A 13C NMR characterization of lysine residues in apolipoprotein B and their role in binding to the low density lipoprotein receptor.
NMR spectroscopy of 13C-labeled human low density lipoprotein (LDL) has been employed to characterize the lysine (Lys) residues in apo B-100. Reductive methylation with [13C]formaldehyde converts up to two-thirds of the Lys to the dimethylamino derivative; this pool of Lys is exposed at the surface of the LDL particle. The [13C]dimethyl-Lys which are visualized exhibit resonances at chemical shifts of 42.8 and 43.2 ppm (pH 7.6) indicating that they exist in two different microenvironments; this is a reflection of the native conformation of apo B associated with lipid, because the labeled, reduced, and alkylated protein gives a single resonance when dissolved in 7 M guanidine hydrochloride. The pH dependences of the Lys chemical shifts indicate that the two types of Lys titrate with different pK values; "active" Lys have a pK of 8.9, while "normal" Lys have a pK of 10.5. About 53 active Lys and 172 normal Lys are exposed on the surface of LDL with the remaining 132 Lys which are present in the human apo B-100 molecule being buried and unavailable for methylation. Addition of paramagnetic ions indicates that the active and normal Lys have different exposures to the aqueous phase; apparently this is a reflection of folding of the apo B molecule. The relative involvement of active and normal Lys in binding of apo B-100 to the LDL receptor on fibroblasts was explored by measuring the decrease in receptor binding as a function of the degree of methylation of the two types of Lys. Upper limits of 21 active and 31 normal Lys in the entire apo B-100 molecule are involved in the binding of LDL to the receptor. It is likely that these Lys are located in domains of apo B which contain clusters of basic amino acid residues and also bind heparin. If the sequence corresponding to apo B-48 (residues 1-2151) which does not bind to the receptor is excluded, then the above limits are halved; an upper limit of 10 active Lys may be particularly involved in receptor binding.
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005292:Ferricyanides; D005557:Formaldehyde; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008077:Lipoproteins, LDL / Q000378:metabolism; D008239:Lysine; D009682:Magnetic Resonance Spectroscopy; D008745:Methylation; D010446:Peptide Fragments / Q000378:metabolism; D011973:Receptors, LDL / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000378:metabolism
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D005292:Ferricyanides; D008077:Lipoproteins, LDL; D010446:Peptide Fragments; D011973:Receptors, LDL; C007931:hexacyanoferrate III; D005557:Formaldehyde; D013917:Thrombin; D008239:Lysine