A.3 Results and Discussion
A.3.4 Titin kinase truncations and mutations
4.6 Structural comparison of PB1 domains
˚ A isgiven,obtainedbySSM(Krissineland Henrick,2004)withthenumberofmatchingCαinparentheses,andinthetoprightthesequenceidentityofthematchingresiduesis givenin%.
72 NBR1 PB1 in complex with p62 PB1
Figure 4.9: 2 Fo-Fc electron density map atσ=1.5 of the OPCA loop in NBR1 PB1. The sidechains of the residues E51, E52 and E54 are not visible in the electron density of the single NBR1 PB1 at 1.55 ˚A resolution (left panel), but are ordered in the PB1 complex at 2.1 ˚A resolution (right panel). The figure was prepared using the program Pymol (DeLano, 2002).
sequence alignment (Figure 4.11). Moreover, this is supported by mutational studies, in which a D50R point mutation, located in the OPCA motif, abrogates the binding of NBR1 to p62. In contrast, the NBR1 PB1 mutation K12A, implying a basic cluster in this position, interacts with p62 (Lamark et al., 2003) and disproves NBR1 PB1 to act as a type-B in the interaction with p62 PB1.
4.3.9 Structure solution and refinement of the PB1 complex
The structure of the PB1 complex was solved using molecular replacement with NBR1 PB1 as search model, and was refined to 2.1 ˚A resolution. Two PB1 complexes are located in the asymmetric unit which are related in a two-fold NCS. The refinement statistics are given in Table 4.7.
Resolution range (˚A) 20.0-2.1
R (%) 20.6
Rfree (%) 26.1
Ramachandran plot (%)
Residues in most favoured region 91.9 Residues in disallowed region 1.0
Number of atoms 2764
Number of water molecules 122 R.m.s.d. from ideal bond length (˚A) 0.015 R.m.s.d. from ideal bond angles (◦) 1.374 Table 4.7: Refinement statistics of PB1 complex.
4.3 Results 73
Figure 4.10: Superimposition of the type-A PB1 domains. The PB1 domain of NBR1 is shown in blue. The OPCA loop points towards the left. A: superimpo-sition with cdc24p (1Q1O) in yellow, B: superimposuperimpo-sition with p40phox (1OEY) in red, C: superimposition with aPKCi (1WMH) in green, D: superimposition with MEK5 (1WI0) in orange.
74 NBR1 PB1 in complex with p62 PB1
E1E2D1E3E4D2E5
A1 A2
NBR1QVTLNVTFK--------NEIQSFLVSDP---------ENT--TWADIEAMVKVSFDL-------NTIQIKYLDEENE-EVSINSQGEYEEALKMAVKQ----------GNQLQMQVHEGcdc24pSILFRISYNNNSNNTTSSEIFTLLVE-----------KVW--NFDDLIMAINSKISNTHNNNISPITKIKYQDEDGD-FVVLGSDEDWNVAKEMLEANNE---------KFLNIRLY/p40phoxTNWLRVYYYEDT----ISTIKDIAVEEDL-------SSTP--LLKDLLELTRREFQR-------EDIALNYRDAEGD-LVRLLSDEDVALMVRQA--RGLPSQKR--LFP-WKLHITQKDNYRVYNTMPMEK5VLVIRIKIP-------NSGAVDWTVHS---------GPQL--LFRDVLDVIGQVLPEA------TTTAFEYEDEDGD-RITVRSDEEMKAMLSYYYSTVMEQQVNGQLIEPLQIFPRACPKCLQVRVKAYYR--------GDIMITHFE-----------PSI--SFEGLCNEVRDMCSFDNE----QLFTMKWIDEEGD-PCTVSSQLELEEAFRLYELNKD---------SELLIHVFPCp62SLTVKAYLLGKE--DAAREIRRFSFCCSPEPEAEAEAAAGPGPCERLLSRVAALFPALR----PGGFQAHYRDADGD-LVAFSSDEELTMAMSYVKD------------DIFRIYIKEKpar6ĮIVEVKSKFD--------AEFRRFALP-----------RASVSGFQEFSRLLRAVHQIPG-----LDVLLGYTDAHGD-LLPLTNDDSLHRALASG-------------PPPLRLLVQKRbem1pTTKIKFYYK--------DDIFALMLK-----------GDT--TYKELRSKIAPRIDTD-------NFKLQTKLFDGS-GEEIKTDSQVSNIIQA----------------KLKISVHDI/p67phoxAYTLKVHYK---------YTVVMKTQ-----------PGL--PYSQVRDMVSKKLELRL-----EHTKLSYRPRDSNELVPLSE-DSMKDAWGQVKN------------YCLTLWCENT B1 B2 B2’ B1
Figure4.11:Structure-basedsequencealignmentofPB1domains.ThesequenceoftheOPCAmotifisboxed.Keyresiduesoftheacidic(A1,A2)andbasic(B1,B2,B2’)clustersarehighlightedinredandinblue,respectively.ThesecondarystructureelementsareindicatedaccordingtoNBR1PB1.
