Structural insight into the environment of the serine/threonine protein kinase domain of titin

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Structural insight into the

environment of the serine/threonine protein kinase domain of titin

Dissertation

zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften

an der Universit ¨at Konstanz

Mathematisch-Naturwissenschaftliche Sektion Fachbereich Biologie

vorgelegt von

Simone M ¨uller

Tag der m ¨undlichen Pr ¨ufung: 30.03.2006 1. Referent: Prof. Dr. Wolfram Welte 2. Referent: PD Dr. Matthias Wilmanns

3. Referent: Prof. Dr. Helmut Plattner

Konstanzer Online-Publikations-System (KOPS) URL: http://www.ub.uni-konstanz.de/kops/volltexte/2006/2203/

URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-22032

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List of Figures

1.1 Striated muscle . . . 2

1.2 Titin . . . 4

1.3 Titin kinase . . . 10

1.4 Titin kinase downstream signalling pathway . . . 12

1.5 Domain architecture of MURF family members . . . 14

2.2 Diffraction patterns of A168-A169-A170 . . . 25

2.3 Crystals of A168-A169 . . . 27

2.4 Overall structure of the tandem Ig domains . . . 29

2.5 Superimposition of the tandem Ig domains . . . 30

2.6 Schematic representation of the continuousβ-strand interaction . 33 2.7 Tandem domain interface . . . 34

2.8 Structure-based sequence alignment of titin Ig-like domains . . . 35

2.9 Superimposition of A169 and 1Flt-D2 . . . 36

3.1 Schematic domain arrangement representation . . . 42

3.3 Crystal of M1 . . . 44

3.4 Ramachandran plot for the M1 structure . . . 47

3.5 Structure-based sequence alignment on M1 . . . 48

3.6 Structure of M1 . . . 48

3.7 Electron density of M1 . . . 49

3.8 Superimposition of M1, telokin, and Ig26 . . . 50

3.9 Model of titin kinase with M1 . . . 52

4.2 Model of PB1 interaction . . . 55

4.3 Crystals of the PB1 domain of NBR1 grown in ammonium sulphate 58 4.4 Crystals of the PB1 complex . . . 63

4.5 SEC of p62 PB1 (DA) and p62 PB1 (DDAA) . . . 66

4.6 SEC of the complex of the two PB1 domains . . . 67

4.7 ITC of NBR1 and p62 (DDAA) . . . 68

4.8 Structure of NBR1 PB1 . . . 70

4.9 Electron density of NBR1 PB1 . . . 72

4.10 Superimposition of PB1 domains . . . 73

4.11 Sequence alignment of PB1 domains . . . 74 v

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vi List of Figures

4.12 Anomalous difference fourier map . . . 75 4.13 Overall structure of the NBR1/p62 PB1/PB1 heterodimer . . . . 76 4.14 Stereo view of the main PB1 interaction site . . . 77 4.15 Electrostatic potential of NBR1 PB1 and p62 PB1 . . . 78 4.16 Molecular interaction between p62 PB1/PB1 heterodimer . . . . 78 A.1 Expression of 170TK and TKM1 . . . 93 A.2 Anion exchange chromatography of 170TK and TKM1 . . . 94 A.3 SDS-PAGE of 170TK and TKM1 . . . 95

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List of Tables

1.1 Interaction partners of p62 . . . 17

2.1 X-ray data collection statistics of A168-A169 . . . 26

2.2 Selenium sites in A168-A169 . . . 27

2.3 Refinement statistics of A168-A169 . . . 28

2.4 Structural comparison of titin Ig domains . . . 31

3.1 X-ray data and structure refinement statistics of M1 . . . 45

3.2 Structural comparison of titin M1 . . . 49

4.1 PDB entries for PB1 domain structures . . . 57

4.2 X-ray data collection statistics of NBR1 PB1 . . . 59

4.3 Primers for cloning of p62 PB1 and mutants . . . 60

4.4 X-ray data collection statistics of the PB1 heterodimer complex . 64 4.5 Refinement statistics of NBR1 PB1 . . . 69

4.6 Structural comparison of PB1 domains . . . 71

4.7 Refinement statistics of the PB1 complex . . . 72

A.1 Primers for titin kinase cloning . . . 89

A.2 Titin kinase constructs . . . 92

A.3 Summary of titin kinase construct preparation . . . 95

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ix

Abbreviations

1 ˚A 10⁻¹⁰ m

aPKC atypical protein kinase C

Bicine N,N-Bis(2-hydroxyethyl) glycine DESY Deutsches Elektronen Synchrotron

E. coli Escherichia coli

DTT dithiothreitol

FnIII fibronectin type-III

HEPES 4-(2-hydroxyethyl), 1-piperazineethane sulphonic acid

IEX Ion Exchange

Ig immunoglobulin

IMAC immobilized metal ion chromatography IPTG isopropyl-β-D-thiogalactopyranoside ITC isothermal titration calorimetry

LB Luria-Bertani

MAD multiwavelength anomalous dispersion

MPD 2-methyl-2, 4-pentanediol

MURF muscle-specific RING finger protein

NBR1 next to BRCA1

PAGE polyacrylamide gel electrophoresis

PB1 phox and bem1

PBS phosphate-buffer saline

PCR polymerase chain reaction

PDB protein data bank

PEG polyethylenglycol

PEG-MME polyethylenglycol-monomethylesther

pI isoelectric point

r.m.s.d. root mean square deviation

rpm rounds per minute

SDS sodium dodecyl sulfate

SEC size exclusion chromatography

TEV tobacco etch virus

TK titin kinase

Tris tris-(hydroxymethyl)-aminoethane

UBA ubiquitin associated

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xi

Summary

The giant muscle protein titin is the largest polypeptide known and constitutes, in addition to actin and myosin, the third filament system in the striated muscle sarcomere. Titin spans half of the sarcomere and interacts with many proteins along all its range. Three regions of accumulated interaction and associated signalling are found in the Z-disc, the I-band and the M-line, respectively.

Among about 300 predicted domains in titin, to date only one has been identified to comprise a catalytic function, a serine/threonine kinase domain within the M-line, referred to as ’titin kinase’. The aim of this work was to unravel the structural, molecular context of titin kinase in terms of adjacent titin domains and downstream signalling domains.

The tandem immunoglobulin domains A168-A169 are located amino-terminal to titin kinase at the end of the A-band within titin in the sarcomere. The structure solved in this work implies that these two domains are tightly connected via a continuousβ-strand by merging of the lastβ-strand of A168 with the first of A169. A bulge is formed between two strands in A169 at a position which is rather uncommon for an insertion in an immunoglobulin (Ig) domain. This insertion is involved in the interaction of A168-A169 with muscle specific RING protein MURF-1. In addition to A168-A169, the carboxy-terminal domain to titin kinase, the Ig domain M1 was solved.

The proteins NBR1 and p62 are substrates of titin kinase, and NBR1 links p62 to titin kinase. The interaction of NBR1 and p62 within the signalling pathway was studied here. Both proteins interact via their N-terminal PB1 domain, a recently identified interaction domain. The structure of NBR1 PB1 was solved as a single domain and in complex with p62 PB1. The complex reveals two patches of positive (p62 PB1) and negative (NBR1 PB1) charge. The affinity of the complex is in the nanomolar range.

This work extends our structural knowledge about titin immunoglobulin domains. Furthermore, it contributes to the understanding of interaction among domains of the titin kinase downstream signalling pathway. Thereby, it may help to unravel the connection between signalling related to titin stretching and transcription control in the context of muscle degradation.

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xiii

Zusammenfassung

Das Muskelprotein Titin ist das größte bekannte Polypeptid und stellt neben Aktin und Myosin das dritte Filamentsystem im Sarkomer des gestreiften Muskels dar. Titin erstreckt sich über die Hälfte des Sarkomers und interagiert mit vielen Proteinen entlang seiner Spannweite. Drei Regionen mit gehäuft vor- kommenden Interaktionen und damit verbundener Signalübertragung befinden sich in der Z-Scheibe, der I-Bande und der M-Linie.

