The results of this study show that residues, which form the intramolecular contact between the β-propeller and LA4/5, play critical roles in lipoprotein uptake by the LDLR (). We tested nine residues at the interface and three residues in the EGF-A/EGF-B modules. All nine interface mutations and one mutation in EGF-B resulted in defective lipoprotein uptake. Most defects fell into one of three categories. Mutations H190Y, K560W, H562Y and K582W impaired both LDL and β-VLDL uptake by reducing the number of surface receptors. The W144A and W193A mutations reduced lipoprotein uptake by impairing lipoprotein binding. This defect manifested as a reduction in the affinity of LDL binding and as reductions in the fraction of surface receptors that were competent to bind LDL and β-VLDL. The W515A, W541A, H562Y and H586Y mutations reduced the ability of the LDLR to release LDL in response to acidic pH. These mutations crippled LDL uptake, but not β-VLDL uptake. The G293S and G375S mutations had no effect on lipoprotein uptake and we observed no defects in receptor distribution, lipoprotein binding, or acid-dependent release with cells expressing LDLRs with these mutations.
The H190Y, G375S and H562Y mutations were originally identified in individuals with FH and our data provide a mechanistic basis for loss-of-function for the H190Y and H562Y mutations. Fibroblasts expressing LDLR variants with these mutations had reduced LDL and β-VLDL uptake that correlated well with the relative number of surface receptors, suggesting that these mutations impair uptake by reducing the surface expression of the LDLR. In the case of H190Y mutation, the effect on surface expression was small and did not quite reach statistical significance (). Synthesis of the H190Y variant appeared to be normal because expression of GFP, which is translated from the same message as the LDLR-H190Y, was normal (); however, somewhat less LDLR-H190Y protein was observed in fibroblasts at steady state (). The H190Y mutation may slightly reduce the fraction of receptors that successfully transit through the ER-Golgi system. This type of defect (Class 2 FH mutation) reduces the total number of receptors through ER-mediated degradation of receptors that fail to achieve a native state. Class 2 FH mutations are the most common type of FH mutation observed in LA5 [34
] and biochemical studies have identified several mutations near H190 that hinder refolding of LA5 [7
]. Any effect of H190Y on folding is minor, however, because we do not observe immature, poorly-glycosylated receptors by immunoblot (), a feature observed with severe Class 2 FH mutations. Cells expressing LDLR-H190Y had normal LDL and β-VLDL binding affinity (), indicating that the LDLR-H190Y, which successfully arrives at the cell surface, is natively folded. Consistent with a minor effect of H190Y on LDLR function, individuals bearing an H190Y LDLR allele do not always exhibit hypercholesterolemia [22
]. In contrast to the H190Y mutation, the H562Y mutation significantly reduced surface expression (). Because total expression was normal, the LDLR-H562Y may spend more time in the endosomal system than normal. The H562Y variant had acid-sensitivity that was as poor as the W515A, W541A and H586Y variants (); however, normalization of LDL uptake to surface receptor number indicates that the abridgement in release was not responsible for the lipoprotein uptake defect observed in cells expressing the H562Y variant (). An extended time in endosomes may give the H562Y variant additional time to release its LDL cargo.
Interestingly, the K560W and K582W variants also had reduced surface expression and the region centered on H562 may play a role in LDLR recycling. As with lipoprotein release, recycling of the LDLR is inhibited when endosomes are unable to acidify [13
]. Recycling is also regulated by the presence of bound lipoprotein in a manner that is dependent upon the EGF-homology region, which encompasses the EGF-A, EGF-B, β-propeller and EGF-C modules [17
]. Point mutations that impair LDLR recycling have been identified throughout the EGF-A, EGF-B and β-propeller modules [34
], suggesting that control of receptor recycling involves coordinated actions of multiple sites. The K560W, H562Y and K582W mutations may highlight one such site, and the decrease in acid-sensitivity observed with the H562Y variant suggests that this site may play a role in coupling lipoprotein release with receptor recycling.
