Chaetomium thermophilum CRM1

C. themophilumCRM1 was expressed recombinantly inE.coli and purified by Dr. Thomas Monecke (AG Ficner, University of Göttingen). A full ProteoPlex screen was performed yielding conditions identical to the successful crystallization conditions (Hepes pH 8.0), which were used for all further analysis. The protein was subjected to the standard GraFix procedure and subsequently analyzed by negative stain EM. An exemplified micrograph is shown in figure 3.15.

A monodisperse field of particle views was obtained allowing standard image analysis techniques to be applied. 300 micrographs with a pixel size of 1.85 Å/px were recorded and 31970 particles were selected. To avoid model bias reference-free class averages were calculated via reference-free alignment usingimagic and 2D maximum-likelihood classifi-cation usingrelion. In figure 3.15 representative class averages are shown. Already at this stage class averages can be found that hardly fit into a closed ring structure as the pre-vious crystal structures suggest. To obtain a valid initial 3D model, several independent

3.2 Structural and Dynamical Insights into CRM1 97

Figure 3.15: Chaetomium CRM1 Raw Data.Raw data and initial class averages ofChaetomium CRM1. Left: A representative negative stain raw micrograph is shown. Right: typical class averages obtained from the data are depicted by standard classification and maximum likelihood classification.

strategies were used in parallel. Firstly, angular reconstitution was used in combination with resampling techniques. Therefore, the obtained 1000 class averages were resampled with replacement into 250 sets of 12 class averages and an angular reconstitution 3D model was calculated. The resulting 3D models were manually inspected and sorted for similarity. The very same class averages were also subjected tosimple PRIME, a software using a reference-free projection matching approach. Both strategies yielded very similar 3D models. In both cases the same two distinct conformations could be identified.

One model resembles the ring shaped, closed form as known from the cargo complexes and the other appears to be an open superhelical, pitched structure, which matches the newly obtained crystal structure model (cp. figure 3.16). Both models were refined until convergence competitively against the full dataset. To assure the validity of both models, a cross validation strategy was used. Therefore, the dataset was split into two groups according to the best fit to one of the two models. Subsequently, images assigned to the closed ring conformation were refined against the open superhelical model and vice versa.

In both cases the resulting model relaxed back from the wrongly assigned model to the original model after several iterations. This way, the final models were obtained directly from the split dataset almost without model bias and can be considered valid. Further verification of the obtained models could be gained by fitting the previously mentioned crystal structures into the models as shown in figure 3.16. To remove model bias, the crystal structures were filtered to a resolution of 20 Å. Both models fitted very well to the crystal structures.

98 3 | Results

Figure 3.16:Chaetomium thermophilumCRM1 conformations.Density models ofChaetomium CRM1 conformations. The resulting two conformations- the compact, closed (grey) and the superhelical, open (yellow) are shown. In both cases the atomic models taken from the PDB were fitted: for the closed model PDB: 3gjx and for the open model: PDB: 4fgv

After the validation of the two models, a first approximation of the energy landscape can be attempted. The distribution of the particle numbers assigned to the model allow an estimation of the energy difference between the two conformations. 18953 particles were assigned to the closed ring shaped model and 13017 to the superhelical model. Using equation 1.1 one can approximate an energy difference of ∆G =−1.5_mol^kJ corresponding to 0.7 kBT. Thus, the energy of the surrounding medium is sufficient to shift the protein from the one conformation to the other.

To gain a better resolution of the energy landscape, it was attempted to resolve more conformational states. As the two independent approaches used to find the initial 3D structures yielded the same two conformations, one can assume that no other major conformational change takes place. This means that further conformations should only be intermediates between the known two. To identify those, new reference models were calculated using a normal mode analysis. This was done using both the obtained EM structure and the crystal structure respectively as ground state model. Interestingly, in

3.2 Structural and Dynamical Insights into CRM1 99 both cases the first non-trivial mode¹ describes exactly the opening movement as it can be seen in figure 3.17. Interestingly, one of the simulated 3D models from the NMA of the open superhelical model matches the experimentally gained closed structure almost perfectly (cp figure 3.17 right)

Figure 3.17: Mode 7 of NMA of CRM1.Mode 7 of the normal mode analysis of CRM1 is shown.