Unter den etwa 300 vorhergesagten Domänen in Titin, wurde bis heute nur eine mit katalytischer Funktion identifiziert, eine Serine/Threonin Kinase Domäne in der M-Linie, auch als ”Titin Kinase” bezeichnet. Das Ziel dieser Arbeit war es, den strukturellen, molekularen Zusammenhang von Titin Kinase bezüglich be- nachbarter Titin Domänen und nachgeschalteter Signaldomänen aufzudecken.

Die Tandem Immunoglobulin Domänen A168-A169 befinden sich N-terminal zu Titin Kinase am Ende der A-Bande in Titin im Sarkomer. Die in dieser Arbeit gelöste Struktur impliziert, dass diese Domänen über ein durchgehendes β-Faltblatt verkn¨upft sind, durch Verschmelzung des letzten β-Faltblattes von A168 mit dem erstenβ-Faltblatt von A169. An einer für eine Immunoglobulin- (Ig) Domäne eher ungewöhnlichen Stelle ist eine Auswölbung zwischen zwei Faltblättern in A169 ausgebildet. Diese Insertion ist an der Interaktion von A168-A169 mit dem muskel-spezifischen RING Protein MURF-1 beteiligt.

Zusätzlich zu A168-A169 wurde die sich C-terminal zu Titin Kinase befind- ende Domäne, die Ig Domäne M1, gelöst.

Die Proteine NBR1 und p62 sind Substrate von Titin Kinase, und NBR1 verbindet p62 mit Titin Kinase. Hier wurde die Interaktion von NBR1 und p62 im Signalweg untersucht. Beide Proteine interagieren über ihre N-terminale PB1 Domäne, eine erst kürzlich identifizierte Domäne. Die Strukturen von NBR1 PB1 als einzelne Domäne sowie im Komplex mit p62 PB1 wurden gelöst.

Der Komplex l¨asst jeweils zwei Stellen positiver (p62 PB1) und negativer (NBR1 PB1) Ladung erkennen. Die Affinit¨at des Komplexes befindet sich im nanomo- laren Bereich.

Die vorliegende Arbeit erweitert damit unser strukturelles Wissen über Im- munoglobulin Domänen von Titin. Darüber hinaus trägt sie zum Verständnis von Interaktionen zwischen Domänen des Titin Kinase Signalweges bei. Dies könnte helfen, die Beziehung von Signalen, die durch Dehnung von Titin aus- gelöst werden, und Transkriptionskontrolle im Zusammenhang von Muskelab- bau aufzudecken.

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Chapter 1

Introduction

Movement is one of the basic phenomena of life including deliberate movements as well as many essential body functions in animals and men. The crucial role in generating motility falls to muscles. Basically three different types of muscle are found in the vertebrate musculature. Smooth muscles are so called non-striated muscles and are mainly responsible for the contraction of hollow organs such as the gastrointestinal tract, the vascular system and the uterus.

Another type of muscle is the striated heart muscle or cardiac muscle. Owing to its prominent task of maintaining blood circulation it features some unique characteristics. These differentiate it from the third type of muscle, the striated skeletal muscle often briefly called striated muscle. As the name implies the skeletal muscle is connected to the skeleton. Its contraction underlies deliberate control and is stimulated by the nervous system, in contrast to the cardiac and smooth muscle. Skeletal muscles generate force applying two specialised types of contraction. These are isotonic contraction for moving and isometric contraction for generating tension. The key to a deeper understanding of muscle functionality lies in their structural assembly, briefly outlined in the following.

1.1 Striated muscle and the structure of the sarcomere

Striated muscle are assembled by bundles of muscle fibres composed of multi- nucleated cells which are typically 10 to 100µm in diameter and several centime- ters in length. The muscle fibres are composed by parallel myofibrils separated by the sarcoplasmic reticulum and transversely running tubules (T tubules).

A repetitive contractile unit in the myofibrils, called the sarcomere, packs end to end from one Z-disc to the next over a distance of 2.0-2.5µm. The repetitive unit emerges from the ordered arrays of myosin (thick) filaments and actin (thin) filaments. In the light microscope, the myofibrils display a characteristic pattern of alternating striation where the dark A-band (anisotrop) and the light I-band (isotrop) reflect the behaviour in polarised light (Figure 1.1).

Cross-sections along the sarcomere show the arrangement of thick and thin filaments. In the part of the A-band where both filaments overlap, the actin filaments adapt a trigonal arrangement to fit to the hexagonal array of myosin

1

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2 Introduction

Figure 1.1: Striated muscle and the components of the sarcomere. Top panel:

electron micrograph of a longitudinal seciton of a skeletal muscle myofibril (adapted from Stryer, 1996). Bottom panel: schematic presentation of the sarcomere with its main components: the thin filament, the thick filament and titin, shown in blue (adapted from Tskhovrebova and Trinick, 2004).

filament, whereas their organisation towards the Z-disc gradually changes into a tetragonal array (Squire, 1997). During muscle contraction the length of the I- band changes, while that of the A-band remains the same. The sliding filament model, which was independently proposed by A. Huxley and R. Niedergerke (1954) and H. Huxley and J. Hanson (1954), explains the generation of force.

During muscle contraction the thick and thin filament slide past one another without changing the length of the filaments. Thereby, force is developed based on the interaction of the actin and myosin filaments during sliding. The mechanism of the so-called cross-bridge cycle is ATP-dependent and describes the movement of the myosin-heads along the actin filament (Geeves and Holmes, 2005).

The thin filament comprises a number of proteins and the globular 42 kDa actin (G-actin) is a major component. Single G-actin (globular) molecules polymerise in an ATP-dependent manner into the double-helical (filament) F- actin. Tropomyosin and three troponin subunits, TnC (Ca²⁺-binding), TnI (inhibitory), and TnT (tropomyosin-binding), represent the other constituents

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1.2 Titin 3

of the thin filament. Tropomyosin extends over seven actin subunits and troponin regulates its binding to myosin. In the presence of Ca²⁺, TnC binds Ca²⁺ resulting in a conformational change and movement of tropomyosin into a position with exposure of the myosin-binding site.

The myosin molecule consists of a globular head region, the S1 fragment, which can interact with actin and possesses ATPase activity. The S2 fragment comprises a flexible region of regulatory and essential myosin light chains (MLC) and a tail which combines via a long coiled-coil region with tails of other molecules. A thick filament is assembled by approximately 300 myosin molecules with the myosin heads pointing away from the M-line where they are anchored.

Besides the thick and the thin filament, a third filament system exists. In contrast to the actin and myosin filament, it is constituted by a single protein called titin. This immensely large protein extends over half of the sarcomere length. Due to its size and location, titin can interact with numerous sarcomeric proteins existing in the different regions (Granzier and Labeit, 2004; Miller et al., 2004). Therefore, it is capable of regulating diverse processes. The giant protein is involved in muscle assembly and elasticity of the sarcomere as well as signalling pathways through interaction with binding partners. Since titin fulfills many diverse functions, it has been entitled a molecular ’control freak’ (Trinick and Tskhovrebova, 1999). Titin will be described in more detail in the following.

1.2 Titin

Titin, also known as connectin, has been identified as a large component of striated muscle (Maruyama et al., 1977; Wang et al., 1979). The flexible molecule spans half of the sarcomere (Nave et al., 1989) from the Z-disc to the M-line and is more than 1µm long. With a molecular weight of up to 3.7 MDa in the skeletal N2A titin, the giant protein titin is the largest polypeptide known (Labeit and Kolmerer, 1995). In human, titin is encoded by a single gene on chromosome 2, region 2q31 (Labeit et al., 1990), containing 363 exons coding together for 38138 residues (GenBank accession code: AJ277892) (Bang et al., 2001).

