Homodimeric solution structure

Accurate docking of two proteins requires both orientational and translational in-formation. Measurement of HN-RDCs in a single alignment medium, leads to four possible orientations of one subunit of CylR2 with respect to the second subunit, when the alignment tensor is not axially symmetric. The four-fold degeneracy is re-solved by intermolecular PRE distances or intermolecular NOE data. PRE distances have the same distance dependence as NOE data and so the XPLOR-protocol for rapid protein-protein docking on the basis of RDCs and intermolecular NOEs and PRE data can be used with the same standard energy function [89].

The performance of this PRE-based rigid-body docking has been tested for the 57 HN-RDCs for each monomer in combination with two different sets of experimental intermolecular restraints: PRE data (Table 5.2a) and PRE together with NOE data (Table 5.2b). In addition, the docking was tested with symmetry distance restraints to imply twofold symmetry [135]. These symmetry distance restraints were either used together with RDC data (Table 5.2c and d) or used to replace RDC data (Ta-ble 5.2e and f). For comparison, the same rigid-body protocol was run with RDCs and intermolecular NOEs as the only experimental restraints (Table 5.2g). These seven rigid-body dockings with different sets of restraints resulted in structures with the same monomer-to-monomer orientation. After simulated annealing with all co-ordinates fixed with the exception of the side-chain atoms of residues contributing to the dimer interface and refinement in explicit solvent, the seven ensembles, com-posed of 20 structures, have a backbone rmsd (residues 3-63) between 0.54 ˚A and

Table 5.2: Statistics for CylR2 dimer solution structure determination.

Most-favored region (%) 88.3 88.0 88.0 88.2 88.5 88.7 87.7

Additionally allowed region (%) 10.4 10.8 10.5 10.3 9.4 9.9 11.2

Generously allowed region (%) 1.0 1.0 1.2 1.3 2.0 1.3 1.0

Disfavoured region (%) 0.2 0.2 0.3 0.2 0.2 0.2 0.1

Rmsd from the mean^b:

Backbone atoms (˚A) 0.59± 0.59± 0.59± 0.61± 0.54± 0.59± 0.65±

0.11 0.08 0.11 0.07 0.09 0.10 0.13

All heavy atoms (˚A) 0.90± 0.89± 0.88± 0.91± 0.86± 0.89± 0.94±

0.10 0.09 0.11 0.09 0.08 0.10 0.13

Rmsd from the X-ray structure^b:

Backbone atoms (˚A) 1.15 1.14 1.18 1.22 1.99 1.23 2.14

All heavy atoms (˚A) 2.08 2.01 2.10 2.07 2.79 2.10 2.92

intermonomer rmsd:

mean NMR structure:

Backbone atoms (˚A) 0.29 0.37 0.34 0.25 0.52 0.33 0.33

All heavy atoms (˚A) 0.72 0.75 0.66 0.88 0.81 0.78 0.76

NMR ensemble (20 structures):

Backbone atoms (˚A) 0.88± 0.79± 0.84± 0.94± 0.73± 0.92± 0.83±

0.28 0.27 0.36 0.28 0.22 0.34 0.42

All heavy atoms (˚A) 1.49± 1.44± 1.47± 1.54± 1.34± 1.54± 1.43±

0.25 0.26 0.4 0.3 0.3 0.32 0.4

intermolecular energy (kcal/mol) -1609.6 -1343.5 -1662.2 -1309.7 -1527.9 -1422.1 -1436.4 a Restrains distance differences to imply twofold symmetry [135]

b determined for residues 3-63 of each monomer

0.65 ˚A (Table5.2). The 10 lowest energy structures of the NMR ensemble calculated with HN-RDCs and PREs is shown in Figure5.7. The small backbone intermonomer

MTSL

MTSL

Figure 5.7: Homodimeric solution structure of CylR2. Superposition of the 10 NMR structures with the lowest energy. Helices and β-strands are shown in magenta and violet, respectively. The calculated average position of MTSL attached to either position N40C (green) or position T55C (orange) is indicated for the left subunit.

rmsd values for the mean structure (<0.6 ˚A) and all structures of the NMR ensemble (<1˚A) indicate the high symmetry of the homodimers and are similar to the backbone intermonomer rmsd value of 0.75 ˚A between the monomers of the X-ray structures.

As expected, the orientation of the monomers within the homodimer can be restrained by experimental RDCs or a symmetry function [135] (Table 5.2). Therefore, in this case, the experimental RDCs were not absolutely required. However, considering the backbone rmsd (residues 3-63) to the X-ray structure, the experimental HN-RDCs perform significantly better in the absence of intermolecular NOEs (1.15 ˚A versus 1.99 ˚A) and slightly better in the presence of intermolecular NOEs (1.14 ˚A versus 1.22 ˚A). This can be explained by the fact that with the HN-RDCs, one of the prin-cipal axis of the alignment tensor must be parallel and the other two orthogonal to the twofold axis [136] while the symmetry function restrains symmetry-related inter-monomer distance differences to 0 without restraining the two axis. As the HN-RDCs restrain the symmetry more stringently than the symmetry function, the docking re-sults are not improved when HN-RDCs are used together with the symmetry function.

This is demonstrated by an almost unchanged backbone rmsd (residues 3-63) to the X-ray structure independent of the use of intermolecular NOEs for the calculations (Table 5.2a,b,e and f).

Decreasing the error bounds to ± 4 ˚A resulted in an increased rmsd and in a larger number of violated intermolecular restraints. Changing τ from 4 ns to 6 ns did not change the docking results because of the small difference in the calculated distances (Figure 5.6). Due to the different sources of errors for the PRE-derived distances (see 5.3.3), the docking did not converge to a unique solution when only intermolec-ular distances for one spin label position together with the HN-RDCs were used.

The backbone rmsd (residues 3-63) between the mean NMR dimer structure gener-ated using intermolecular PRE distances as the only intermolecular restraints together with HN-RDCs and the X-ray structure is 1.15 ˚A (Table 5.2a). The precision of the structure was only insignificantly improved by addition of 24 manually assigned in-termolecular NOEs as indicated by the backbone rmsd (residues 3-63) to the X-ray structure of 1.14 ˚A (Table 5.2b). Moreover, the combination of HN-RDCs and

inter-molecular NOEs is not sufficient to define the dimer structure as accurately as the intermolecular PREs in combination with HN-RDCs. This is indicated by a backbone rmsd of 2.14 ˚A relative to the X-ray structure (Table 5.2g).

There are small differences between the orientation of the two monomers in the NMR and the X-ray structure as the rmsd values are overall higher for the dimer than for the monomer (Figures 5.1B and 5.8B). Most notable are the differences for the

B A

0 10 20 30 40 50 60

residue 0

1 2 3

RMSD [Å]

α5 α1

α2 α3

α3 α2

α4

α1

α4 β1

β2

Figure 5.8: Comparison of the solution and crystal structure of CylR2. Secondary structure elements are indicated. (A) Mean structure of the NMR ensemble (blue) superimposed on the X-ray structure (green/red). (B) Average backbone rmsd per residue between the mean NMR-structure and the 20 NMR-structures (solid line) and the X-ray structure (dashed line).

longest helixα4 (residues 43-52) that contributes strongly to the dimer interface and the loop connecting helix α3 and α4 involved in the DNA binding. Within this loop the flexible residue Ser42 is found. Structural flexibility in this region is probably important for DNA binding (see 4.3.4 and 4.3.5).