Biological relevance of the NBR1/p62 heterodimer complex 81

Despite its discovery already in 1994, little was known about the protein NBR1, the cDNA of which was originally isolated from a serum directed against ovar-ian tumor antigen CA125 (Campbell et al., 1994). However, it was shown that the antigen CA125 is the mucin MUC16, which is entirely different from

82 NBR1 PB1 in complex with p62 PB1

NBR1 (Yin and Llyod, 2001). A potential role in ovarian and breast cancer could not be demonstrated for NBR1 so far, albeit it was speculated due to its location head-to-head with the BRCA1 gene (Dimitrov et al., 2001). Then, an interaction was detected between NBR1 PB1 and p62 PB1 (Lamark et al., 2003), a protein which mainly associates with cytosolic inclusions of ubiqui-tinylated proteins. However, the biological function of this interaction was only spotted, when a connection emerged between the kinase domain in the giant muscle protein titin and a downstream signalling pathway transducing the sig-nal to the nucleus (Lange et al., 2005a). In this pathway, NBR1 acts as a key scaffold protein, which connects p62 to the serine/threonine protein kinase domain through its amino-terminal part.

Chapter 5

Conclusions

Binding sites of titin ligands occur mainly at three positions along the titin filament at distinct spots in the Z-disc, the I-band, and the M-line (Granzier and Labeit, 2004; Granzier and Labeit, 2005). In this study, the region around titin kinase at the transition to the M-line was at the focus of the investigations.

Several domains in this area were studied. The two domains A168-A169, known to bind to the muscle specific RING finger protein MURF-1 from yeast two-hybrid studies (Centner et al., 2001), were analysed structurally (Chapter 2).

These tandem domains do not occur as two independent modules with a high degree of flexibility but rather they form a rigid entity. Connection between the domains is provided by merging of the last β-strand of A168 with the first β-strand of A169. This close linkage of the domains is supported by the lack of linking residues between the Ig domains. Binding of MURF-1 to this titin tandem domain is mediated via a special characteristic found in A169.

An insertion between the strands A and A’ in A169, which is unique among the Ig domains with known three-dimensional structure, bulges out along the longest side of the Ig domain. The basic residue E107 is exposed in this bulge and has been shown to be involved in binding to MURF-1 (S. Lange, personal communication). A deeper insight in the interaction between titin A168-A169 is desirable. This can be gained by mapping potential other residues on titin which support the binding to MURF-1, e.g. in a similar way as the residues on MURF-1, involved in binding to A168-A169, were mapped (Witt et al., 2005).

Clearly, the structure of MURF-1 and particularly the structure of the complex with titin A168-A169 would give insight to the complex formation. Moreover, structural analysis of MURF-1 is of special interest since its versatile interaction with other sarcomeric proteins beyond titin and even nuclear proteins allocates MURF-1 as an important adaptor in linking titin to the nucleus. Owing to this property, MURF-1 appears to be not only of significance in binding to titin and, thus, contributing to signalling in proximity to titin kinase, but also has been shown to play a role in the concept of muscle atrophy by upregulation of specific genes.

The proteins NBR1 and p62 are linked to titin as substrates of the ser-ine/threonine kinase domain titin kinase. Particularly, the N-terminus of

83

84 Conclusions

NBR1 (PB1 domain and ZZ domain) has been reported to bind to a trun-cated titin kinase construct. This construct is supposed to represent an active state kinase (Lange et al., 2005a) which is assumed to arise from stretching of titin (Gr¨ater et al., 2005). The N-terminal NBR1 PB1 has been another subject of this study (Chapter 4).