The sequence of human titin was first published in 1995 (Labeit and Kolmerer, 1995) and was completed by additional PEVK exons and three unique I-band exons (termed novex-1 to -3) in 2001 (Bang et al., 2001). The sequence revealed several splicing alternatives leading to different lengths of the titin I-band. Hence, size differences in splice variants are manifested above all in the shorter cardiac titin isoforms with a molecular weight of approximately 3 MDa. The larger skeletal isoforms show sizes from 3.3 MDa in psoas muscle up to 3.7 MDa in soleus muscle (Maruyama, 1997).

Besides titin occurring in striated muscle of vertebrates, titin-like proteins are also present in invertebrate muscle, however, with a lower molecular weight.

These members of the titin family (Tskhovrebova and Trinick, 2003) are

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4 Introduction

Figure1.2:Domainstructureofthegiantmuscleproteintitin,mainlycomposedofimmunoglobulin(Ig)domains(red)andfibronectintypeIII(FnIII)domains(white)(adaptedfromGregorioetal.,1999).

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1.3 Structure and function of titin 5

known as I-connectin (from crayfish, 2 MDa; Fukuzawa et al., 2001), D-titin (from Drosophila melanogaster, 2 MDa; Machado and Andrew, 2000; Zhang et al., 2000), kettin (about 0.5-0.7 MDa; Lakey et al., 1993; Hakeda et al., 2000; Kolmerer et al., 2000), stretchin (Caenorhabditis elegans; Champagne et al., 2000), twitchin (Benian et al., 1989) and projectin (0.8-1.0 MDa; Ayme- Southgate et al., 1991).

Titin is a modular protein and 90 % of its length consists of two modules, each comprising about 90 to 100 residues. The type I are the fibronectin III (FnIII) and type II are the immunoglobulin (Ig) domains (Labeit et al., 1990). These form repetitive patterns, characteristic for their distinct occurrence within titin in the sarcomere. The designated unique sequences and other elements con- stitute the remaining 10 %, among it a single serine/threonine kinase domain (Figure 1.2).

1.3 Structure and function of titin

Titin can be divided functionally and structurally according to the location of its regions within the sarcomere.

1.3.1 Z-disc and anchoring of titin

The amino-terminal part of titin is anchored in the Z-disc by overlapping of molecules of two neighbouring sarcomeres. Titin in the Z-disc consists of Ig- like domains and a tissue-dependent number of the 45-residue motif called Z- repeat, which is differentially spliced (Sorimachi et al., 1997; Gautel et al., 1996). A characteristic protein of the Z-disc isα-actinin which cross-links actin- filaments of opposing polarity through its actin-binding domain (Schroeter et al., 1996; Blanchard et al., 1989) with titin binding to the Z-repeats via its C-terminal EF-hand (Sorimachi et al., 1997). Additionally, α-actinin inter- acts with other Z-disc proteins such as FATZ (Faulkner et al., 2000), actinin- associated LIM-protein (Xia et al., 1997) and ZASP (Faulkner et al., 1999).

Another protein which interacts with titin (Z1 and Z2, Ig domains) in the Z- disc is telethonin (Mues et al., 1998), also called T-cap (Gregorio et al., 1998), which presumably plays a role in the anchoring of the N-terminus of titin.

Functional connections beyond the contractile unit, laterally to the sarcomere or longitudinally to the neighbouring sarcomere, are transmitted via protein networks at the costameres and the intercalated discs, respectively, which are linked to the Z-disc (Miller et al., 2004).

1.3.2 I-band and elasticity of the sarcomere

The extensibility of the sarcomere is physiologically important for muscle contraction. Therefore, an elastic connection between the thick and thin filaments is required, and this task is maintained by titin (Horowits et al., 1986). Together with the passive tension, a force that restores the sarcomere to its slack length, titin has been assigned as ’molecular spring’ with the basis in the I-band of titin.

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6 Introduction

The I-band titin is composed of different segments: a constitutive I-band region consisting of tandem Ig domains, the N2-region, and the PEVK region, which is rich in proline (P), glutamic acid (E), valine (V) and lysine (K). The PEVK region itself comprises of pattern of the PPAK motifs (pI about 10) and polyE segments (pI about 3-4) which despite large differences, results in a pI of about 5 (Greaser, 2001). Tissue-specific alternative splicing leads to the large size differences in the titin isoforms, predominantly caused by a differing amount of tandem Ig domains in the non-constitutive region of up to 53 extra Ig domains in the skeletal muscle (Freiburg et al., 2000). Furthermore, the PEVK region in the stiffer cardiac muscle is much shorter (163 residues) than in the skeletal muscle (up to 2174 residues), implying importance in elasticity and passive tension. Differential splicing results in the N2A, the N2B and the combination N2BA isoform. The N2A isoform usually occurs in skeletal muscle, whereas the N2B and N2BA isoforms are found exclusively in cardiac muscle (Labeit and Kolmerer, 1995; Freiburg et al., 2000). Coexpression of the cardiac N2A and N2BA isoforms at different ratios have been described to extend independently in the half-sarcomere (Trombit´as et al., 2001).

The components of the I-band act as a molecular spring (Labeit and Kolmerer, 1995; Linke and Granzier, 1998; Granzier and Labeit, 2002) generating passive tension during extension. Extension beyond the resting length entails first in a straightening of the tilted tandem Ig domains, followed by an unfolding of the PEVK sequence and N2 regions. The PEVK region has long been assumed to present a coiled unstructured conformation. However, some evidence of a preferred conformation of repeats of a 28-residue motif within the PEVK, which adapt a polyproline II helix, has been provided (Greaser, 2001;

Ma et al., 2001).

1.3.3 A-band and the thick filament

In the A-band, titin is involved in the control of the thick filament assembly and its centering in the A-band (Whiting et al., 1989). Hence, it has been ascribed to act as a template in the sarcomere assembly. The A-band constitutes the largest part of titin with almost 2 MDa and a length of approximately 0.8µm (Labeit and Kolmerer, 1995). Notably, this is the only part in titin, where the FnIII domains are found. The FnIII domains are embedded in two types of super- repeats together with the Ig-like domains (Gautel et al., 1996; Gautel, 1996).

In the D-zone (Figure 1.2) the super-repeat is composed of 5 FnIII and 2 Ig- like domains with a pattern of Ig-FnIII-FnIII-Ig-FnIII-FnIII-FnIII-FnIII. This pattern is repeated seven times (Gautel, 1996; Gregorio et al., 1999).

In the C-zone (Figure 1.2) of the A-band (Labeit and Kolmerer, 1995), however, the eleven times repeated pattern Ig-FnIII-FnIII-Ig-FnIII-FnIII-FnIII-Ig- FnIII-FnIII-FnIII consists of 11 domains, seven FnIII and four Ig-like domains, resulting in a length of 43 nm as evidenced by electron microscopy (EM) and immunofluorenscence. The myosin binding proteins (MyBP) as ’accessory’ proteins of myosin show eleven repeats with a periodicity of about 43 nm (F¨urst

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1.4 Modules in titin: Ig and FnIII domains 7

et al., 1989; Trinick, 1996). The correlation with titin A-band super-repeats suggests a role as a scaffold involved in arrangement of the thick filaments and definition of their length (Trinick, 1996). Therefore, titin was attributed as a ’molecular ruler’ or template in the sarcomere (Whiting et al., 1989). The interaction of titin and MyBP-C is directed by titin Ig domains (Freiburg and Gautel, 1996). The P-zone (Figure 1.2) comprises of a unique patch of Ig-Ig- FnIII-FnIII-Ig-Ig-FnIII and a kinase domain. Together with the M-line region of titin, the D-zone, C-zone, P-zone reflect the zones of myosin in the A-band.

