Figure 2.4: Overall structure of the tandem Ig domains A168-A169. The domain A168 is shown in red and A169 in dark red. The continuous strand is highlighted in blue and the extended loop between strand A and A’ in yellow.
ing to the reference domain) was calculated. A new coordination frame was defined with the eigenvectors of the inertia tensor as the new coordinate axes and the center of mass of this domain as the origin. The z-axis is the longest axis of the module and directs toward the C-terminus of that domain. The tilt describes the angle between the z-axis of the rotated domain and the refer-ence domain. The twist angle is the angle of rotation around the z-axis which is required to align the two domains. The interdomain geometry was deter-mined (Bork et al., 1996) and the values were calculated using the program
’mod22’ written and kindly provided by Bruno Kieffer (unpublished).
2.3 Results
2.3.1 Overall structure of titin A168-A169
The structure of A168-A169 has been solved to 1.99 ˚A and 2.48 ˚A resolution in the space groups I222 and C222 with one and two molecules per asymmetric unit, respectively.
Both Ig domains adopt the topology of a greek keyβ-sandwich. They consist each of two β-sheets composed of nine β-strands, the sheets ABED of which form one strand and A’GFCC’ the other. In the second sheet all strands are an-tiparallel, except for the A’ strand which is parallel to the G strand. Comparison of these structural features like number and length of the strands, the connec-tion between the strands as well as the presence of I-set key residues allows to classify A168 as well as A169 to the I-set (intermediate set) of immunoglobulin
30 The A-band immunoglobulin domains A168 and A169
Figure 2.5: Superimposition of the tandem Ig domains A168 (purple) and A169 (red) shows a large overall resemblance with other titin Ig domains. The inser-tion in A169 between strand A and A’ emerges strikingly as a unique feature among the available three-dimensional titin Ig domains, which are Z1 (orange), Z2 (yellow), I1 (light green), I27 (dark green), M1 (blue) and M5 (teal).
domains (Harpaz and Chothia, 1994). A 310 turn is present between the E and the F strand at residues D69-D71 and R168-D170 in domain A168 and A169, respectively.
2.3.2 Comparison of the Ig domains Inserted bulge in A169
The structures of the Ig domains A168 and A169 have an overall r.m.s.d. of 1.2 ˚A (91 Cα) and a sequence identity of 20.5 % (Table 2.4). Superimposition of the two domains, however, displays an extended loop in the immunoglobulin domain A169 between the strands A and A’. The insertion in A169 in compari-son to A168 comprises five additional residues, TLEGM, in the connecting loop between strand A and A’, which form a bulge (Figure 2.5). This bulge is located along the longest side of the domain. Apart from the bulge in domain A169, the single tandem Ig domains resemble both a typical I-set immunoglobulin domain.
Remarkably, this insertion in A169 not only stands out in comparison with A168, but also in comparison with the other titin Ig domains with known
2.3 Results 31
A168 A169 Z1 Z2 I1 I27 M1 M5
A168 20.5 35.6 37.1 31.0 20.5 24.4 21.3 A169 1.20 26.8 26.2 26.9 16.7 21.8 18.8
(91)
Table 2.4: Structural comparison of titin Ig domains. In the top right, numbers represent sequence identity (in %) between the Ig domains. In the bottom left, the r.m.s.d. in ˚A is given, obtained by LSQMAN (Kleywegt et al., 1997) with a cut-off of 3.5 ˚A and the number of matching Cα in parentheses.
three-dimensional structure. A structure-based sequence alignment (Figure 2.8) against other Ig structures of titin clearly displays the bulge region in A169, which is, however, not present in the other structures. For comparison, avail-able Ig domain structures of different regions in titin were analysed. Two Ig domains from the Z-disc, Z1 and Z2 (Z1-Z2 in complex with telethonin, X-ray structure solved by N. Pinotsis), two Ig domains from the I-band, I1 (PDB code: 1G1C) and I27 (X-ray structure solved by C. Vega), and two Ig domains of the M-line, M1 (see chapter 3) and M5 (PDB code: 1TNM) were compared with A168 and A169 from the A-band. Interestingly, the bulge is not emerging in sequence alignments (ClustalW, T-Coffee) based only on sequences.
