(1324-A) Insights into the dynamics of PCSK9/LDLR interaction: effect of gain-of-function mutations and pH-dependent affinity
Monday, February 5, 2024
12:00 PM – 1:00 PM EST
Location: Exhibit Halls AB
Abstract: Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates circulating cholesterol levels by binding to low-density lipid receptor family members (LDLR) expressed on cell surfaces. This interaction leads to internalization of the PCSK9-LDLR complex, priming LDLR for degradation in lysosomes and reducing LDLR numbers. Gain-of-function (GOF) mutations in PCSK9 such as D374T or D374Y elevate levels of circulating LDL, increasing the risk of cardiovascular disease, while loss-of-function mutations confer protection. Neutralizing antibodies preventing PCSK9/LDLR binding have emerged as a promising therapeutic class, decreasing heart attack and stroke.
An ELISA (Enzyme-linked immunosorbent assay) was designed to evaluate the effect of candidate drugs on PCSK9/LDLR binding. We optimized experimental parameters and buffer compositions to detect conformational effects of pH and PCSK9 mutations.
The effect of GOF mutations D374T and D374Y on PCSK9 binding to the ectodomain of LDLR was compared between endosomal pH (pH 6) and physiological pH (pH 7.4). PCSK9 showed higher affinity for LDLR at acidic pH compared to neutral pH, indicating that PCSK9 binds more avidly to the LDLR protein in the lysosomal/endosomal compartments. Notably, the PCSK9 (D374T) and PCSK9 (D374Y) GOF mutants had higher affinity for LDLR than wild-type PCSK9 at neutral pH (7.4) and at acidic pH (6.0). The D374T mutation increased PCSK9-LDLR binding by ~2-3-fold, while D374Y led to at least a 40-fold increase in LDLR binding compared with wild type PCSK9, accounting for the severe hypercholesterolemia phenotype in patients with this mutation.
These results were confirmed using AlphaLISA® assays. Again, the affinity of PCSK9 (wild type and mutants) for LDLR was enhanced at acidic pH (from 13 to 38-fold increase) and the mutation D374Y had a much greater affinity (8-fold increase) than the wild type PCSK9 for LDLR at neutral pH. A similar trend was observed using Bio-layer interferometry (BLI). D374Y mutation showed the highest affinity (Kd = 2.97E-09), followed by D374T (Kd = 4.66E-09) and WT (Kd = 6.41E-09).
Thus, PCSK9/LDLR assays can assess pH-dependent differences in LDLR binding affinities of PCSK9 wild-type and mutant proteins and allow for a comprehensive understanding of binding dynamics. Our study independently confirms that the PCSK9-D374Y mutant binds LDLR with greater affinity than does wild-type PCSK9 with a similar order of potency at both acidic and neutral pH (WT < D374T << D374Y). Our results are also in an agreement that the acidic environment of endosomes substantially enhances the affinity of PCSK9 for LDLR (possibly due to formation or exposition of additional sites of interaction). These observations are consistent with the literature on PCSK9 mutations and relevant to the clinical consequences of gain of function PCSK9 mutations.