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 apolipoprotein (apo) E are crucial for the interaction between apoE and the low density lipoprotein receptor (LDLR). To explore the roles of amphipathic alpha-helix and basic residue organization in the binding process, we performed site-directed mutagenesis on the 22-kDa fragment of apoE (amino acids 1-191). Exchange of lysine and arginine at positions 143, 146, and 147 demonstrated that a positive charge rather than a specific basic residue is required at these 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 decrease in free energy of binding of approximately 600 cal/mol, consistent with the 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 reduced when K143 and K146 were both mutated to arginine (K143R + K146R), indicating that more than the side-chain positive charge can be important.Exchanging lysines and leucines indicated that the amphipathic alpha-helical structure of amino acids 140-150 is critical for normal binding to the low density lipoprotein receptor.
Zaiou
M
M
Biochemistry Department, M. C. P. Hahnemann University, Philadelphia, PA 19129, USA.
Arnold
K S
KS
Newhouse
Y M
YM
Innerarity
T L
TL
Weisgraber
K H
KH
Segall
M L
ML
Phillips
M C
MC
Lund-Katz
S
S
D000595:Amino Acid Sequence; D000599:Amino Acids, Diamino / Q000737:chemistry; D001057:Apolipoproteins E / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011973:Receptors, LDL / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D000599:Amino Acids, Diamino; D001057:Apolipoproteins E; D008024:Ligands; D010446:Peptide Fragments; D011973:Receptors, LDL; D011994:Recombinant Proteins