F362 may also participate in receptor recycling. The F362A variant had slightly reduced sensitivity to acidic pH (); however, the release defect does not appear to be causal in uptake impairment, because β-VLDL uptake was also reduced in cells expressing this variant. Indeed, the defect in β-VLDL uptake was more severe than the defect in LDL uptake (). The F362A mutation increased the surface expression of the LDLR (), suggesting that this mutation may augment receptor recycling to the cell surface. Augmented recycling may increase the fraction of receptors that recycle before releasing lipoprotein. Normally, this “retro-endocytosis” accounts for approximately 10% of the lipoprotein internalized by the LDLR, but a variety of perturbations can increase the fraction of lipoprotein retro-endocytosed [37
]. The F362A mutation may impair β-VLDL uptake more than LDL uptake because β-VLDL binds with higher affinity than LDL () and is more resistant to release than LDL [17
One surprise in our findings is that neither the G293S nor the G375S mutations had any detectable effect on lipoprotein uptake. Mutations at both glycines have been implicated in FH and G375S is an FH mutation [23
]. We expected a substantial defect with the G375S mutation, because the G375S mutation was identified in an individual with hypercholesterolemia that is typical of individuals heterozygous for a loss-of-function LDLR allele [29
]. One possibility is that these mutations impair LDLR function in hepatocytes, but not fibroblasts. Fibroblasts are routinely used to characterize FH mutations [34
]; however, a few FH mutations have been identified that impair aspects of LDLR function that are not relevant in fibroblasts. For example, the G823D mutation disrupts basolateral targeting of the LDLR, a function that is required for LDLR function in hepatocytes, which have both apical and basolateral surfaces, but not in fibroblasts, which are not polarized [39
]. A second example is the H306Y mutation, which promotes binding of PCSK9 to the LDLR [40
]. PCSK9 binding drives degradation of the LDLR in hepatocytes, but not in fibroblasts [41
]. Mutations at G293 and G375 may cause FH by impacting unique aspects of LDLR function in the liver, but have little influence on LDLR function in cells such as fibroblasts in the periphery.
The reduced ability of cells expressing the W515A, W541A and H586Y variants to accumulate LDL correlated with reduced ability of these variants to release LDL in response to acidic pH ( and ), suggesting that these mutations impair LDL uptake by impairing endosomal release of LDL. Given the severity of the reduction in LDL uptake, it was remarkable how modest a reduction was observed in acid-sensitivity with the W515A, W541A and H586Y variants as compared to ΔBC variant (). This observation suggests either that these three mutations have additional effects not detected in our assays or that LDL uptake is highly sensitive to even small changes in the acid-sensitivity of LDL release. The effect of these three mutations was specific for LDL because no effect was observed with β-VLDL. This specificity is consistent with prior work showing that LDL uptake requires a β-propeller-driven release process, while β-VLDL uptake involves calcium extraction from the LDLR [17
Cells expressing the W144A and W193A variants failed to support normal uptake of both LDL and β-VLDL ( and ) and this failure correlated with a reduced ability of these variants to bind lipoprotein (). The principal defect in binding was reduced binding capacity (). This reduction in lipoprotein binding capacity contrasted with the normal levels of surface receptors expressed on these cells (), indicating that the W144A and W193A variants impair lipoprotein binding by reducing the fraction of surface receptors that are competent to bind lipoprotein. The division of the surface receptors into multiple populations with different ability to bind lipoprotein suggests that W144 and W193 play a role in the proper folding of LA4 and LA5.
The questions of how the LDLR binds to lipoprotein and how acidic pH releases lipoprotein are key to understanding how the LDLR supports lipoprotein uptake. These questions are intertwined because acidic pH drives release by accelerating lipoprotein dissociation [26
]. Binding affinity is the ratio of the association (on) rate and the dissociation (off) rate. A change in off rate implies an allosteric mechanism, which in turn implies that protonation of the LDLR or the apolipoproteins to which the LDLR binds results in conformational changes that weaken the interaction between the LDLR and lipoprotein. Acid-dependent release requires the β-propeller module [17
], indicating that protonation of the LDLR is the primary driver of acid-dependent release. Consistent with this conclusion, acidic pH causes the LDLR to undergo a conformational change from an extended state to a more compact state in which the β-propeller forms an intramolecular contact with LA 4/5 [19
]. Whether this contact is causal in release has not been established.
If the β-propeller contact is causal in release, then LA4/5 should have structural differences in the presence and absence of the β-propeller and acidic pH. To test this possibility, we aligned the structure of LA5 alone at neutral pH (pdb 1AJJ) [43
] with LA5 as part of the ectodomain at acidic pH (pdb 1N7D) using the alpha carbons of the two structures. The C-terminal half of LA5 aligned well in both structures; however, the N-terminal half of LA5 did not (). In addition to differences in the main chain, significant side chain differences include a twist in W193, a displacement of H190 toward the LA4/β-propeller interface and a flip in the orientation of the sidechain of E187. These differences suggest that the β-propeller drives structural changes in LA4/5.
A key test of whether the β-propeller contact with LA4/5 is causal in release is to determine where apolipoproteins bind on the LA4/5. If the β-propeller contact is causal then the apolipoproteins should bind to a surface that changes conformation in response to docking of the β-propeller and apolipoproteins must bind to a different surface than the β-propeller in order for the β-propeller to induce the necessary conformational changes in LA4/5. If the β-propeller contact with LA4/5 is not causal in release, but rather acts to prevent rebinding of lipoprotein after release [28
], then lipoproteins may bind to the same surface as the β-propeller.
