Figure 4.

Structure of the CNNM4_cNMP dimer. (A) (left) Association of two CNNM4_cNMP domains (blue and orange) in the crystal. The presence of such dimers in solution was confirmed by SAXS data that indicate a shape and volume consistent with the shown bead model (grey spheres). (A) (right) Residues (mostly hydrophobic) at the dimerization interface between CNNM4_cNMP subunits, formed primarily by their strands β4 and β5. (B) SEC-MALS analysis of CNNM4_cNMP (left) and CNNM4_{BAT-cNMP-Ctail} (right). CNNM4_cNMP (grey; Mw = 20936.78 Da monomer) yields an apparent Mw of 24,740 ± 60 Da and 33,920 ± 50 Da at concentrations of 1 mg/mL (dotted lines) and 10 mg/mL (solid lines), respectively, indicating prevalence of the dimer at higher concentration. For the F631A mutant (orange), smaller apparent M_W of 21,730 ± 50 Da and 26,440 ± 50 Da are observed for 1 and 10 mg/mL, respectively, indicating that this mutation strongly impedes dimerization. CNNM4_{BAT-cNMP-Ctail} (grey; M_W = 47,861.17 Da monomer) similarly yields an apparent Mw of 51,120 ± 60 Da and 63,610 ± 100 Da for 1 and 10 mg/mL, respectively, again indicating prevalence of the dimer at higher concentration. For its F631A mutant (yellow), very similar M_W of 49,230 ± 80 Da and 60,920 ± 90 Da are derived, indicating a negligible effect on CNNM4_{BAT-cNMP-Ctail} dimerization. Thus, the disruption of CNNM4_cNMP dimerization by this mutation (see left) is overridden by dimerization via the still intact Bateman module. (C) SAXS analysis of CNNM4_cNMP. (left) Experimental SAXS data (orange) and scatter curve (black) for the best-fit bead model (Χ² = 2.93) of CNNM4_cNMP dimers in solution (shown in (A)). (right) The dimensionless Kratky plot (orange) indicates a well-folded, flexible, and elongated arrangement for CNNM4_cNMP. The corresponding BSA reference peak is also shown.