The interaction of NBR1 PB1 or the elongated NBR1 N-terminus (PB1 and ZZ domain) with the truncated titin kinase may be subject of further investi-gations. These could provide the ’structurally’ missing link between titin and NBR1. First attempts towards obtaining active state titin kinase constructs have been made (Appendix A). Eukaryotic expression (bacculovirus/insect cell) has proven to be problematic with the active kinase constructs. A change to a prokaryotic expression system (E. coli) was attempted. So far, an expression and purification protocol has been established for two autoinhibited titin kinase constructs, bearing in addition to the kinase domain the FnIII domain A170 ahead (A170TK) and the preceding Ig domain M1 (TKM1), respectively. A demonstration of how titin kinase together with M1 may look like is illustrated in Chapter 3. These extended titin kinase constructs and the constructs mim-icking the active state of the kinase were intended to further investigate the activation mechanism by crystallisation and co-crystallisation with substrate peptide and ATP-analogues. However, crystallisation has not been achieved yet. Posttranslational modification as provided in the eukaryotic expression system may be a key to obtain protein suitable for crystallisation. Another possibility to obtain an active titin kinase might be by expression of these au-toinhibited constructs in the prokaryotic expression system with an internal cleavage site for removal of the regulatory tail. Studies on the binding of NBR1 to titin kinase can also be performed with synthetic peptides representing the αR1 helix of titin kinase. This part of titin kinase was assumed to be the bind-ing site for NBR1 (Lange et al., 2005a). The mappbind-ing of the exact bindbind-ing site on titin kinase as well as on NBR1 will be a valuable piece of information in linking the downstream signalling to titin.

The PB1 domain has previously been identified as an interaction domain which mainly forms heterotypic interactions with other PB1 domains (Noda et al., 2003; Wilson et al., 2003). As for p62 homotypic interactions have been reported. In this study, this interaction property was shown in particular for NBR1 PB1 and p62 PB1 (Chapter 4). In order to prevent self-oligomerisation of p62 PB1, two mutations (D69A and D73A) in one of the interacting motifs, the OPCA motif, were introduced, which do not avoid the binding to PB1 NBR1. Thus, complex formation and thermodynamics of the binding could be studied by ITC. The crystal structure revealed the residues which contribute to the interaction of both proteins.

In summary, titin domains from the titin kinase region and domains of the associated titin kinase signalling pathway have been investigated in this study.

This work has shed some light on the interactions occurring in this area of titin. This represents a further step towards elucidating the concept of M-line signalling in particular and titin signalling in general on a strucutral basis.

Conclusions 85

More structural investigations on the entity of this region are required. Due to limitations in crystallisation of the generally large and flexible titin, other methods may be applied to obtain more structural knowledge. Further studies by methods such as small angle X-ray scattering or electron microscopy on titin fragments encompassing several domains, e.g. the domains A168 to M1 may provide insight if and how the overall more rigid structure of A168-A169 can combine with the titin kinase which is assumed to be stretched during muscle activity.

Appendix A

Titin kinase

A.1 Introduction

The structure of titin kinase was solved previously (Mayans et al., 1998). It explains the basis for activation achieved by tyrosine phosphorylation and cal-cium/calmodulin binding. Two potential models for the release from autoin-hibition by the pseudosubstrate have been suggested (Wilmanns et al., 2000).

Structures of titin kinase mutants and truncations should give further insight into the activation mechanism. Initially, titin kinase used for crystallisation was isolated from the bacculovirus/insect cell system (Mayans et al., 1998; Mayans and Wilmanns, 1999). However, for mutants and truncations of titin kinase, the insert introduced into the bacculovirus vector was lost after two to three cell generations (P. Zou, personal communication). The instability of the vector in correlation with the physiologically problematic presence of excess titin kinase in the active state disabled the expression in eukaryotic cells. Thus, a change to the prokaryotic expression system was carried out. Several mutation constructs had been prepared for expression in E. coli (P. Zou). Starting from these con-structs, expression and purification protocols were tested on autoinhibited titin kinase and some mutants which were designed to mimic the active kinase. Only one of the mutants (TKYD, description see below) was expressed and purified at a low yield of about 0.1 mg purified protein from ten liters of culture. Hence, as new strategy, the design of new constructs in a different vector was chosen.

A.2 Materials and Methods