1.3.4 M-line and anchoring function

The M-line region corresponds to the carboxy-terminal part of titin with a kinase domain at its periphery to the A-band (Obermann et al., 1996). Besides, the M-line titin consists predominantly of Ig-like domains interspersed with unique sequences such as the KSP-repeats between Ig modules M5 and M6, phosphorylated by titin KSP kinase during muscle differentiation (Gautel et al., 1993).

Comparable to the anchoring of titin in the Z-disc, the titin filaments also overlap in the M-line with those titin filaments from opposite sarcomeres (Ober- mann et al., 1997). The M-line Ig domain M5 interacts with myomesin (Ober- mann et al., 1997), a protein mainly comprised of Ig-like and FnIII domains.

Myomesin has been suggested to crosslink myosin, as an antiparallel dimer, with titin by binding to myosin with its N-terminal domain. Furthermore, myosin was shown to interact with its central part with two antiparallel overlapping titin molecules (Lange et al., 2005b; Agarkova and Perriard, 2005).

An additional M-band component, the M-protein, is related with myomesin in domain composition of Ig-like and FnIII-domains. In contrast to myomesin, it is not equally expressed in different muscle types (Agarkova et al., 2003). M- protein as well as myomesin bind to myosin filaments (Bahler et al., 1985) and to titin (Nave et al., 1989) in isolated muscle. The M-protein bridges myosin filaments at the M-line (Obermann et al., 1996). The recent three-dimensional model of the M-band network presents the arrangement of titin, myosin and myomesin within the M-band (Lange et al., 2005b).

Expression of a truncated titin devoid of the kinase domain and the MURF-1 binding sites, resulted in sarcomere disassembly (Gotthardt et al., 2003). In- terestingly, in the truncated M-line titin, expression of CARP (cardiac ankyrin repeat protein) and ankrd2, a CARP-like protein, are upregulated. Both have been implicated in linking between the sarcomere and the nucleus (Miller et al., 2003; Kojic et al., 2004).

1.4 Modules in titin: Ig and FnIII domains

The Ig-like domains and the FnIII domains appear like beads on a string in the electron micrograph images of the sarcomere (Trinick et al., 1984). The number of Ig- and FnIII domains varies from 244 to 297 (Witt et al., 1998),

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8 Introduction

due to alternative splicing in different muscle tissue. The Ig-like domains make up about 112 to 166 of the titin domains.

According to differences in sequence similarities and a resulting variable number and length of the strands in Ig domain structures, Ig domains are classified in the V-(variable), C1-, C2-(constant) and I-(intermediate) set. It is still unclear, whether the Ig domains evolved by convergence to a stable fold, or by divergence from one ancestral domain (Williams and Barclay, 1988; Bork et al., 1994; Kenny et al., 1999). Assuming a common ancestor within the Ig domain sets, diverse options of the primordial domain have been discussed (Williams and Barclay, 1988; Hunkapiller and Hood, 1989; Smith and Xue, 1997).

The I-set of Ig domain was introduced based on sequence comparisons and the structure of telokin, a muscle member of the immunoglobulin superfamily (Harpaz and Chothia, 1994). Accordingly, Ig domains of sarcomeric proteins were predicted to belong to the I-set (Kenny et al., 1999; Improta et al., 1996). The first structure of a titin Ig domain, M5 of the M-band, confirmed its classification as an I-set Ig domain (Pfuhl and Pastore, 1995). Although the sequence identity of the overall Ig domain is about 20-35 % and the similarity about 50 % (Fraternali and Pastore, 1999), some subgroups with a high similarity of about 90 % exist (Tskhovrebova and Trinick, 2004). Among the different Ig-like domains of titin, there are groups of closer related domains. In the I-band, where the Ig-like domains are arranged mainly in tandem domains,

”proximal” and ”distal” to the PEVK segment differences in their consensus sequence exist. In the A-band, the FnIII and Ig-like domains are assembled in the 7- and 11-domain super-repeats, repeated six and eleven times, respectively (Gautel, 1996), in which domains at similar positions have higher sequence homology (Amodeo et al., 2001a; Muhle-Goll et al., 1998; Fraternali and Pastore, 1999).

In titin, FnIII domains are exclusively found in the A-band arranged in the super-repeats (Tskhovrebova and Trinick, 2004). Similarly to Ig domains, the FnIII domains in titin are less than 50 % conserved within the family. The atomic structures of FnIII and Ig domains are similar; both share a greek- key superfold with a two β-sheet sandwich structure. Of the seven strands in the FnIII domains, strands A, B, E compose one β-sheet and strands G, F, C and C’ the other. Based on the structural similarity of the overall fold with the different types of Ig domains, these domains were all joint in the immunoglobulin fold family IgFF, including the FnIII domains (Halaby et al., 1999). Superimposition of Ig and FnIII domains reveals that strand C’ is on two differentβ-sheets on the two domain types (Erickson, 1994). Despite their overall structural similarity, the consensus sequence of the Ig and FnIII domains is different. The structure of the first intracellular FnIII domain, A71 of the titin A-band, confirmed the characteristic FnIII fold (Muhle-Goll et al., 1998).

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1.5 Titin kinase 9

1.5 Titin kinase

The catalytic domain of titin, a serine/threonine kinase termed titin kinase, is located in the transition between the A-band and the M-line of titin (Labeit et al., 1992). In general protein kinases are involved in diverse, important biological functions where they control the reversible process of phosporylation.

The reaction which they catalyse is the transfer of the γ-phosphate of ATP to the hydroxyl group of a serine, threonine, or tyrosine within the substrate protein. Phosphorylation can evoke distinct responses, which are comparable to molecular switches, where an on or off signal results in a reverse molecular reaction such as conversion of enzyme activation to inhibition (Johnson and O’Reilly, 1996). The class of serine/threonine kinases is a well conserved superfamily which is reflected both in the sequence similarity and in the overall structural fold of the activated kinase owing to chemical constraints of the catalysed reaction (Huse and Kuriyan, 2002). The kinases can exhibit conformational differences between the active or ’on’ state and the inactive, also called

’off’ state. The regulation mechanism of the kinases which can be determined by interaction with specific regulatory domains or proteins, e.g. in response to second messengers (Johnson et al., 1996), generates a conformational plasticity of the inactive kinases (Huse and Kuriyan, 2002).

Comparison with other kinases and biochemical assays (Mayans et al., 1998) revealed an assignment of titin kinase to the subfamily of Ca²⁺/calmodulin (calcium modulating protein, abbreviated as CaM) regulated kinases. Regulation of the activation of the kinase by binding of Ca²⁺/CaM or the related protein S100 is typical for kinases like the CaMK family (Ca²⁺-calmodulin-dependent protein kinase (CaMK) family), the MLCK (myosin light chain kinase), and the twitchin kinase, another muscle kinase but found in invertebrate smooth muscle. In absence of calmodulin, the substrate binding site is blocked by an intrinsic autoinhibition mechanism (Kobe et al., 1996). Calmodulin is an ubiquitously expressed, highly conserved protein with four binding sites for calcium. Two EF-hands at each end which are connected by a short linker can each bind a calcium ion. Upon calcium binding, calmodulin undergoes a conformational change resulting in a dumbbell-shaped structure. When calmodulin binds to a synthetic calmodulin recognition peptide (Vetter and Leclerc, 2003;

Hoeflich and Ikura, 2002) which covers the calmodulin binding site of for ex- ample, titin kinase (Amodeo et al., 2001b), smMLCK (Meador et al., 1992), CaMKIIα (Meador et al., 1993), or CaMKK (Kurokawa et al., 2001), it forms a globular structure wrapped around the substrate helix by bending the central linker. However, in complex with a Ca²⁺-pump (Elshorst et al., 1999), a K⁺-channel (Schumacher et al., 2001) and the anthrax adenylate cyclase exo- toxin (Drum et al., 2002) calmodulin shows an extended conformation.

