Structural basis for the specific inhibition of glycoprotein Ibα shedding by an inhibitory antibody

Ectodomain shedding of glycoprotein (GP) Ib alpha is thought to mediate the clearance of activated, aged or damaged platelets. A monoclonal antibody, 5G6, has been developed recently to specifically bind to the GPIb alpha shedding cleavage site and to inhibit its shedding. However, the molecular mechanism underlying antigen recognition and inhibitory specificity is not clear. To elucidate the structural basis for 5G6 binding to GPIb alpha, we determined the crystal structure of 5G6 Fab fragment in complex with its epitope peptide KL10 (GPIb alpha residues 461-470, KLRGVLQGHL), to 2.4-angstrom resolution. Key residues in both 5G6 and KL10 were mutated to validate their effects in antibody binding by using isothermal titration calorimetry. The 5G6 Fab-KL10 peptide complex structure confirmed the direct association of 5G6 with its target GPIb alpha residues and elucidated the molecular basis underlying its binding specificity and high affinity. The similar binding properties of 5G6 Fab fragment to GPIb alpha on human platelets as those to KL10 suggests that such an interaction may not be affected by the plasma membrane or nearby GPIb beta. This structural information may facilitate further antibody optimization and humanization.