Figure 5. Stereochemical analysis of modulator preferences.

(A) The clustering of CALP-bound P⁻⁵-substituted iCAL36 peptides (stick figures) is shown following main-chain superposition of the CALP B-protomers. Peptides are colored by sequence: iCAL36 (W; green carbons), F- (red), Y- (black), H- (yellow), A- (cyan), L- (orange), and V- (purple). RMSD offsets at the P⁰-P⁻³ residues were within coordinate error, while modest variations were observed at the P⁻⁴ and P⁻⁵ positions (black arrow). The largest difference is 3.2 Å, between the C_α of the L- and C_α of the A-substituted peptides.

(B) Each substituted P⁻⁵ side chain (colored stick figure) is shown, as docked against its CALP binding interface (surface, cartoon and stick figure). The residues that contact the P⁻⁵ residue (His309, Gly310, Val311) are labeled in the CALP:F-iCAL36 structure. Each panel is labeled with the P⁻⁵ residue identity. See also Figure S6.

(C) The electrostatic potential surface of WT CALP (left) reveals a highly polarized distribution on either side of the peptide binding cleft. The electrostatic potential surface of CALP-E317A (right) shows a reduced, although still negative, interaction surface for the P⁻³ Asp residue of iCAL36_QDTRL (insets). See also Figure S7.

(D) Electrostatic interactions are shown for P⁻¹ Arg (left) and P⁻³ Asp (right) residues of the iCAL36_QDTRL peptide (stick figure, yellow carbons) bound to CALP (gray C_α trace). At the top, nearby charged CALP residues are shown (stick figures, green carbons). The P⁻¹ Arg forms a hydrogen bond (dashed line) with Gln³¹⁴. The closets charged residue for the P⁻³ Asp is Glu³¹⁷ (d > 5 Å). In the lower panels, the corresponding electrostatic potential surfaces are shown (rendered at 10 k_BT/e) underscoring the negative electrostatic environment of both the P⁻¹ and the P⁻³ side chains.