Comparison with telokin and twitchin Ig26

Since the position of the titin M1 Ig domain proximal to titin kinase corresponds to the domain arrangement found in MLCK and titin related proteins, available structures from similar positions were considered for structural comparison.

Two structures which meet this criterium, were the structure of the Ig domain C-terminal to the C. elegans twitchin kinase (1KOA) comprising residues 375-472 and the high resolution structure of 2.0 ˚A resolution of telokin (1FHG) from Meleagris gallopavo(turkey).

50 The titin M-band immunoglobulin domain M1

Figure 3.8: Superimposition of M1, telokin (1FHG), and Ig26 (1KOA). The domains are colour-coded: M1 in red, telokin in blue and Ig26 in green.

A structure-based sequence alignment is given in Figure 3.5. Supplementary to the predominately conserved I-set key residues (Harpaz and Chothia, 1994), six additional conserved residues were found in the three structures which are highlighted in blue in the alignment. A structural comparison with telokin and Ig26 is given in Table 3.2. Superimposition of the M1, telokin and twitchin Ig26 structure (Figure 3.8) illustrates the large structural agreement of Ig domains from different proteins, but related positions within. Worth mentioning is the deviation at the site of the N-terminus in the B-C loop which forms contacts in Ig26 with twitchin kinase.

3.4 Discussion

In titin, twitchin, MLCK and related proteins a conservation of the domain arrangement particularly close to the kinase domains has been stated (Kamm and Stull, 2001; Champagne et al., 2000). The overall structures of titin ki-nase (Mayans et al., 1998) and twitchin kiki-nase from Aplysia (Hu et al., 1994) andC. elegans(Kobe et al., 1996) are similar. They match in a remarkable reg-ulatory tail, association of which with the kinase core explains the autoinhibited character of the kinases (Kobe et al., 1996; Mayans et al., 1998). Truncation of this regulatory segment leads to activation of the kinases (Mayans et al., 1998; Heierhorst et al., 1995; Heierhorst et al., 1996b; Lei et al., 1994). More-over, binding of an effector protein (calmodulin, and the structurally related SA100Al₂) to a segment of the autoregulatory tail of titin kinase and twitchin kinase is part of the activation mechanism (Mayans et al., 1998; Heierhorst

3.4 Discussion 51

et al., 1996a). The high structural and functional similarities may be related to a conservation in the arrangement of these kinase domains and their adja-cent modules. Interdomain associations, even though less analysed so far, may reflect this as well. Between the twitchin kinase and Ig26 from C. elegans a relatively rigid interdomain relationship has been proposed due to the large buried surface area and the hydrophobic and partially charged character of the interface (Kobe et al., 1996). Based on these data, an assembly model of the titin kinase to M1 domain is given, obtained by superimposition (Figure 3.9).

The largest deviation between Ig26 and M1 is found in the B-C loop, which is involved in interdomain interaction with twitchin kinase. Although, differences in the interaction interface may originate therein, a different conformation of this loop could be formed in M1, when attached to titin kinase, resembling that of Ig26. The overall higher B-factors and the lower resolution of Ig26 cannot be neglected. However, the overall shape and, thus, the conformation permit-ting the interaction between the domains may be maintained. Similarly, the largest differences between the kinases are found in the last strand. However, the presence of the other domains can result in a slight conformation change.

The hydrophobic and charged composition is less conserved in the titin kinase and M1 domain interface and may not support a connection as strong and rigid as in twitchin kinase from C. elegans. A stretch-based mechanism was pro-posed for titin kinase to encompass the active form (Gr¨ater et al., 2005). This would additionally require a rupture of the kinase and Ig domain interface prior to detaching the last β-strand from hydrogen-bridging with anotherβ-strand, provided that this interaction would be existent and relevant.

Further experimental evidence on the basis of the shape of titin kinase-M1 could be obtained by small-angle X-ray scattering (SAXS) studies of the two domain construct TKM1 that consists of titin kinase and M1 (Appendix A).

Figure 3.9: Model of titin kinase with M1. Superimposed structures of titin kinase (1TKI) and M1 with twitchin kinase with the adjacent Ig domain Ig26 (1KOA) are basis for the presented interdomain arrangement. Ribbon repre-sentation of titin kinase inblue with the regulatory tail ingreenand M1 inred and surface representation; twitchin kinase and Ig26 are not shown.

Chapter 4

NBR1 PB1 in complex with p62 PB1

4.1 Introduction

Protein-protein interactions mediated by scaffold proteins play a pivotal role in signal transduction. In cellular signalling, scaffold proteins are involved in coupling signalling pathways regulating cell polarity, survival, and differentia-tion (Moscat and Diaz-Meco, 2000; Ohno, 2001). Protein interacdifferentia-tion occurs via specific interaction domains recognising dedicated patterns and other domains.

A lot is known about the basis of interaction of domains such as the SH2-, SH3,-or WW-domain, which were studied intensively. Constantly, new domains are discovered and their molecular basis of interaction is unravelled. One of these recently identified domains is the PB1 domain.

In the titin kinase downstream signalling pathway, the interaction of two scaffold proteins through their PB1 domain plays a key role. Both proteins are substrates of titin kinase. While NBR1 directly binds to titin kinase, p62 is linked via the PB1 domain interaction (Lange et al., 2005a). The schematic representation of the titin kinase signalling pathway (Figure 4.1) highlights the PB1 domains, which are subject of this work.