Structural basis for the specific inhibition of glycoprotein Ibα shedding by an inhibitory antibody.
Ectodomain shedding of glycoprotein (GP) Ibα 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 GPIbα 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 GPIbα, we determined the crystal structure of 5G6 Fab fragment in complex with its epitope peptide KL10 (GPIbα residues 461-470, KLRGVLQGHL), to 2.4-Å 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 GPIbα residues and elucidated the molecular basis underlying its binding specificity and high affinity. The similar binding properties of 5G6 Fab fragment to GPIbα on human platelets as those to KL10 suggests that such an interaction may not be affected by the plasma membrane or nearby GPIbβ. This structural information may facilitate further antibody optimization and humanization.
Key Laboratory of Pediatric Hematology &Oncology Ministry of Health, Pediatric Translational Medicine Institute, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Yue
Y
Tao
Key Laboratory of Pediatric Hematology &Oncology Ministry of Health, Pediatric Translational Medicine Institute, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Xiaoqin
X
Zhang
Aflac Cancer and Blood Disorders Center, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA.
Xin
X
Liang
Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences; University of Science and Technology of China, Hefei, China.|Key Laboratory of Structural Biology; Chinese Academy of Sciences, Hefei, China.
Jianye
J
Zang
Key Laboratory of Pediatric Hematology &Oncology Ministry of Health, Pediatric Translational Medicine Institute, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Xi
X
Mo
Aflac Cancer and Blood Disorders Center, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA.
Renhao
R
Li
D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D001792:Blood Platelets / Q000378:metabolism; D002151:Calorimetry; D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D006801:Humans; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding
D000911:Antibodies, Monoclonal; D019038:Platelet Glycoprotein GPIb-IX Complex