Structurally, protein kinases present the typical bilobal fold, with a smaller N lobe consisting of a five-stranded β-sheet and one prominent α-helix αC, and a larger predominately helical C lobe. The ATP binding site is situated in the cleft between the two lobes beneath the conserved phosphate binding P

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10 Introduction

Figure 1.3: The ribbon presentation of titin kinase (Mayans et al., 1998) illus- trates the typical protein kinase fold of a bilobal structure. The regulatory tail in the C-terminal lobe is shown in red (with the helices αR1 and αR2 and the strand βR1), the catalytic loop in violet and the activation segment in green which is including the P+1 loop in yellow. The catalytic residue D127 and residue Y170, which is phosphorylated in the activation process, are displayed in grey.

loop (GXGXΦG with Φ=Y or F) connecting β1 and β2 of the N lobe. The substrate binds to the activation loop or activation segment. In many kinases, the activation segment needs to be activated by phosphorylation. In the C lobe, the catalytic loop with the catalytic site, an invariant aspartate, is found.

Kinases that are regulated by phosphorylation in the activation segment are all so-called RD-kinases, in which the catalytic aspartate is preceded by an arginine residue (Johnson et al., 1996). Titin kinase, however, with a phenylalanine instead of the arginine, was the first described non-RD kinase which is activated by phosphorylation (Mayans et al., 1998).

Studies on titin kinase revealed that both phosphorylation of tyrosine 170 (Y170) in the P+1 loop and the binding of Ca²⁺/CaM are required to acti- vate the kinase (Mayans et al., 1998). The structure of titin’s kinase domain

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1.6 Titin kinase signalling pathway 11

has been solved (Mayans et al., 1998) and it revealed an autoinhibited conformation, in which the catalytic residue aspartate 127 (D127) is embedded in a hydrogen-network with the residues R129 (in the catalytic loop), Q150 (in the activation segment), and Y170 (in the P+1 loop, which in other kinases typically forms a substrate binding site for the P+1 position, when the substrate is phosphorylated at position P=0). Since these residues block the active site from substrate binding, the basis in the autoinhibition was assumed to lay therein.

A dual activation mechanism was proposed involving the phosphorylation of Y170. Thereby, the P+1 loop is released from the substrate-binding site and, in addition, the adjacent Y169 is set free from the interaction with theαR1-helix to which Ca²⁺/CaM binds (Gautel et al., 1995; Amodeo et al., 2001b) upon a conformational change that includes a release of the pseudosubstrate-like αR2 helix (Figure 1.3). Two different models of the activation mechanism have been proposed (Wilmanns et al., 2000). One model describes the complete release of the regulatory tail consisting of αR1, αR2, and the βR1 (”fall-apart”), while according to the second model the αR1 and the βR1 are anchored and solely theαR2 is ”looping-out” (Wilmanns et al., 2000). Results of molecular dynam- ics studies suggested that titin kinase acts as a force sensor in the sarcomere for conversion of mechanical stress into a biochemical signal, where rupture of theβR1-βC10 sheet and theβR1-βC11 sheet gives rise to rearrangement of the autoinhibitory tail and, thus, opens the active site for kinase activity (Gr¨ater et al., 2005). Hence, this would favour the ”fall-apart” over a ”looping-out” mechanism, but the precise activation mechanism still remains to be elucidated.

Interestingly, telethonin which binds in differentiated muscle to the Z-disc of the sarcomere (Mues et al., 1998; Gregorio et al., 1998) has been found to be a substrate of titin kinase. Since telethonin is phosphorylated in developing muscle, a role of titin kinase in myofibrillogenesis has been assumed (Mayans et al., 1998). Two more substrates of titin kinase, NBR1 (phosphorylated at S115 or S116) and p62, have been identified recently, involved in a titin kinase signalling pathway, which will be considered in the next section (Lange et al., 2005a).

1.6 Titin kinase signalling pathway

Mechanical tension during muscle contraction and stretching opens the active site of titin kinase (Gr¨ater et al., 2005) which allows the interaction with NBR1 (Lange et al., 2005a). NBR1 is phosphorylated at S115 or S116, and targets the titin kinase substrate p62 to the sarcomere by interaction via the PB1 domains. MURF-1 and -2 bind to the Ig-like domains A168-A169 in close proximity to the titin kinase (Centner et al., 2001; Pizon et al., 2002). Furthermore, the RING B-box domain of MURF-2 interacts with the C-terminal ubiquitin- association (UBA) domain of p62 (Lange et al., 2005a). Under atrophic conditions as induced mechanical arrest, MURF shuttles to the nucleus (Pizon et al., 2002; Bodine et al., 2001). In the nucleus, the RING domain of MURF is responsible for the interaction with nuclear components. The transactivation

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12 Introduction

Figure 1.4: Titin kinase (TK) downstream signalling pathway. The proteins titin (domains A168 to M1), NBR1, p62 and MURF are presented schematically by their domain composition and are described in the text. Ig domains are presented in oval shape and in red, while the FnIII domain is in white. The domains are as follows: B-box = type of zinc finger, CC = Coiled coil, MFC = MURF family conserved, NBR1 = next to breast cancer 1, PB1 = Phox and Bem1p, RING = really interesting new gene, UBA = ubiquitin association, ZZ

= type of zinc finger.

domain of the serum response factor (SRF) has been identified as nuclear lig- and of MURF-2 (Lange et al., 2005a). Hypertrophic stimuli, however, result in SRF-driven transcription of immediate-early genes. Thus, MURF is involved in regulation of the myogenic transcription (Li et al., 2005). Titin kinase activity, encompassed by the hypertrophy marker brain natriuretic peptide upregulation, can compensate for mechanical arrest and disturb the inhibitory influence of MURF-2 towards SRF (Lange et al., 2005a).

1.7 Disease association of a titin kinase mutation

A mutation mapped in the titin kinase αR1 helix (R279W) is associated with the hereditary myopathy with early respiratory failure (HMERF) (Lange et al., 2005a). Due to the mutation, the binding of NBR1 to titin kinase is abrogated resulting in an abnormal NBR1 localisation. Consequentially, p62 and MURF are also not assembled in the signal complex at the titin kinase. Disruption of the signalling pathway could be the cause for the HMERF, which is characterised by structural disorder of the sarcomeres (Edstrom et al., 1990) leading to death by respiratory failure.

Proteins involved in the pathway (Figure 1.4), such as the scaffold proteins NBR1 and p62, and the RING finger-containing protein MURF are described in more detail in the following.

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1.8 Muscle-specific RING finger protein MURF 13

1.8 Muscle-specific RING finger protein MURF

The muscle-specific RING finger protein MURF is connected to the titin signalling pathway (Figure 1.4). The three family members MURF-1 (Dai and Liew, 2001; Centner et al., 2001), MURF-2 (Pizon et al., 2002; McElhinny et al., 2002) and MURF-3 (Spencer et al., 2000) are found on distinct chromosome loci (1p31.1-p33, 8p12-13, 2q16-21) (Centner et al., 2001). All share a similar domain architecture (Figure 1.5), with an N-terminal RING finger domain, a B-box, a leucine-rich coiled-coil domain and a C-terminal acidic domain. More- over, a typical MURF family-conserved domain (MFC) of 30 amino acids sepa- rates the RING finger and the B-box domain (Centner et al., 2001; McElhinny et al., 2004; Witt et al., 2005). The RING finger domain (forReallyInteresting NewGene) (Lovering et al., 1993) is a widespread domain in proteins of diverse cellular distribution and organisms (Saurin, 1996). Proteins which contain a RING domain mediate protein-protein interaction in large macromolecular scaf- folds and have recently been implicated in the process of ubiquitylation and sumoylation (Freemont, 2000; Borden, 2000). The RING finger detected in the MURF sequence belongs to the C₃HC₄ finger (Freemont et al., 1991) in which two zinc ions are coordinated in a so-called ’cross-brace’ arrangement with a conserved distance of 14 ˚A (Borden and Freemont, 1996). Due to the evolu- tionary conserved tripartite organisation of RING finger, B-box, and coiled-coil domain, the MURF family is classified as a RBCC (RING-B-box-coiled coil) subclass of the RING proteins (Borden, 1998).

