by complex formation.

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Figure 3.4.: Conformational alterations in Zuo upon complex formation (a) Time- dependent incorporation of deuterons in full length Zuo in the absence (open circle) and presence (open triangle) of its complex partner Ssz. (b)Mass spectra of representative peptic peptides of Zuo incubated in H2O (0%) or for 2 min in D2O in the uncomplexed state (Zuo), in the complexed state (Zuo(RAC)), and in the reconstituted complex (Zuo(Ssz)). 100%

designates a control spectrum for the same peptides obtained from fully deuterated RAC. (c) Difference of deuteron incorporation between Zuo and Zuo(RAC) after different incubation times (10 s to 30 min) in D2O. The data were resolved to individual peptic peptides as indicated by the start and end residue numbers of the corresponding segments(d)Kinetics of deuteron incorporation into selected segments of Zuo in the absence (filled circle) and the presence of Ssz (Zuo(RAC), filled square; Zuo(Ssz), filled triangle).

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3.2.4. Localization of complex formation-induced conformational alterations in Zuo

The next demand was to identify regions of Zuo involved in Ssz binding and therefore to compare Zuo with Zuo(RAC) or Zuo in the complex assembled in vitro (Zuo(Ssz)) in HX pepsination experiments. Interestingly, the N-terminal region, covering residues 1 - 51, which showed very fast HX exchange in uncomplexed Zuo (Fig 3.2 c) became much less dynamic in the presence of Ssz (Fig 3.4b). In particular, peptides covering aa 11-35 showed a strong decrease in HX exchange (Fig 3.4 c,d; peptide 11-35). It is most likely that this region constitutes the potential binding site for Ssz. In contrast, peptides covering aa 52 - 79 did not show any change in their deuterium exchange pattern in Zuo compared to Zuo(RAC) (Fig 3.4 d; peptide 52 - 74). This indicates a very flexible region which exchanges almost 100% of its hydrogens within 10 sec, and is not affected by complex formation. Further, but less pronounced, protection effects were observed in the the C-terminus of Zuo (e.g. peptides covering aa 394 - 428)(Fig 3.4 b). However, so far no functional relevance of these changes can be assigned. However, a third category (see section 3.2.2) of peptides can be found by comparison of peptide HX kinetics: (III) peptides that incorporated more deuterons in Zuo(RAC) than in uncomplexed Zuo. This HX deprotection was localized exclusively in the J-domain of Zuo. Specifically, peptides covering the region of residues 99 - 168 showed a bimodal HX behavior (Fig 3.4 c), indicating that opening of the structure in this region is slower than the hydrogen exchange reaction itself. In the presence of Ssz, the rate of opening of the Zuo J-domain was increased, suggesting an activation mechanism for Zuo upon complex formation.

3.2.5. Influence of ATP on the complex

Since ATP had a strong influence on Ssz, it was of interest to find out whether it also influences the complex. Therefore, full length measurements of Ssz(RAC) were done in the presence and absence of ATP. Comparison of the respective HX kinetics showed that Ssz(RAC) was also stabilized by the addition of nucleotides in its complexed form (Fig 3.5 a). Since full length measurements of Ssz(RAC) showed such a significant effect of ATP, the next step was to localize the region stabilized within Ssz(RAC) in the presence of nucleotide. Therefore HX kinetics of respective peptides of Ssz(RAC) in the presence and absence of 0.6 mM ATP were compared.

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Figure 3.5.: Influence of the presence of nucleotide on RAC (a) Time-dependent incorporation of deuterons in full length Ssz(RAC) in the presence (filled triangle) and absence (open triangle) of ATP. (b)Time-dependent incorporation of deuterons in full length Zuo in the absence (open triangle) and presence (filled triangle) of ATP. (c) Difference in deuteron incorporation between Ssz in the absence and the presence of nucleotide (gray bars) and difference in deuteron incorporation between Ssz(RAC) in the presence and absence of nucleotide (black bars) after incubation in D₂O for 10 s. (d) Difference in deuteron incorporation between Ssz and Ssz(RAC) in the presence of nucleotide for different incubation times in D2O (10 s and 2 h), the individual peptides are indicated by the start and end residue numbers of the corresponding segments.

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A similar protection pattern as for Ssz, in the presence of ATP was found for short HX incubation times (10s) (Fig 3.5 c; black bars). Almost exclusively the lobe II of the NBD is strongly HX protected whereas little protection is found in lobe I of the NBD and no effects were observed in the SBD of Ssz. The protected segments do not coincide with any regions affected by Zuo binding (Fig 3.3 c). Thus ATP binding predominantly affects the ATPase domain of Ssz, in complex as well as uncomplexed, involving regions that are distinct from the segments which are stabilized by Zuo binding. Interestingly, complex formation has an ATP and time dependent influence on the NBD of Ssz. For short HX times (10 s) complex formation has only minor influence on the NBD, since this domain is clearly protected by the bound nucleotide (Fig 3.5 d; black bars). However, for longer incubation times the pattern changes significantly. While in Ssz the NBD gets more and more unprotected due to the transient binding of ATP, the NBD in Ssz(RAC) remains considerably protected (Fig 3.5 d, gray bars). In contrast to Ssz, the presence of ATP affected neither Zuo alone nor in complex with Ssz (Fig 3.5 b).

3.2.6. Discussion of HX-MS data

This study revealed new insights into both, the conformational dynamics and the complex interface of RAC. Although Zuo and Ssz are members of the Hsp40 and Hsp70 chaperone family, respectively, they display a number of atypical features compared with their canonical homologs. In particular, the formation of a stable Hps70/40 complex is unique and was yet not understood.

The subunits Ssz and Zuo were found to be highly dynamic individually, but are strongly stabilized upon complex formation.

Ssz is stabilized both, by ATP binding and by Zuo binding. Complex formation affects mostly the SBD whereas binding of ATP to Ssz leads to a compaction of the ATPase domain. This suggests that the respective ligands affect distinct and different regions within Ssz. ATP stabilized the ATPase domain of Ssz when uncomplexed and also when in complex with Zuo. This was not the case for Zuo; ATP binding in Ssz seems not to be recognized by Zuo. Therefore distinct functions of the binding of Zuo and ATP to Ssz can be concluded.

The pattern of nucleotide-induced HX protection in Ssz differs from those observed for classical Hsp70s, such as DnaK (Rist et al., 2006; Andreasson et al., 2008a,b). The

Characterization of the ribosome-associated complex RAC from S. cerevisiae