4.3 Results 75
Figure 4.12: Anomalous difference fourier map at σ = 4.0. In the NBR1/p62 complex structure to 2.1 ˚A resolution, cadmium is coordinated by H66 of p62 PB1 and three waters. Here, the cadmium site with a 10.8σpeak in the anoma-lous difference fourier map is shown. The water molecules are in hydrogen distance from the cadmium ion.
4.3.10 Cadmium chloride bound to p62 PB1
Presence of 10 mM cadmium chloride dihydrate facilitated crystal growth to en-able data collection. The cadmium signal for the data collected atλ= 0.9201 ˚A (with f” = 1.9 at this wavelength) can be detected, but is not strong enough for solving the structure. In the anomalous fourier map, two strong cadmium peaks appear with 10.8σ and 7.6σ for the two cadium sites. An anomalous dif-ference fourier map is shown atσ = 4.0 for the first site in Figure 4.12. During structure refinement the cadmium was refined anisotropically. The cadmium is bound on the surface of the structure, in a tetra-coordination in each complex of the asymmetric unit. The heavy metal cadmium is coordinated by H66 of p62 PB1 and three water molecules, which are in hydrogen distance.
4.3.11 Overall structure of the heterodimer
The overall structure of the complex of the PB1 domains of NBR1 and p62 is given in Figure 4.13. Both domains adopt a β-grasp like fold comprising 5 β-strands and 2 α-helices as described for the single NBR1 PB1 domain (see above). The OPCA motif of NBR1 PB1 with residues T43 to M70 which is involved in the interaction is coloured in violet. Hence, NBR1 PB1 is categorised as type-A and p62 PB1 as type-B in this interaction. Some residues in p62 in the elongated loops between strands β1 and β2 and strand β1 and α1 are disordered and, therefore, they are not shown in Figure 4.13. These residues are E14 and D15 and C27 to G40, which make 16 out of 187 residues in the complex. Moreover, the two N-terminal residues of NBR1 PB1 are not visible in the electron density. The NBR1 PB1 domain is generally better defined than p62 PB1. Moreover, a higher hydration of NBR1 PB1 is observed.
76 NBR1 PB1 in complex with p62 PB1
Figure 4.13: Ribbon presentation of the NBR1 PB1/p62 PB1 heterodimer. The NBR1 PB1 is shown in blue with the OPCA motif indicated in violet, and the p62 PB1 is rendered in green. Parts of two loops in p62 PB1 are not visible in the electron density, comprising E14 and D15 (in the β1-β2 loop) and C27 to G40 (in the long β2-α1 loop).
Since the structure of NBR1 PB1 has been solved as a single molecule and in complex with p62 PB1, a comparison of the two different states can be made.