Homo- and heterodimers among the MURF proteins are formed via the coiled-coil domain (Spencer et al., 2000; Centner et al., 2001) except for cardiac muscle MURF-2/p27 which lacks this coiled-coil domain. Beyond this interaction, a number of diverse proteins interact with MURF. First identification of MURF emerged from the binding of MURF-3 to the serum response factor SRF, although the biological relevance was ambiguous to the authors (Spencer et al., 2000). MURF interaction with the carboxy-terminal region of titin (A168-169) in proximity to the M-line has been shown for MURF-1 (Centner et al., 2001) and a transient interaction of MURF-2 and -3 with this part of titin unravelled later (Centner et al., 2001; Pizon et al., 2002). Both, MURF-1 and -2, have been described to bind to the ubiquitin-conjugating enzyme 9 (Ubc9) and isopepti- dase T-1 (ISOT-3), involved in SUMO modification (McElhinny et al., 2002).

The RING domain of MURF-1 (previously called SMRZ for striated muscle RING zinc finger) was mapped to be responsible for interaction with SMT3b (Suppressor of MIF (migration inhibitory factor) Two 3 homolog 2) (Dai and Liew, 2001), a ubiquitin-related protein, now termed SUMO-2 (small ubiquitin- like modifier) (Dai and Liew, 2001). Moreover, MURF has been reported to bind to the nuclear glucocorticoid modulatory-element binding protein GMEB- 1 that regulates transcription as response to changes in cellular glucocorticoid levels (McElhinny et al., 2002). Further myofibrillar proteins, proteins of the energy metabolism, mitochondrial proteins, and some unknown proteins were identified to interact with MURF-1. Most of them also interacted with MURF-

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14 Introduction

Figure 1.5: Domain architecture of MURF family members. Adapted from Centner et al. (2001) and Pizon et al. (2002). Accession codes for the different MURFs with the first of two given MURF-2 variants having an N-terminal extension of 16 residues, and sequence differences in a few residues: MURF- 1 (AJ291713), MURF-2/p27 (AJ277493), MURF-2/p50 (AJ243488, AJ291712 short), MURF-2/p60 (AJ243489, AJ291712 long), MURF-2/p60B (AJ431704), MURF-3 (AJ291714 short), MURF-3’ (AJ291714). Differential splicing has been reported for MURF-3 (Centner et al., 2001) and MURF-2 (p27, p50 and p60), while a frameshift in the reading frame causes an alternative C-terminus for the longest isoform (p60B) of MURF-2 (Pizon et al., 2002).

2 as well, in a yeast two-hybrid screen on adult skeletal muscle cDNA (Witt et al., 2005). Thus, proteins like RACK-1 (Arya et al., 2004) and p62 (Lange et al., 2005a) were identified as MURF binding partners as well as troponin I, troponin T3, telethonin (also called T-cap), nebulin, NRAP, myotilin, and MLC-2 (Witt et al., 2005). The localisation of the muscle protein MURF is reflected by the diversity of MURF-interacting proteins. Interestingly, apart from the M-line and the Z-disc in muscle sarcomere as well as its association to micro- tubules (Pizon et al., 2002; Spencer et al., 2000) and myosin (Pizon et al., 2002), MURF is also found in the nucleus (Dai and Liew, 2001). Allocation to the nucleus is controlled by the RING-finger domain, whereas the central part of MURF targets to the M-line and Z-disc (McElhinny et al., 2002). Muscle under atrophic conditions upregulates the ubiquitin ligase MURF (Bodine et al., 2001). As the inhibition of discrete ubiquitin ligases could decrease muscle loss due to atrophy-inducing stimuli, MURF-1 has been proposed as potential drug target (Glass, 2003).

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1.9 NBR1 15

1.9 NBR1

NBR1 was isolated from serum directed against ovarian tumor antigen CA125 (Campbell et al., 1994), which is used for monitoring ovarian cancer (Lavin et al., 1987). The genenbr1is located on the chromosome region 17q21 (Campbell et al., 1994) and lies head-to-head with the the BRCA1 (Breast cancer type 1 susceptibility protein) gene, that is transcripted in opposite directions (Dimitrov et al., 2001). Due to the location of the NBR1 gene close to the BRCA1 gene, the originally termed 1A1.3B product was called NBR1, i.e. next to BRCA1.

Three alternative splicing variants have been identified (Dimitrov et al., 2001).

A potential role in ovarian and breast cancer has not been demonstrated for NBR1 so far. The antigen CA125 has been identified as the mucin MUC16, which was shown to be entirely different from NBR1 (Yin and Llyod, 2001).

NBR1 contains a PB1 domain, a ZZ domain and an UBA domain. Thus it has a similar domain composition as the human p62, the rat homologue ZIP, or ref(2)p from Drosophila. The ZZ domain is a putative zinc-finger domain, coordinating two Zn²⁺ per motif, with a signature motif Asp-Tyr-Asp-Leu and a core consensus sequence C-x₂-C-x₅-C-x₂-C similar to the B-box (Ponting et al., 1996).

The ubiquitin-association domain (UBA) is associated with ubiquitination.

Binding of ubiquitin has been demonstrated for p62 (Vadlamudi et al., 1996), but this is not a general feature of the domain. Conferring target specificity to enzymes of the ubiquitination system was proposed (Hofmann and Bucher, 1996), and also interaction with proteins lacking ubiquitin-like domains or any link to ubiquitin have been found (Buchberger, 2002). Generally, the diverse proteins that contain a UBA domain are involved in the ubiquitin-proteasome pathway, DNA excision repair, and cell signalling via protein kinases (Hofmann and Bucher, 1996). The domain consists of a sequence motif comprising about 40-45 residues. Structurally, the UBA domain forms a compact three helix bundle (Dieckmann et al., 1998).

NBR1 interacts with fasciculation and elongation protein zeta-1 (FEZ1), a PKCζ-interacting protein, and a calcium- and integrin- binding protein (CIB).

Thereupon, a role in signal transduction in neural development has been proposed (Whitehouse et al., 2002). Interaction of the PB1 domains of NBR1 and p62 has been described, although the function remained unclear (Lamark et al., 2003). Self-interaction of NBR1 is not mediated by the PB1 domain, but by a region C-terminal to it (Whitehouse et al., 2002; Lamark et al., 2003).

Formation of NBR1 dimers via a coiled-coil domain and via binding of the N- terminal PB1 domain of NBR1 to titin kinase has been presumed recently and a function as a scaffold protein linking titin kinase to p62 and MURF-2 has been suggested (Lange et al., 2005a).

