(A) Domain overview of L. major SAS-6. Lines indicate constructs that were used in this work. (B and C) Left: ribbon presentation of the head-to-head dimers of L. major SAS-6’s N-terminal domain present in the SAS-697–274 crystal. Shown are the dimers formed between chain B and chain C (B) and chain A and symmetry-related chain A (C). α-helices (α) and β-sheets (β) are numbered sequentially. Right: detailed views of the corresponding dimerization interfaces. Interface residues are labelled and shown in sticks, dotted orange lines indicate hydrogen bonds. The two dimers show largely identical side-chain orientations in their interfaces. Note, however, that F257 (ringed in blue) in the B–C dimer inserts into a hydrophobic pocket, while in the A–A dimer Y215 is flipped into this pocket and displaces F257. To better illustrate this, a semi-transparent molecular surface of one of the subunits is also presented (grey). (D) Sedimentation-equilibrium analytical ultracentrifugation data for 400 µM L. major SAS-697–274 wild-type (blue circles) and F257E mutant (green circles) and Y215K mutant (red circles) obtained at 11300, 17000, and 21200 rpm. Data for the F257E mutant were fitted to an ideal single-species model (solid line). Analysis of multiple concentrations gave a molecular weight of 17,727 ± 219 Da, close to the expected molecular weight for the monomer of 19,640 Da. As initial fits to a similar model for the WT and Y215K data gave higher molecular weights of 27,589 ± 209 Da and 30,951 ± 595 respectively, the data were fitted to a monomer–dimer equilibrium model (solid line) giving dissociation constants, KD, of 622 ± 70 µM for the WT and 190 ± 27 µM for Y215K mutant. The plots on the right show the residuals of the fits to the data for the wild-type (blue circles) and the corresponding F257E mutant (green circles) and Y215K mutant (red circles). (E) Left: ribbon presentation of the L. major SAS-697–320 F257E coiled-coil dimer structure (chain A: red, chain B: green). Right: detailed view of the region boxed on the left. Interaction interface between N-terminal head domain and the coiled-coil stalk. Residues that make contact are labelled and are shown as sticks, dotted orange lines indicate hydrogen bonds.