We have expressed the ECDs of the human

We have expressed the ECDs of the human α1, β1, γ and ɛ AChR subunits in the past using the methylotropic yeast Pichia pastoris as soluble glycosylated proteins [15], [16]. Further work with the γ subunit ECD revealed a marked improvement of the solubility and yield following the mutation of hydrophobic APETx2 or regions [17]. In the present study, we proceeded to mutate the ECDs of the remaining muscle AChR subunits, namely α1, β1, δ and ɛ, in an attempt to improve their characteristics. Furthermore, since the mature AChR is a heteropentamer, in which the interface among adjacent subunits is important for many physiological (e.g. ligand binding) and immunological characteristics (autoantibody binding), we linked the ECDs in tandem by a flexible peptide to produce concatamers, thus aiming to create a model of the extracellular part of the whole AChR. Such constructs would provide valuable information for structural and pathophysiological attributes of the muscle AChR. Overall, we show that the mutant ECDs display greatly improved characteristics, are suitable for structural studies, and we present our promising initial crystallization efforts.
Materials and methods
Results
Discussion In this paper we describe the expression and characterization of mutant ECDs of the human muscle AChR α1, β1, δ and ɛ subunits in the P. pastoris expression system. All the wild type subunit ECDs had been expressed in our lab in the past as soluble secreted polypeptides [16], [18]. However, the expression levels were low and the extended oligomerization/aggregation of most ECDs posed as a serious drawback for structural studies or large-scale therapeutic applications. All the ECDs have also been expressed in Escherichia coli with much higher yields, but lacking posttranslational modifications, while they were found mainly in the insoluble fraction and had to be purified under denaturing conditions [20]. We, therefore, aimed at creating mutant ECDs of the muscle AChR that would have improved characteristics and yield. A detailed analysis of the γ subunit ECD revealed that certain mutations could have a pronounced impact [17]. The most striking effect was seen when the Cys-loop of the γ-wt ECD was exchanged for the Cys-loop of the AChBP, a protein homologous to the AChR ECD that forms soluble homopentamers. Previously, the same mutation had also been shown to improve the solubility of the neuronal AChR α7 ECD [21]. Consequently, we exchanged the Cys-loops of all the other muscle subunit ECDs to create the BPloop mutants. These showed a profound improvement of their solubility and expression yield, which increased from 2 to 43-fold with respect to the wild type (Table 1). An improvement was also observed in the size exclusion chromatography analysis of the BPloop mutants, which showed minimal aggregation for most ECDs, sharper peaks and elution at larger volumes, suggesting the presence of monomers or oligomers rather than multimers. The size exclusion chromatography data agree with the DLS analysis of the BPloop ECDs. The α1-BPloop, α1-BPloopAsn141 and β1-BPloop ECDs showed the smallest sizes and polydispersities, in accordance with their excellent chromatograms. On the other hand, the ɛ-BPloop and δ-BPloop ECDs, which form dimers/oligomers, presented with larger size measurements on DLS, as well as considerably higher polydispersity values. The enduring presence of oligomers/multimers for the ɛ and δ ECDs could be due to the presence of cysteine residues (other than the pair flanking the Cys-loop), or additional hydrophobic surfaces, which might be responsible for intermolecular interactions. Indeed, both the ɛ and δ ECDs have one extra cysteine each, at position Cys190 and Cys108, respectively. On the other hand, the monomeric α1 and β1 BPloop mutants do not have similar additional cysteine residues (with the exception of the ACh-binding cysteine pair at 192–193 on α1), suggesting that these actually might be responsible for the aggregation/multimerization seen in the ɛ and δ ECDs. A study on the neuronal α7 ECD showed that mutation of a similar residue at Cys116 had a significant effect on aggregation and structure stabilization [22]. Similarly, mutation of the additional cysteine residues in the γ ECD (Cys106 and Cys115) resulted in improved solubility, albeit only in the BPloop mutant background [17]. It is now possible, therefore, to consider equivalent mutations for the ɛ and δ BPloop ECDs as they may improve further the characteristics of these two ECDs.