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16 Introduction

1.10 The protein p62

The protein p62 with a molecular weight of 62 kDa, is involved in a variety of different biological processes and has been given diverse names. p62 has been primarily identified as a ligand of p56^lck binding to the SH2 domain of p56^lck (Park et al., 1995). As ubiquitin-binding protein sequestosome 1 (SQSTM1), it is associated with the Paget disease of bone (Laurin et al., 2002;

Hocking et al., 2002). The counterpart of human p62 in rat is known as zeta- interation protein (ZIP), which has been assigned to act as scaffold protein for linking aPKCζ to protein tyrosine kinases and cytokine receptors (Puls et al., 1997). A170 from murine peritoneal macrophages is induced upon oxida- tive stress (Ishii et al., 1996). Moreover, the signal transduction and adaptor protein STAP from the mouse osteoblastic cell line MC3T3-E1 is identical to A170 (Okazaki et al., 1999). The 60 kDa EBIAP, Epstein-Barr virus (EBV)- induced gene 3 (EBI3) association protein, corresponds to p62. It associates with the hematopoietin receptor EBI3, which is induced in Epstein-Barr virus- infected B lymphocytes (Devergne et al., 1996). Chicken ovalbumin upstream promotor transcription factor (COUP-TF) is an orphan member of the nuclear hormone receptor superfamily which regulates transcription and binds presumably via DNA to ORCA, orphan receptor coactivator, again identical to p62 (Marcus et al., 1996).

The human p62 encodes a 440 amino acid protein and is highly conserved, particularly in the functional domains. These domains include an N- terminal PB1 domain (3-102), a ZZ zinc finger domain (122-163), two PEST sequences (266-294 and 345-377), and a ubiquitin association domain UBA (396-431) (Geetha and Wooten, 2002). Both, the PB1 domain and the ZZ zinc finger, mediate protein-protein interaction. The UBA domain confers non- covalent ubiquitin binding to mono- and poly-ubiquitin chains and interaction with other proteins (Hofmann and Bucher, 1996; Buchberger, 2002). The solution structure of the UBA domain of p62 has been solved recently (Ciani et al., 2003). The PEST sequences, rich in proline (P), glutamic acid (E), serine (S), and threonine (T), are a characteristic degradation signal in proteins with a short life time (Rechsteiner and Rogers, 1996) and occur in key regulatory proteins (Okazaki et al., 1999).

Due to several functional domains (see above), p62 is capable of interacting with a variety of different proteins (see Table 1.1).

Human p62 is ubiquitously expressed and is found within the cell in the cy- tosol and nucleus. The widespread location within the cell and interaction with diverse proteins define p62 as a multifunctional protein. The presumably best characterised function of p62 is its role as scaffold protein in a number of signalling pathways (Geetha and Wooten, 2002). A regulatory role in the ubiquitin proteasomal degradation is derived from p62’s property to bind non-covalently to ubiquitin and signalling proteins (Vadlamudi et al., 1996; Pridgeon et al., 2003). p62 has been implicated to shuttle and anchor ubiquitinated targets for proteolytic degradation (Seibenhener et al., 2004; Shin, 1998). p62 itself and in

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1.11 Aim of the work 17

Protein Citation

aPKCι/λ Puls et al., 1997; Avila et al., 2002 MEK5 Lamark et al., 2003; Noda et al., 2003 NBR1 Lamark et al., 2003

PAR-4 Chang et al., 2002 p38 MAPK Sudo et al., 2000 p120 ras-GAP Ellis et al., 1990

TrkA Geetha and Wooten, 2003 RIP Sanz et al., 1999

TRAF6 Sanz et al., 2000 p56^lck Park et al., 1995 Grb14 Cariou et al., 2002 Kvβ2 Gong et al., 1999 GABA_C Croci et al., 2003 COUP-TFII Marcus et al., 1996 ubiquitin Vladlamundi et al., 1996

p62 Lamark et al., 2003; Wilson et al., 2003

Table 1.1: Interaction partners of p62. Abbreviation of the proteins are given in the following: aPKC = atypical protein kinase C, MEK5 = mitogen-activated protein kinase kinase 5, PAR-4 = prostate apoptosis response-4, MAPK = mitogen-activated protein kinase, p120 ras-GAP = p120 ras-specific GTPase- activating protein, TrkA = neurotrophic tyrosine kinase receptor, RIP = receptor-interacting protein, TRAF6 = TNF (tumor necrosis factor) receptor- associated factor 6, p56^lck= lymphocyte-specific protein tyrosine kinase, Grb14

= growth factor receptor-bound protein 14, Kvβ2 = potassium channelβ2 sub- unit, GABA_C = gamma-aminobutyric acid receptor C, COUP-TFII = chicken ovalbumin upstream promotor transcription factor

association with ubiquitin-protein conjugates constitutes the so-called sequestosome, an alternative storage of cytoplasmic ubiquitinylated proteins which are not degradated by the proteasome in the first place (Shin, 1998).

1.11 Aim of the work

The enormous size of titin, extending over half of the sarcomere, and its location enables titin to interact with a variety of different muscle proteins, and hence get involved in signal transduction. This diversity in interactions together with its various functions makes titin a particularly interesting protein to study.

The immense size by its assembly of about 300 domains entail the flexible nature of this protein, which is obviously not suitable for crystallisation. In or- der to obtain structural information about titin, segments encompassing single or several domains are studied.

In this study, the region of titin in proximity to titin kinase as well as a connected signalling pathway are considered. Several domains of titin, namely

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18 Introduction

the Ig-like domains A168 and A169 of the A-band region, M1 in the M-line and extended constructs of titin kinase are investigated. The work on titin connected pathways focuses on the proteins NBR1 and p62 and their interaction via the lately identified PB1 domain. Both proteins are a substrate of titin kinase and are linked to titin. Throughout all of the work, a special emphasis is placed on interactions of the studied domains.

Since the era of characterisation of the molecular and biochemical properties of signalling pathways in titin has just started, this work intends to shed some light on the molecular structure of domains and their interactions involved in the titin kinase downstream signalling pathway.

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Chapter 2

The A-band immunoglobulin domains A168 and A169

2.1 Introduction

The assembly of large proteins from small building blocks or modules is considered a common practice for generating a diversity of multifunctional proteins (Spitzfaden et al., 1997; Bork et al., 1996). These mosaic proteins are primarily extracellular proteins which excel in the immune system in recognition and in cell-cell interactions. But, also intracellular proteins exhibit the modular arrangement of contiguous domains such as for structural organisation of the muscle. Typically, modules of theβ-fold emerge – often with their N-and C-terminus at opposite ends – which provide the basis for easy array assembly and spacer formation. The most abundant protein module is the immunoglobulin (Ig) domain (Doolittle and Bork, 1993) with a large functional, structural and sequential diversity (Bork et al., 1994).

2.1.1 Immunoglobulin domains

Antibodies (immunoglobulins) were eponymous for the immunoglobulin domains, first discovered in the immune system. Due to their wide distribution and great number, proteins containing Ig domains cover diverse biological functions, such as recognition, growth, development, signalling, and carbohydrate recognition (Srinivasan and Roeske, 2005).

Ig domains display no enzymatic activity but are good in being recog- nised (Barclay, 2003). The stable fold that is resistant to proteolysis, has the ability to interact by formation of homo- and heterodimers either along theβ- strands or through loops. Their broad range of high affinity interaction resides mainly in the hypervariable loops, which are located at one end of the ellipsoid domain (Barclay, 2003).

The fold topology of Ig-like domains follows the Greek key fold with two twisted β-sheets comprising seven to nine antiparallel β-strands which form a β-sandwich structure (Richardson, 1981; Lesk and Chothia, 1982). Despite the

19

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20 The A-band immunoglobulin domains A168 and A169

conserved common fold, the Ig-like domains represent distant sequence similarity. Similar residues within the module of about 100 residues were detected and a conserved disulphide-bridge has long been considered as the hallmark of the Ig-like domains (Williams and Barclay, 1988). Typically, it is formed in the extracellular Ig domains connecting two cysteines between strand B and F. However, many intracellular and also some extracellular Ig-like domains are devoid of disulphide bridges. If the disulphide bridge is present, it packs against an invariant tryptophan in the hydrophobic core forming the so-called ’pin’ motif (Lesk and Chothia, 1982). Overall, the loops intervening the strands show variability in length and particularly the B-C and F-G loops play a key role in protein recognition as well as in determining the size of the domain.