The overall fold is similar with an r.m.s.d. of 1.03 ˚A (86 Cα) as determined by LSQMAN (Kleywegt et al., 1997) with a cut-off of 3.5 ˚A. The density of the OPCA loop is defined in the PB1 heterodimer presumably due to the par-ticipation of these residues in the interaction (Figure 4.9, right panel) and is illustrated in comparison with the single NBR1 PB1 (Figure 4.9, left panel).
The largest differences are found in the loop from S21 to N26, residues of which are involved in crystal contacts with that loop from another molecule in the complex structure.
4.3.12 Heterodimeric PB1 domain interface
The front-to-back interaction of NBR1 PB1 and p62 PB1 is mediated by two areas on the surface of each domain. NBR1 PB1 interacts as the type-A domain with residues from the OPCA motif, which are D50, E52 and E54. P62 PB1 contributes as a type-B domain to the interaction with the ’basic back’ lysine
4.3 Results 77
Figure 4.14: Stereo view of the NBR1/p62 PB1 interaction site at the cluster A1-B1. The residues of the NBR1 PB1 are shown in blue, and those of the p62 PB1 in green, which make up the A1-B1 interaction site. The lysine K9 (p62 PB1) interacts with D50, E52 and E54 (on NBR1 PB1), and Y9 and R96 (p62 PB1) support this interaction. The residue R22 (p62 PB1) cannot be seen in this orientation. The 2 Fo-Fcmap is contoured at σ= 1.3.
K7. Moreover, the binding is further supported by Y9 and R96 from p62 PB1 (Figure 4.14). This leads to a particular strong interaction in the first cluster A1-B1, which is mainly built up by salt bridges between the residues.
The second acidic-basic cluster in this complex, A2-B2, is formed by E63 and N59 (NBR1 PB1) representing cluster A2, and R21 (p62 PB1) representing cluster B2. The residue R21 is stabilised by interdomain interaction with residue E19. While R21 participates in the A2-B2 cluster the preceding R22 points toward the opposite site of the strand β2 and interacts with D50 form the A1-B1 cluster.
The contributing residues to this cluster are shaded in red (for the A1) and in blue (for the B1) in the sequence alignment (Figure 4.11).
The total accessible surface area buried in the interface is 1146 ˚A, which is less than the usually observed value in structures of dimers (Lo Conte et al., 1999).
The electrostatic surface potential for the complex is shown in an open-book style in Figure 4.15. On the left the electrostatic potential is given for NBR1 PB1 and on the right p62 PB1 is shown. NBR1 PB1 displays the acidic patches as discussed for the single domain, and p62 PB1 carries the basic patches in agreement with the sequence comparison (Figure 4.11) and surface patterns from other PB1 domain complexes (Hirano et al., 2005). In Figure 4.16 the molecules NBR1 PB1 and p62 PB1 are represented as ribbon model in the same orientation as in Figure 4.15 with the residues involved in the interaction highlighted in a stick presentation. The two patches on each molecule are encircled.
78 NBR1 PB1 in complex with p62 PB1
Figure 4.15: Electrostatic potential of the PB1 domain of NBR1 and p62 in an open-book style. On the left, NBR1 PB1 is shown with residues of the acidic cluster A1 (D50, E52, E54) and A2 (E63). On the right, p62 PB1 is given with the residues of the basic cluster B1 (K7, R22, R96) and B2 (R21). The surface is coloured by electrostatic potential in blue (positive) and red (negative). The figure was prepared using the program GRASP (Nicholls et al., 1991).
Figure 4.16: Molecular interaction between p62 PB1/PB1 heterodimer. The ribbon and stick presentation is given in an open-book style in the same orien-tation as Figure 4.15. Residues from the acidic regions in NBR1 PB1 and from the basic regions in p62 PB1 are encircled.
