Fig. 5: Identification of key residues in MtSerB2 and analysis of their implication in tetramerization ability by mutagenesis and size-exclusion chromatography (SEC-UV).

a Sequence alignment of MtSerB2, MaSerB and MmSerB2 focused on the hinge-loop region. The residues are coloured according to their degree of conservation between the three sequences according to the legend. The position of residue Q92 in MtSerB2 is indicated by an arrow. b Superimposition of chromatograms obtained during the SEC-UV analysis of MtSerB2 Q92E variant and native MtSerB2. Area under curve percentages (%AUC) are indicated next to each peak to quantify the change in the dimer (D)/tetramer (T) ratio between the variant and the native enzyme. MtSerB2 Q92E forms less tetramer than native MtSerB2. A more visual indication of that change is shown in the inset where the UV absorbance at 280 nm is normalised to the dimer peak. c Sequence alignment of MtSerB2, MaSerB and MmSerB2 focused on the second alpha helix (α₅) of the ACT2 domain. Residues C148, V149, G150 and I154 differentiate MtSerB2 from MaSerB and MmSerB2 by their distinct physicochemical properties (low conservation score) and their positions are indicated by arrows. d Superimposition of chromatograms obtained during the SEC-UV analysis of MtSerB2 Q92E C148T V149Y G150R I154T variant and native MtSerB2. Variant MtSerB2 is essentially dimeric. e Three-dimensional representation of the location of residues A150, D151, E152 and T156 (homologous to key residues C148, V149, G150 and I154 for tetramerization in MtSerB2) of helix α₅ of the ACT2 domain in MaSerB crystal structure¹⁴ (PDB: 3P96). The residues are exposed to solvent and not engaged in intramolecular interactions. The difference in numbering comes from the fact that MaSerB bears two more residues than MtSerB2 at the N-term.