Apolipoprotein E-low density lipoprotein receptor interaction: influences of basic residue and amphipathic alpha-helix organization in the ligand

Conserved lysines and arginines within amino acids 140-150 of apolipoprotei n (apo) E are crucial for the interaction between apoE and the low density lipoprotein receptor (LDLR). To explore the roles of amphipathic alpha-heli x and basic residue organization in the binding process, we performed site- directed mutagenesis on the 22-kDa fragment of apoE (amino acids 1-191). Ex change of lysine and arginine at postions 143, 146, and 147 demonstrated th at a positive charge rather than a specific basic residue is required at th ese positions. Consistent with this finding, substitution of neutral amino acids for the lysines at positions 143 and 146 reduced the binding affinity to about 30% of the wild-type value. This reduction corresponds to a decre ase in free energy of binding of similar to 600 cal/mol, consistent with th e elimination of a hydrogen-bonded ion pair (salt bridge) between a lysine on apoE and an acidic residue on the LDLR. Binding activity was similarly r educed when K143 and K146 were both mutated to arginine (K143R + K146R), in dicating that more than the side-chain positive charge can be important. Ex changing lysines and leucines indicated that the amphipathic or-helical str ucture of amino acids 140-150 is critical for normal binding to the low den sity lipoprotein receptor.