Structure comparisons and the sequence identity between the analysed struc-tures are given in Table 2.4. For both domains, A168 and A169, the titin Ig domain structure with the lowest deviation is Z1, with an r.m.s.d. of 0.83 ˚A (92 Cα) and 1.08 ˚A (89 Cα) for A168 and A169, respectively.
Loop and crystal contacts
The bulge is involved in crystal contacts with the symmetry related molecule.
This can be observed in both crystal forms. The interactions found in the crystal contacts are mainly hydrogen bonds, but also a salt bridge between E107 of one and K54 of the other molecule is found. The interactions are restricted to the residues P103, K104, T105, and E107 on the side of the loop contribution in
32 The A-band immunoglobulin domains A168 and A169
domain A169. These residues are interacting with the residues A45, I52, Q53, E54, and K56 located in the D strand of domain A168 of the second molecule.
In addition, one water molecule is involved in the interaction. Interestingly, in both crystal forms (all three molecules) a glycerol is found stabilizing the bulge. The B-factors of the bulge do not emerge by high values, but rather adopt values in the range of the stable core region.
2.3.3 Comparison with other tandem Ig domains Continuousβ-strand
The two Ig domains are connected by a continuous β-strand. Owing to the extension of the G strand of A168 into the A strand of A169, the two domains are tightly connected. The strand G of A168 with residues G84 to E95 merges in the domain A169 in the residues V96 to I100. The secondary structure elements as derived from DSSP (Definition of secondary structure of proteins) (Kabsch and Sanders, 1983) assign residues G84 to I100 to an extended β-strand in the structure with 1.99 ˚A resolution in space group I222. While the amino acids G84 to S91 bridge in an antiparallel way with the residues Q81 to V74 (F strand of A168), residues L92 to E95 form bonds to the parallel oriented strand A’ of A168 with the amino acids L12 to R15 (Figure 2.6). The antiparallel strand B of A169 participates in β-bridging with residues F126, S127, and K129 to the end of the continuous β-strand, of which V96, K99, and I100 are involved.
One chain of the lower resolution structure is not attributed as a continuousβ -strand, since it shows an interruption of the strand at residue E95 according to DSSP. In the higher resolution structure, P97 and A98 do not form hydrogen-bonds to anotherβ-strand, despite their assignment to the continuousβ-strand.
Residues S85, T89, and A98 are assigned according to DSSP to the chirality of a right-handed α-helix in contrast to other residues in this β-strand labelled as ideally twisted β-strand. In the continuously attributed β-strand, not all residues comply with all requirements of a continuous β-strand.
No hydrogen bonds are formed between the backbone Cα of one domain with those of the other domain. Backbone hydrogen-bonding occurs between E95 of domain A168 with V14 and R15, and between V96 of domain A169 with K129 (Figure 2.7). A few weak interactions are found in the interface, but an inter-domain interaction stabilizing the two domains could not be observed.
The continuous β-strand is found in the structures refined in space group I222 and in one molecule refined in space group C222, while the other shows an interruption in the strand in one residue between the two domains at residue E95. The three molecules superimpose well with an r.m.s.d. of 1.08 ˚A (193 Cα), 0.86 ˚A (195 Cα), and 0.84 ˚A (194 Cα) for I222 and A-chain of C222, I222 and B-chain of C222, and A-and B-chain of C222, respectively. The values of the B-factors are increased both in the linking region between the domains as well as in the regions where the strands are connected within the domain.
2.3 Results 33
Figure 2.6: Schematic representation of the continuous β-strand interaction.
The continuous β-strand is shown in blue. Hydrogen-bonding by the strands A’ and F from domain A168 (in red) and by the strand B of domain A169 (dark red) are represented.
34 The A-band immunoglobulin domains A168 and A169
Figure 2.7: Tandem domain interface. Backbone hydrogen bonding of E95 with V14 and Y16 in domain A168 (red), and of V96 with K129 in domain A169 (dark red). The continuous β-strand is shown in blue.
Tandem domain-domain arrangement
The tilt angle and twist angle were calculated according to Borket al. (1996). A plain interdomain linkage with a tilt angle between the two domains of 23◦and a twist angle of 137◦ in the structure in space group I222 are found. The two chains in space group C222 display slightly different values: chain A has a tilt of 23◦ and a twist angle of 125◦ and the chain B has 26◦and 132◦, respectively.