Starting from the open CRM1 crystal structure an NMA was calculated. Intermediates of the first non-trivial mode (mode 7) are shown. On the right the superimposition simulated closed state (orange) and the experimentally determined closed state (green) is shown.

This further indicates, that the opening and closing of the ring is an energetically favoured movement which occurs rapidly, because the NMA modes are by default sorted with respect to increasing energy. To identify intermediate states in the dataset, 10 equally spaced structures from the NMA trajectory were used as references for a competitive alignment against the full dataset. To minimize the model bias, structures were calculated by angular reconstitution from class averages not by projection matching in the first few iterations. The resulting structures were subjected to three further rounds of projection matching. Five of the ten tested conformations yielded reasonable structures. Thus, the 13000 closed state particles are split in 7500 closed and 5500 almost closed particles, while the 18000 which aligned to the open conformation were in three almost equally populated states (see figure 3.18).

The newly found almost closed conformation of CRM1 is a compact ring structure, which shows a small but significant gap between the N- and C-terminal regions. MD simulations performed in the group of Helmut Grubmüller (Max Planck Institute for biophysical Chemistry, Göttingen, Germany) support the existence this intermediate.

In general, this more detailed analysis provides a more accurate description of the energy landscape of conformational transitions. While the various open conformations are mostly equally populated, meaning there is almost no energy barrier between them, the closed conformation is slightly higher populated and therefore slightly stabilized. The free energy difference between the open and the closed state ensemble can now be better estimated as 0.4kBT = 0.9_mol^kJ .

1The first six modes gained from an NMA describe the three possible translations and rotations of the full molecule. They are thus called trivial since they contain no information on the molecules flexibility.

100 3 | Results

Figure 3.18: Energy LandscapeC.thermophilum CRM1.An approximation on the energy land-scape of free CRM1 is shown. Ten equally spaced intermediate structures from NMA mode 7 were produced and all particles images were refined against these models. Particles were assigned according to the best fitting model. The number of particles in the individual substates is plotted against the state as conformational coordinate. Further, the free energy was calculated following equation 1.1 to gain a first estimate on the conformational landscape. For the significantly populated states calculated structures are depicted.

Even though this energy difference is rather small, it is significant and most probably important for CRM1’s conformational cycle. Thus, it is necessary to find the molecular reason for this difference. In principal, it can either arise from an entropic or enthalpic contribution or for both. Entropically the open state is much more favored since more different conformational states can be populated. Thus, the energy difference has to be explained by the enthalpic contribution. Most likely it is the binding energy of the two termini to each other. The crystal structures determined by Thomas Monecke predict that the major determinant for the two conformations is a C-terminal helix. This expands to the NES-cleft in the open conformation, but is folded back to the terminus in the closed conformation, most likely to facilitate binding of the termini (see figure 3.19 left).

To investigate this assumption, a variant of the protein deleting this helix was produced.

While the helix itself is invisible at the current resolution range, the energy landscape can still be analyzed. It has to be noted that the overall quality of the sample got much worse due to the deletion and significant aggregation of CRM1 particles was observed in the micrographs. Nevertheless, through manual selection of the isolated particles the full computational analysis could be performed. Here, the resulting energy landscape was flat, meaning the particles were rather evenly distributed among all of the conformations (see figure 3.19 right). The deletion of the helix does result in open and closed structures that do no longer differ significantly in energy, confirming the hypothesis.

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Figure 3.19: Significance of the C-terminal helix. Left: The conformational change of the C-terminal helix (orange) is shown in the crystal structures. In the open conformation, the helix expands towards the acidic loop (green) and NES-cleft (blue) while it folds back to the interaction surface between the termini in the closed conformation. Right: An representation of the energy landscape of free CRM1 with deleted C-terminus is shown. Ten equally spaced intermediate structures from the described NMA mode 7 were produced and all particles images were refined against these models. Particles were assigned to the model they fitted best to. The number of particles in the individual substates is plotted against the state as reaction coordinate. Further, the free energy was calculated following equation 1.1 to gain a first estimate on the conformational landscape. For the significantly populated states, the refined structures are shown.

To finalize the analysis, higher order NMA modes were analyzed in the same manner.

This, however, resulted in no significantly different structures. Thus, the energy landscape of the C. thermophilum CRM1 seems to be almost fully described by the opening and closing movement, which seems to be purely driven by thermal energy.