Folding and binding integrity of variants of a prototype ligand-binding module from the LDL receptor possessing multiple alanine substitutions.
The LA repeats that comprise the ligand-binding domain of the LDL receptor are among the most common autonomously structured extracellular modules found in the nonredundant protein sequence database. Here, we investigate the information content of the amino acid sequence of a typical LA module by constructing sequences with alanine residues at nonconserved positions in the module. Starting with the sequence of the fifth ligand-binding repeat of the LDL receptor (LA5), we created generic LA modules with alanine substitutions of nonconserved residues in only the N-terminal lobe, only the C-terminal lobe, and throughout both lobes of the module. LA variants with alanine residues at as many as 18 of 37 positions fold to a preferred disulfide isomer in the presence of calcium. Indeed, the six cysteines, the C-terminal calcium coordinating residues, two hydrophobic residues involved in packing, two glycines, and five other residues that form side chain-intramodule hydrogen bonds are alone sufficient to specify the fold of an LA module when alanine residues are present at all other positions. The LA variants with multiple alanines in either the N- or C-terminal lobe were then exploited to identify residues of LA5 that contribute to the binding of apoE-containing ligands in LDL receptor-derived "minireceptors", implicating nonconserved residues of the N-terminal lobe of LA5 in recognition of apoE-DMPC. Our library of LA modules with multiple alanine substitutions should be generally useful for probing the roles of nonconserved side chains in ligand recognition by proteins of the LDL receptor family.
Abdul-Aziz
Dunia
D
Department of Pathology, Harvard Medical School and Brigham & Women's Hospital, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA.
Fisher
Carl
C
Beglova
Natalia
N
Blacklow
Stephen C
SC
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001057:Apolipoproteins E / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism; D014644:Genetic Variation; D006801:Humans; D006860:Hydrogen Bonding; D066298:In Vitro Techniques; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011973:Receptors, LDL / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D001057:Apolipoproteins E; D008024:Ligands; D011973:Receptors, LDL; D011994:Recombinant Proteins; D000409:Alanine; D002118:Calcium; D004134:Dimyristoylphosphatidylcholine