Ig domains of giant proteins such as titin have been grouped to the I-set class (Harpaz and Chothia, 1994). The first structure of a muscle Ig-like domain, termed telokin, was solved from theC. eleganstwitchin protein (Holden et al., 1992) and became the representative of this group.

2.1.2 Interaction of titin A168-A169 and MURF

The muscle-specific RING protein MURF interacts with diverse proteins in- and outside the nucleus. One of the binding partners is the titin tandem Ig domain A168-A169. The interaction has been demonstrated for MURF-1 using yeast two-hybrid assays and pulldown experiments of in vitro translated protein (Centner et al., 2001). Investigations regarding the binding capability of the MURF family members MURF-2 and MURF-3 to titin A168-A169 have been inconsistent so far (Centner et al., 2001; Witt et al., 2005; Pizon et al., 2002). As MURF-1 was found to bind to A168-A169, a potential role in modulating the activity of titin kinase was suggested due to the close proximity of titin kinase and A168-A169 (Centner et al., 2001). Several regions of MURF-1 were mapped to contribute in binding to titin A168-A169 (Witt et al., 2005).

MURF-1 plays an important physiological role in regulating muscle degradation (Bodine et al., 2001). Under atrophic conditions in muscle, upregulation of MURF-1 and muscle atrophy F-box protein MAFbx was detected. Fur- thermore, MURF-1 knock-out mice were described to be resistant to muscle wasting (Bodine et al., 2001).

MURF-1 has E3-like ubiquitin ligase activity and may thereby be involved in sumoylation (Dai and Liew, 2001; McElhinny et al., 2002) or ubiquitination processes involved in proteasome-dependent proteolysis of muscle proteins (Bo- dine et al., 2001; Kedar et al., 2004; Witt et al., 2005).

Location of MURF-1 in the nucleus, where it acts in transcription regulation (Dai and Liew, 2001; McElhinny et al., 2002), and its interaction with titin (Centner et al., 2001) and other sarcomeric proteins (Witt et al., 2005;

Kedar et al., 2004) suggest a dynamic role as adaptor, linking myofibrillar signalling with transcription control.

To structurally elucidate the basis for the interaction of the titin A168-A169 and MURF-1, the structure of A168-A169 has been solved. Since some studies

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2.2 Materials and Methods 21

Figure 2.1: Titin kinase downstream signalling pathway. The proteins titin (domains A168 to M1), NBR1, p62, and MURF are presented schematically by their domain composition. The domains mainly analysed in this chapter, the tandem Ig domain A168 and A169, are highlighted in red.

on the interaction between titin and MURF include the FnIII domain A170, analysis of A168-A169-A170 has also been enclosed in this chapter. In Figure 2.1 the domains A168-A169 are highlighted in red with respect to their location in titin.

2.2 Materials and Methods

2.2.1 Purification of A168-A169

The sequences encoding the two A-band Ig domains 168 and 169 of titin, corre- sponding to titin residues 24429-24623 (accession code: Q10466), were amplified from a human cardiac titin library (G. Stier, EMBL Heidelberg, Germany) by polymerase chain reaction (PCR) using the oligonucleotides A168/169 NcoI (5’-AAACCATGGC ACCACACTTT AAAGAGGAA-3’) and A168/169 KpnI (5’-AAAGGTACCT CAATCAGCCA CATCCAGTTC AAC-3’) as forward and reverse primers, respectively. The DNA fragments were subcloned with NcoI and KpnI restriction sites into vector pETM11 (based on pET24d (Novagen), modified by G. Stier, EMBL) containing a polyhistidine tag cleavable by TEV protease. The construct was verified by sequencing (MWG Biotech). In the following, the protein sequence of A168-A169 has been renumbered in 1-195.

The protein was expressed in BL21(DE3) CodonPlus RIL cells grown at 37^◦C to an optical density (λ=600 nm) of 1.0 and expression was induced by adding 1 mM IPTG. After growth for three hours at 37^◦C, the cells were harvested by centrifugation (6000 rpm (JLA 8.1000, Beckman), at 4^◦C for 10 min) and frozen at -20^◦C. The pellet of 1 liter cells was resuspended in 25 ml lysis buffer (25 mM Tris pH 8.0, 300 mM NaCl, 5 mM imidazol and 5 mM β-mercaptoethanol) and

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22 The A-band immunoglobulin domains A168 and A169

lysed by sonication (Bandelin electronic) for 2-3 min (pulses of 0.7 s and pauses of 0.3 s). After centrifugation (18000 rpm (SS-34, Sorvall), 30 min, 4^◦C) the supernatant was passed through a 0.45µm filter to 1 ml pre-equilibrated Ni- NTA beads (Qiagen). The resin was washed with lysis buffer and the protein was eluted with 400 mM imidazol in buffer (25 mM Tris pH 8.0, 300 mM NaCl).

To cleave the polyhistidine-tag, TEV protease (in a molar ratio of about 1:20) and 2 mM DTT were added and incubated over night at room temperature. The protein was concentrated and further purified by gel filtration chromatography using a Superdex 75 10/30 (GE Healthcare) equilibrated in 25 mM HEPES pH 7.5, 150 mM NaCl and 5 mM DTT. The protein sequence was confirmed by mass spectrometry (T. Franz and X. Li, Proteomics Core Facility, EMBL).

2.2.2 Preparation of Selenomethionine incorporated A168-A169

For substitution of the methionines M1 and M109 by selenomethionine (Hen- drickson et al., 1990), the A168-A169 containing plasmid was transformed in the methionine auxotrophicE. colistrain B834(DE3) (Novagen). Selenomethionine- labelled protein was prepared according to a protocol described elsewhere (van Duyne et al., 1993) with minor modifications. A colony was picked and plated on an agar plate with the appropriate antibiotics. Several of these new colonies were inoculated in the preculture consisting of M9 minimal medium (Sam- brook et al., 1989) supplemented with a mixture of L-amino acids (all except L-Selenomethionine; each 40 mg/l), 60 mg/l L-selenomethionine, 1×trace elements, 0.4 % glucose, 1 mM MgSO₄, 0.3 mM CaCl₂, 1 mg/l Biotin, 1 mg/l Thi- amine and 50µg/ml kanamycin, and grown for about 20 hours at 37^◦C. The trace elements solution (100×) contained 5 g EDTA, 0.83 g FeCl₃ × 6 H₂O, 84 mg ZnCl₂, 13 mg CuCl₂ × 2 H₂O, 10 mg CoCl₂ × 6 H₂O, 10 mg H₃BO₃ and 1.6 mg MnCl₂ x 6 H₂O in 1 liter with the pH adjusted to pH 7.5. The cells were centrifuged at 4000 rpm and 4^◦C for 10 min and resuspended in M9 medium. The medium of the main culture was composed of the same sup- plements in the M9 minimal medium as the preculture medium with 60 mg/l L-selenomethionine, in which the preculture was diluted at a ratio of 1:10. A 2 liter baffled flask was filled with 0.5 liter medium to allow better aeration.

The cells were grown to an optical density of 0.6-0.7 before the induction of the expression by adding 1 mM IPTG and an incubation time of about 10 hours at 37^◦C. The cells were harvested by centrifugation at 6000 rpm (JLA 8.1000, Beckman) and 4^◦C for 10 min, washed in PBS, pelleted, and frozen at -20^◦C.

The selenomethionine-incorporated protein was purified using the same protocol as for the native protein. Incorporation of the selenomethionine was confirmed by mass spectrometry (T. Franz and X. Li, Proteomics Core facility, EMBL).

2.2.3 Purification of titin A168-A169-A170

Human titin encoding the three A-band domains A168-A169-A170 correspond- ing to residues 24429-24730 were amplified by PCR from the TOPO con-

Structural insight into the environment of the serine/threonine protein kinase domain of titin