Considerable work has examined the interaction of the LDLR and LDLR family members with ApoE. ApoE can adopt at least two conformations: a poorly lipidated, four-helix bundle state and a highly-lipidated, extended state in which the residues that form the protein core in the four-helix bundle state instead interact with lipids in lipoproteins [44
]. The LDLR binds with high affinity to the highly-lipidated state, but not the poorly-lipidated state of ApoE [35
]. By contrast, the LDLR family member, LRP1, binds with high affinity to the poorly-lipidated state, but not the highly-lipidated state [46
]. Saturation mutagenesis in LA5 of the LDLR suggests that the N-terminal half of LA5 is required for ApoE binding [47
]. Consistent with this observation, swapping residues 2809–2816 of LA18 of LRP1 with residues 186–193 of LA5 confers to an LA16-18 recombinant protein the ability to bind to the highly-lipidated form of ApoE [35
]. E187 may play a key role in the interaction because the E187K mutation reduces the affinity of the LDLR for β-VLDL 7-fold [26
]. Others have suggested that H190 and W193 are also involved in binding to ApoE-lipoproteins [35
]; however, while we see reduced β-VLDL binding capacity by LDLRs with mutations at these residues, we do not observe affinity differences (), suggesting that H190 and W193 do not contact ApoE directly. The β-propeller contacts only H190, S191 and W193 within the N-terminal half of LA5, suggesting that the residues involved on LA5 in the apoE interaction are not occluded by the interaction with the β-propeller.
LA5 is not sufficient for apoE binding by the LDLR, but inclusion of LA4 suffices for normal binding [4
]. Deletion of LA4 from the LDLR does not impair binding to β-VLDL [2
], suggesting that LA3 and LA4 share a determinant that can support the ability of LA5 to bind apoE. The ability of cells expressing W144A to bind β-VLDL with normal affinity suggests that this determinant does not require direct contacts with W144. Consistent with this conclusion, LA3 lacks a tryptophan at the position corresponding to W144 in LA4. Which aspects of LA3 and LA4 promote apoE binding is not clear; however, not all LA repeats can replace LA4 because exchange of LA2 with LA5 does not confer normal ability to bind apoE-lipoproteins [48
The binding surface on the LDLR for apoB100 is less clear. While the sites for ApoB100 and ApoE overlap because LDL and β-VLDL compete for binding to the LDLR [49
], the available data indicates that sites for ApoB100 and ApoE are not identical. Single domain deletions have shown that only deletion of LA5 cripples β-VLDL binding, while deletions of LA3, LA4, LA5, the linker between LA4 and LA5, LA6, LA7 or the EGF-A module all impair LDL binding [2
]. Several point mutations in LA4 and LA5 have likewise shown different effects on LDL and β-VLDL binding. For example, the D203K and E208K mutations weaken the binding affinity for β-VLDL, but not for LDL [26
], while the W144A and W193A mutations reduce LDL binding affinity, but not β-VLDL binding affinity (). The reduction in LDL binding affinity suggests that W144 and W193 may contact apoB100. These tryptophans are partially occluded by interaction with the β-propeller; however, a recent structure of a tethered ApoE peptide bound to LA17 of LRP provides an example of how an apolipoprotein can interact with tryptophans using a surface different than that occupied by the β-propeller [51
]. Thus, apoB100 may interact with W144 and W193, but the surface involved in the interaction may not be occluded by the β-propeller contact surface. It is notable that LDL release is far more sensitive to acidic pH than is β-VLDL release [17
]. While the difference in sensitivities is likely due in part to differences in binding affinities of the two lipoproteins, contact of the β-propeller with W144 and W193 may shift these residues away from an optimal conformation for the interaction with apoB100, thereby weakening the ability of the LDLR to bind LDL.
We propose a new model for how the β-propeller drives release. We propose that the N-terminal loop of LA5 provides a critical contact site for both ApoB100 on LDL and ApoE on β-VLDL and that this contact site is what the β-propeller regulates (). During release, acidic pH causes the LDLR to adopt a closed conformation, placing the β-propeller in position to make contact with LA4. The strength of this contact depends upon hydrophobic contacts involving P141, W144, W515 and W541 and ionic contacts involving D149, H562 and H586. The combination of LA4 and the β-propeller generates a surface that then recruits LA5. The interaction of LA5 with this surface strains the structure of the N-terminal loop of LA5. This strain shifts residues of the N-terminal loop that contact apolipoproteins, thereby weakening the ability of the LDLR to bind lipoprotein and increasing the rate of lipoprotein dissociation. E187 may play a key role in binding and release because the E187K mutation weakens the binding affinity of the LDLR for both LDL and β-VLDL and the side chain of E187 points in opposite directions in the structures of LA5 alone and LA5 in the ectodomain structure (). In this model, the W515A, W541A, H562Y and H586Y mutations impair acid-dependent release by weakening the initial contact with LA4. Consistent with this model is the observation that strengthening the contact by replacing the three histidines with charged residues makes lipoprotein release more sensitive to acidic pH [26
]. Future work will test this model by identifying residues necessary for normal lipoprotein binding affinity on LA4 and LA5.
Fig. 8 Model for β-propeller driven lipoprotein release - When acidic pH places the β-propeller in position to bind LA4/5, the β-propeller binds first to LA4 through hydrophobic contacts involving P141, W144, W515 and W541 and through (more ...)