Fig. 1.
Binding and activity of LukGH wild-type and mutants to CD11b-I and crystal structure of LukGH-CD11b-I. (A) Steady-state analysis of LukGH wild-type binding to moCD11b-I. The steady state Kd is shown in the Inset. (B) Binding of LukGH to hu- or moCD11b-I expressed as response units (mean of 2 to 10 independent experiments ±SEM) and Kd (mean of 2 to 10 independent experiments ±SD). EC50 values of LukGH mutants toward differentiated HL-60 cells or mouse PMNs assessed in a luminescent cell viability assay measuring cellular ATP content (mean of 2 to 8 independent experiments ±SEM). For variants that had limited or no cytotoxicity (could not kill >75% of cells at the highest toxin concentration used), EC50 is not shown. (C) Cytotoxicity of LukGH, LukGHK319A, and LukED toward mouse PMNs assessed in a luminescent cell viability assay measuring cellular ATP content at cytotoxin concentrations of 30 µM, 20 µM, and 100 nM, respectively (mean of 3 independent experiments ±SEM). (D) Front and top view of LukGHK319A–moCD11b-I crystal structure. Dark blue and light green cartoons represent LukH and LukG from dimer 1 and dark green and light blue cartoon represent LukG and LukH from dimer 2, respectively. moCD11b-I is shown as an orange cartoon. Other dimers forming the octamer pore and bound CD11b-I molecules, are shown as a gray cartoon. Red spheres represent bound DMSO molecules from one asymmetric unit (dark red sphere represents DMSO 2). Comparison of moCD11b-I secondary structure (E) and MIDAS residues (F) from LukGHK319A–moCD11b-I structure (orange ribbon) with the active (1IDO, light pink ribbon) and inactive (1JLM, light gray ribbon) form of huCD11b-I. C-terminal α-helix is shown as light pink cartoon (1IDO) and gray cartoon (1JLM). Structures are aligned on moCD11b-I and MIDAS residues in E and F, respectively. The metal ions from the moCD11b-I structure and the inactive form of CD11b-I (1JLM) are shown as orange and gray spheres, respectively.