4.3 Results 79
4.3.13 Comparison with other heterodimeric PB1 complexes
The crystal structures of the PB1 complexes p67phox-p40phoxand aPKCι-par6α have been solved previously (Wilson et al., 2003; Hirano et al., 2005). In addition, the solution structures of the PB1 domains of bem1p (Terasawa et al., 2001) and cdc24p (Yoshinaga et al., 2003), which are also forming a complex, were determined by NMR as single domains.
Comparison of the residues contributing to the interaction within cluster A1-B1 shows that the lysine is the key residue on the type-B domain. In the structure-based sequence alignment the residues involved in interaction are accentuated. In NBR1-p62, Y9, R96, and R22 further strengthen the binding in addition to the lysine. This interaction by at least three residues on both domains in the first cluster is not present in the other complexes with known structure. In the p40phox-p67phoxcomplex, a single residue in the type-B domain p67phox, the conserved lysine, contributes to the interaction with the type-A OPCtype-A motif. In aPKCι-par6α, in addition to the lysine (here K19), R89 in par6α PB1 is involved in the binding at an equivalent position to R96 in p62 PB1. Although no complex structure of bem1p and cdc24p is available, comparison of residues at equivalent positions reveals that besides the lysine on the type-B domain, a tyrosine (Y18) at a corresponding position to Y9 in p62 PB1 is found, which could be involved in binding. The equivalent residue to R96 (p62 PB1) is K79 (bem1p). Although this residue is not an arginine, participation in the binding owing to the basic character of that residue may be possible and cannot be excluded at this point.
In contrary to the cluster A1-B1, the acidic-basic cluster A2-B2/B2’ is less strictly defined in terms of conserved residues on the type-B domain. Location at two different positions in the sequence (see Figure 4.11) has been identi-fied (Hirano et al., 2005). The complex NBR1-p62 belongs to the A2-B2 cate-gory with the residues in the second acidic-basic cluster located in the strand β2. The constellation with residue R21 mediating a salt bridge formation in one cluster and R22 with the other cluster has also been described for the com-plex interface of aPKCι-par6α. There, the second basic site comprises residue R27 (par6α PB1, corresponding to R21 in p62 PB1) in interaction with S72 and E76 (aPKCιPB1, corresponding to N59 and E63 (NBR1 PB1)). The first basic cluster built up by R28 (par6α PB1) interacts with D63 (aPKCι PB1, corresponding to D50 (NBR1 PB1)). Residue E17 of the same molecule (par6α PB1) which is involved in the interaction does not have a correspondence in p62 PB1. The complex p40phox-p67phoxdoes not show this pattern of interaction in the second basic cluster, since residue K382 (p67phoxPB1) in helixα1 mediates mainly the interaction with D302 (p40phox PB1).
80 NBR1 PB1 in complex with p62 PB1
4.4 Discussion
4.4.1 Cadmium bound to H66 in p62 PB1
During optimisation of PB1 complex crystal growth, cadmium chloride was found to be an effective additive to improve crystal size. In the structure, a cadmium atom is tetra-coordinated at H66 and three water molecules at the surface of the p62 PB1. Cadmium has been investigated as additive in tallisation and has been reported to be the cation of choice for protein crys-tallisation (Trakhanov and Quiocho, 1995). Diverse coordination of Cd2+ have been described, such as tetra-, penta- and hexacoordination even within the same structure, e.g. of the histidine-binding protein from E. coli (Trakhanov and Quiocho, 1995). Generally, cadmium ions are positioned at the interface between two neighbouring protein molecules. These metal ions can form a com-plex with the electron donors such as histidine and cysteine but also glutamate, aspartate, arginine, or lysine (Trakhanov et al., 1998). Hence, coordination usually forms cadmium bridges across the interface of protein molecules. Here, bridging of two molecules could not be observed since contribution from amino acids in the coordination is limited to H66 (p62 PB1) and the three water molecules. For one of the cadmium ions in the asymmetric unit, the coordi-nation of one water could be replaced by D81 (p62 PB1) from a symmetry molecule due to vicinity of D81 to the cadmium. However, the sidechain of D81 (p62 PB1) is disordered and hence it was not modelled. A clear bridging of symmetry molecules could not be observed, however, the binding of cadmium to p62 PB1 may cover patches on the surface which unfavour crystallisation.
