Abstract
Lysine residues are implicated in driving the ligand binding to the LDL receptor family. However, it has remained unclear how specificity is regulated. Using coagulation factor VIII as a model ligand, we now study the contribution of individual lysine residues in the interaction with the largest member of the LDL receptor family, low-density lipoprotein receptor-related protein (LRP1). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and SPR interaction analysis on a library of lysine replacement variants as two independent approaches, we demonstrate that the interaction between factor VIII (FVIII) and LRP1 occurs over an extended surface containing multiple lysine residues. None of the individual lysine residues account completely for LRP1 binding, suggesting an additive binding model. Together with structural docking studies, our data suggest that FVIII interacts with LRP1 via an extended surface of multiple lysine residues that starts at the bottom of the C1 domain and winds around the FVIII molecule.
Highlights
It is unclear how the LDL receptor family binds large protein ligands
Using coagulation factor VIII as a model ligand, we study the contribution of individual lysine residues in the interaction with the largest member of the LDL receptor family, low-density lipoprotein receptor-related protein (LRP1)
Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and SPR interaction analysis on a library of lysine replacement variants as two independent approaches, we demonstrate that the interaction between factor VIII (FVIII) and lipoprotein receptor-related protein 1 (LRP1) occurs over an extended surface containing multiple lysine residues
Summary
It is unclear how the LDL receptor family binds large protein ligands. Results: HDX and lysine scanning identified factor (F)VIII regions and specific lysine residues binding low-density lipoprotein receptor-related protein 1 (LRP1). The acidic necklace model is consistent with previous observations that lysine residues are implicated in the binding of ligands other than RAP to the LDL receptor family [21,22,23,24,25,26,27,28], it has remained unclear how large ligands containing multiple lysine residues interact with the LDL receptor family members This is relevant because experiments using single CR domains and simple (modified) amino acids have shown that arginine and protonated histidine, especially pairs of proximal charges, can interact well with CR domains [29]. These combined studies reveal important novel aspects of the interaction between FVIII and LRP1
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