Sequence conservation of Xpo6 in evolutionary distant species

In parallel to the secondary structure predictions, we also analyzed the sequence conservation of Xpo6 in other species. We used the position-specific-iterated BLAST (PSI-BLAST) tool of NCBI, which was more powerful than normal BLAST tools in identifying the Xpo6 sequences with low overall conservation. We specifically looked at the conservation of the predicted loop regions. Loops that are not directly involved in cargo recognition are more likely to be divergent. Indeed, we found that the conservation in these regions were lower than the regions that corresponded to predicted helices.

Especially in evolutionary distant species like the tunicate Ciona intestinalis and the slime mold Dictyostelium species, these loop regions were significantly shorter. Also in the Xpo6 sequences from Xenopus laevis and Xenopus tropicalis, the loop regions are strikingly less conserved compared to the rest of the protein. Also second loop region has an insertion in all Xenopus isoforms we analyzed. This was a further indication that the deletion regions corresponded to loops that are not essential for Xpo6 function. Xpo6 from birds and mammals showed quite high conservation throughout the entire sequence. Figure 3-27 shows the Xpo6 alignments from selected species in comparison to human Xpo6.

Our loop truncations on Xpo6 might be very crude and have negative effect on the overall fold of the protein, and may hinder crystallization, rather than improving. Using an Xpo6 version that is trimmed over millions of years by evolution might be a more elegant approach to solve the problem with flexibility. We have chosen Xpo6 of two species, Dictyostelium purpureum and Dictyostelium fasciculatum for later crystallization studies.

Figure 3-27 Sequence conservation of Xpo6 in different species

A) Alignment of Xpo6 sequences from human, X. laevis and X. tropicalis. The predicted loop regions were shown in red boxes. Note the low conservation in these regions compared to the rest of the sequences. Also there is an insertion in the loop 2 regions in frog sequences. B) Alignment of Xpo6 sequences from human chicken and D. purpureum and D.

fasciculatum. The predicted loop regions were shown in red boxes. Note the missing sequences in slime mold Xpo6 versions, corresponding to the predicted loop regions. Xpo6 from chicken is highly similar to human Xpo6 throughout the entire sequence.

Using shorter versions of a protein is a frequently used approach in crystallization. Another approach is using proteins from thermophilic organisms. Proteins from thermophiles are evolved to withstand high temperatures, and are therefore more rigid in ambient or low temperatures used for crystallization, compared to their mesophilic counterparts. Since NTRs are only present in eukaryotes, thermophilic fungus Chaetomium thermophilum is the favorite choice for NTR crystallization studies (Monecke et al., 2013). However, Xpo6 being absent in fungi, this species is not a likely solution for us. We therefore decided to use Xpo6 from species with slightly higher body temperatures than humans. Birds with an average 41ºC body temperature were the first choice. We decided to use Xpo6 from chicken, and would also like to test whether chicken Xpo6 works better with chicken skeletal actin. We expressed and purified Xpo6 from chicken (ggXpo6), D. purpureum (dpXpo6) and D. fasciculatum (dfXpo6) and tested them in a complex formation assay with RanGTP and profilactin in comparison to human Xpo6. The binding assay was performed as described in 3.5.1. We also used Xpo6 from Xenopus laevis as another positive control. ggXpo6 was indistinguishable from human and Xenopus Xpo6 in terms of

A) Sequence alignment of human and Xenopus Xpo6

B) Sequence alignment of human, chicken and Dictyostelium Xpo6

loop regions 1 and 2

complex formation. However, neither dpXpo6 nor dfXpo6 was bound to profilactin in this setup (Figure 3-28-A). Before giving up hopes on our new species, we wanted to test them on a phenyl sepharose matrix that is now known to increase the stability of the actin export complex.

Figure 3-28 Export complex formation with new Xpo6 species

Xpo6 from human, frog, chicken and slime mold were compared for their ability to form an export complex. The buffer for each binding reaction was the same: 10 mM Tris pH 7.5, 20 mM NaAc, 1 mM DTT A) Complex was immobilized through profilin, and the amount of Xpo6RanGTP bound to profilactin was used to judge the complex stability.

Experimental setup was identical to Figure 3-26. Note that human frog and chicken were similar, whereas the Xpo6 from slime molds did not form a complex. B) Complex formation was tested on phenyl sepharose matrix. Experimental setup was identical to Figure 3-4. The Note that human and chicken Xpo6 are similar, whereas the Xpo6 from slime molds bind weaker to RanGTP and profilactin. However, the binding is specific and significantly higher than background as shown by the control: without any Xpo6.

Indeed both dpXpo6 and dfXpo6 bound RanGTP and profilactin. The binding was weaker compared to human and chicken Xpo6, but it was clearly stronger than background control (Figure 3-28-B). The difference between a weak binding and no binding is very significant.

We can say that Xpo6 from these two evolutionary distant species still retain the ability to bind Ran, actin and profilin from human. The binding may be enhanced if the binding partners are chosen form the same species. We will use these new Xpo6 constructs for crystallization studies of the free Xpo6, as well as for the crystallization of the actin export complex.