4.4.2 The three classes of PB1 domains
The PB1 complex of NBR1-p62 constitutes the third complex structure of a PB1 complex. NBR1 PB1 represents the type-A domain in this interaction and the mutated p62 (DDAA) PB1 the type-B domain. NBR1 PB1 represents a typical domain of the type-A category according to sequence and structure.
Wild type p62 PB1 is known to homo-oligomerise and, thus, arrays of p62 PB1 domains are formed which can create large aggregation of p62. Hence, p62 PB1 was classified as type-AB domain. p62 PB1 exerts its role as a type-B domain in the NBR1/p62 heterodimer due to the introduced mutations D69A and D73A which prevent self-oligomerisation. In the basic cluster B1, a remarkably strong interaction was detected, based on mainly four residues (K7, Y9, R22 and R96).
4.4.3 Model of the p62 PB1 homodimer
Superimposition of a p62 (DDAA) PB1 with NBR1 PB1 demonstrates how wild type p62 PB1 would homodimerise. The residues of the OPCA loop backbone adopt a similar location as in NBR PB1, and the corresponding residue of E63 (NBR1 PB1) which is D82 is in vicinity to R21 (p62 PB1). Only the distance of S78 (equivalent of N59) to R21 in the direct superimposition is too large for
4.4 Discussion 81
interaction, but a change towards a similar arrangement as seen in the aPKCι-par6α complex with S72 (aPKCι, corresponding to S78 (p62 PB1)) and R27 (par6α, corresponding to R21 in the other p62 PB1 molecule) upon binding is conceivable.
Further investigations of the p62 PB1 homodimer without aggregation may provide a second mutation construct of p62 PB1. Instead of mutating the propensities of the type-A domain, the characteristic residues of the type-B domain can be mutated. Mutation of residue R21 (Wilson et al., 2003) and eventually E19, which stabilises R21, will result in a type-A domain. This can be used to study the affinity by ITC as shown here or for structural investigations which may also visualise the loop region between β2 and α1.
4.4.4 Affinity of the NBR1/p62 PB1/PB1 heterodimer complex
The affinity of the heterodimer complex has been studied by ITC and the dissociation constant determined to be Kd = 12 nM ± 1 nM. PB1 complex formation has been studied using ITC by other groups before. For the PB1 complex of p40phox-p67phox a dissociation coefficient Kd = 10 nM has been re-ported (Lapouge et al., 2002). The low value of the dissociation constants Kd (Kd = 1/Ka with Ka being the equilibrium binding coefficient) in both com-plexes indicates a high affinity of PB1 domains towards each other and a tight complex formation.
4.4.5 p62 interactions
In addition to the NBR1/p62 PB1 heterodimer and the discussed p62 ho-modimer complex formation, interaction of p62 PB1 occurs with MEK5 PB1 (Lamark et al., 2003) and with aPKCζ PB1 (Wilson et al., 2003). The ev-idenced association of p62 PB1 as the type-B domains with MEK5 and aPKCζ may occur under similar prerequisites as with NBR1. A reminiscent compact structure devoid of additional insertions has been found in all three structures (Figure 4.10 C, D). The r.m.s.d. of these PB1 domains is low (Table 4.6). The overall structural appearance of MEK5 PB1 and aPKCζ PB1 and particular the conservation of the OPCA motif permit binding of these domains to p62 PB1 in a similar manner as NBR1 PB1. Large structural differences were not detected which may explain potential favouring of p62 PB1 for one domain or another. Owing to the fact that p62 is a ubiquitously distributed protein, in-teraction of p62 PB1 with the according binding partners NBR1, MEK5, and aPKC depends on the spatial and temporal distribution and availability.
4.4.6 Biological relevance of the NBR1/p62 heterodimer complex
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
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