Ca 2+ -binding of C 2 F-mutants with mutations affecting hearing

Two mutations - known to affect hearing - in the C2F-domain of the protein were also analyzed for their ability to bind Ca²⁺. Mice which carry the pga mutation within the C2F-domain of otoferlin are deaf [54]. In a loop region located at the opposite site of the Ca²⁺-binding region an aspartate residue was replaced by a glycine (D1767G) [20]. The position of this amino acid indicates that the mutation has no influence on Ca²⁺-binding. Whether or not the exchange of this amino acid in a flexible loop region is enough to influence the overall structure of the C2-domain is questionable. Due to the position in a flexible bottom loop it is unlikely that this mutation is able to cause such a change in the structure that the Ca²⁺-binding is influenced. The performed MicroScale Thermophoresis measurements showed a decrease of Ca²⁺-affinity for the pga-fragment in comparison with the wild type C2F (Figure 3.17–16). With a higher

4.2 Ca2+-binding of otoferlin C2-domains

Kd value as the wild type the pga-mutant is not able to bind Ca²⁺ under physiological conditions [86]. Ramakrishnan et al. as well reported a diminished Ca²⁺-binding for C2F-pga [35].

The E1804del-mutation was found in the human C2F-domain of otoferlin. The deletion of glutamic acid at position 1804 leads to temperature sensitive deafness [17]. For MicroScale Thermophoresis this mutation was inserted into the C2F-domain.

Two different temperatures were tested in MST measurements. The first sample was measured at 22 °C for comparability with the wild type and other measured C2-domains. In this case a binding curve for Ca²⁺ was obtained. In consideration that this deletion leads to temperature sensitive deafness above 38 °C the measurement was repeated at 39 °C. As well a Ca²⁺-binding were detected. The obtained Kd for 39 °C (8.4 ± 0.3 mM) is a little bit lower than the value for 22 °C (10.2 ± 0.3 mM). In comparison to the wild type curve (Kd = 290 +/- 14 µM) (Figure 3.17–18) both E1804del curves display a much lower affinity for Ca²⁺. Due to the physiological Ca²⁺-concentrations in the order of a few tens of µM [86] the E1804del-mutant is not able to bind Ca²⁺ neither at 22 °C nor 39 °C.

In both cases it has to be considered that the native protein would likely contain different post-translational modifications which can lead to a different folding of the domain and consequently to a different behavior. In this case as well interactions with phospholipids or other proteins might have an effect as well.

In the following Table 4.2-1 all measured Kd values for the wild type and the different mutations of the C2F-domain are listed. It seems that all tested mutations affect the C2F-domain in a similar manner. Typically, it would be expected that the aspartate mutants have a much higher effect on the Ca²⁺-binding than the pga-mutant but all obtained Kd values are in the same order of magnitude. Except for the wild type domain none of the mutants seems able to bind Ca²⁺ under physiological conditions.

4.2 Ca2+-binding of otoferlin C2-domains Table 4.2-1 Overview of the obtained Kd values of the different C2F-mutants. All measurements took place in a sample buffer containing 10 mM HEPES pH 7.4, 150 mM NaCl and 0.1 % Tween at a temperature of 22 °C. A final protein concentration of 1 µM was used and the highest used Ca²⁺-concentration was set to 200 mM.

a The highest Ca²⁺-concentration was changed to 20 mM.

b This measurement was performed in the presence of 300 mM NaCl.

c For the E1804del-mutant a second set of samples at 39 °C were measured.

C2-domain-fragment Dissociation constant (Kd) C2F wild type 449 ± 20.8 µM^a (290 ± 14.4 µM^b) specific aspartates without any influence on the high affinity binding site [36]. Based on the possibility to bind more than one Ca²⁺-ion it might be the case that the obtained Kd value of the wild type comprise two binding events which cannot be detected separately using MicroScale Thermophoresis. The resulted Kd values of the tested aspartate mutants in this work lead to the hypothesis that all aspartates share a similar role within the binding process in otoferlin. The mutation of one aspartate could affect the whole Ca²⁺-binding process. It is likely that all present aspartates are interconnected to each other and the required conformational change of the involved aspartates for Ca²⁺ coordination is disturbed by a single mutation.

The influence of at least the two pseudo-phosphorylation sites integrated in the β-strands of the C2F-domain probably cause a slightly different folding than for the wild type. Therefore it seems likely that the positions of the top loops are changed to an arrangement in which no Ca²⁺-binding under physiological conditions would be possible.

For the pga-mutant another hypothesis was made. According to predictions based on homology between C2-domains of closely related proteins the pga-mutation is located in the flexible bottom loop of the C2-domain. It is known that mutations which are not

4.2 Ca2+-binding of otoferlin C2-domains

part of the active site, interaction areas or binding sites often affect the protein fold or decrease the stability of the functional conformation [94]. The incorrect folding can lead to degradation or aggregation of the protein. In comparison to the other expressed and purified C2F-fragments the pga-mutant showed a much lower expression level. This leads to the assumption that the exchanged amino acid is relevant for the correct folding of the domain. Furthermore a misfolding of the protein is likely to cause a different conformation of the top loops which might not be able to bind Ca²⁺ anymore. Moreover the made predictions could be different to the actual structure of the native C2F-domain. Because no structure of the otoferlin C2F-domain is known so far the location of the pga-mutation in a structured part of the domain cannot be excluded.

The before mentioned problems during folding can also be caused by in-frame deletions like the E1804del which is positioned in a bottom loop of the domain. The misfolding resulting from this deletion would explain why no changes in the Ca²⁺-affinity could be detected performing MST measurements at different temperatures. The temperature sensitive mutant should show a different behavior at temperatures higher than the body temperature in comparison to the tested 22 °C.

But if the protein was folded incorrectly during expression it is no wonder that we did not observe two different affinities at the two tested temperatures.

In summary all tested mutant C2F-domains seem to be present in conformations which are different to the wild type C2F-domain. It is unlikely that the aspartate mutants influence the folding of the β-sheet consisting core component of the C2-domain. The change in Ca²⁺-binding probably results from a rearrangement of the top loops. The inserted pseudo-phosphorylation sites as well as the mutation/deletion known to affect hearing presumably cause a different folding of the entire domain. All tested mutations have in common that no Ca²⁺-binding occurs under physiological conditions.