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Journal of Lipid Research, Vol. 42, 894-901, June 2001
Copyright © 2001 by Lipid Research, Inc.

Research Articles

Effects of polymorphism on the microenvironment of the LDL receptor-binding region of human apoE

Sissel Lund-Katza, Suzanne Wehrlia, Mohamed Zaioub, Yvonne Newhousec, Karl H. Weisgraberc,d,e, and Michael C. Phillipsa
a Joseph Stokes, Jr. Research Institute, ARC, Suite 302, Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, PA 19104
b Department of Biochemistry, MCP Hahnemann University, Philadelphia, PA 19129
c Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, San Francisco, CA 94110
d Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94110
e Department of Pathology, University of California, San Francisco, San Francisco, CA 94110

Correspondence to: Sissel Lund-Katz, To whom correspondence should be addressed.


  ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL METHODS
 RESULTS
 DISCUSSION
 REFERENCES

To understand the molecular basis for the differences in receptor-binding activity of the three common human apolipoprotein E (apoE) isoforms, we characterized the microenvironments of their LDL receptor (LDLR)-binding regions (residues 136;–150). When present in dimyristoyl phosphatidylcholine (DMPC) complexes, the 22-kDa amino-terminal fragments (residues 1;–191) of apoE3 and apoE4 bound to the LDLR with ~100-fold greater affinity than the 22-kDa fragment of apoE2. The pKa values of lysines (K) at positions 143 and 146 in the LDLR-binding region in DMPC-associated 22-kDa apoE fragments were 9.4 and 9.9 in apoE2, 9.5 and 9.2 in apoE3, and 9.9 and 9.4 in apoE4, respectively. The increased pKa of K146 in apoE2 relative to apoE3 arises from a reduction in the positive electrostatic potential in its microenvironment. This effect occurs because C158 in apoE2, unlike R158 in apoE3, rearranges the intrahelical salt bridges along the polar face of the amphipathic {alpha}-helix spanning the LDLR-binding region, reducing the effect of the R150 positive charge on K146 and concomitantly decreasing LDLR-binding affinity.

The C112R mutation in apoE4 that differentiates it from apoE3 did not perturb the pKa of K146 significantly, but it increased the pKa of K143 in apoE4 by 0.4 pH unit. This change did not alter LDLR-binding affinity. Therefore, maintaining the appropriate positive charge at the C-terminal end of the receptor-binding region is particularly critical for effective interaction with acidic residues on the LDLR. — Lund-Katz, S., S. Wehrli, M. Zaiou, Y. Newhouse, K. H. Weisgraber, and M. C. Phillips. Effects of polymorphism on the microenvironment of the LDL receptor-binding region of human apoE. J. Lipid Res. 2001. 42: 894;–901.

Supplementary key words: apolipoprotein E2, apolipoprotein E3, apolipoprotein E4, lysine pKa, amphipathic {alpha}-helix, receptor-binding affinity


  INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL METHODS
 RESULTS
 DISCUSSION
 REFERENCES

Apolipoprotein E (apoE), a constituent of several plasma lipoproteins, mediates the catabolism of lipoprotein particles by binding to receptors of the LDL receptor (LDLR) family and to cell surface heparan sulfate proteoglycans (1). It also participates in the lipolytic conversion of VLDL and its intermediates and promotes VLDL triglyceride production (2). ApoE contains two structural domains that also define functional domains: the amino-terminal domain (residues 1;–191) contains the LDLR-binding region (vicinity of residues 136;–150), and the carboxyl-terminal domain (residues 218;–299) contains the major lipoprotein-binding elements (3).

Human apoE is a polymorphic protein with three common isoforms. ApoE3, the most common, has cysteine at position 112 (C112) and arginine at position 158 (R158), whereas apoE2 has cysteine at both positions and apoE4 has arginine (3). These differences have profound functional consequences. ApoE2 binds defectively to the LDLR (4) and is associated with type III hyperlipoproteinemia, a disorder characterized by the accumulation of chylomicron remnants in plasma and increased atherosclerosis (5). ApoE4 binds to the LDLR with the same affinity as apoE3 (4) but is associated with high plasma concentrations of cholesterol and LDL and increased risk of coronary heart disease (6) (7) (8); these effects are thought to reflect the preferential binding of apoE4 for VLDL. In contrast, apoE3 binds preferentially to HDL (3) (9). ApoE4 is also a major risk factor for Alzheimer's disease and poor outcome from stroke and head trauma (10).

The LDLR-binding region of apoE is highly enriched in basic amino acids (11). The positive electrostatic potential created by these residues gives rise to electrostatic attractions to acidic residues on the LDLR. Subtle changes within the receptor-binding region of apoE lead to defective receptor activity. For example, C158 in apoE2 causes defective binding to the LDLR although this residue lies outside the receptor-binding region. The primary cause for the defective binding is that the R158;–D154 salt bridge in apoE3 is replaced by an R150;–D154 bridge in apoE2, which pulls R150 out of the LDLR-binding region, effectively reducing the positive electrostatic potential (12). In addition, the C-terminal domain in apoE2 interacts with the N-terminal domain, which further decreases receptor-binding activity (13).

Our understanding of the molecular basis for the functional differences in the apoE isoforms is largely based on analyses of their 22-kDa amino-terminal domains in the lipid-free state by X-ray crystallography. In all three isoforms, this domain consists of a four-helix bundle of elongated amphipathic {alpha} helices (12) (14) (15) (16). However, apoE in the lipid-free state is not recognized by the receptor (17). Only when apoE interacts with a phospholipid does the four-helix bundle open and adopt a receptor-active conformation (17) (18) (19).

To investigate the enhanced binding affinity to the LDLR induced by interaction of apoE with phospholipid, we used NMR to study the microenvironments of the eight lysines (K) in the 22-kDa fragment of apoE3 in the lipid-free and lipid-associated states (20). When the protein was complexed with dimyristoyl phosphatidylcholine (DMPC), K143 and K146 (both in the LDLR-binding region) had unusually low pKa values (9.5 and 9.2, respectively), reflecting local increases in positive electrostatic potential caused by reorganization of the helices on lipid association. The increased electrostatic potential, coupled with enhanced exposure to the aqueous phase of the polar face of the amphipathic {alpha}-helix containing residues 136;–150, seems to explain why lipid association is required for high affinity binding of apoE to the LDLR.

In the present study, we characterized the microenvironments of lysines in LDLR-binding regions of apoE2 and apoE4. The goal of the study was to gain additional insight into the structural basis of differences between the apoE3 and apoE4 domains, which bind normally to receptors, and the apoE2 domain, which does not.


  EXPERIMENTAL METHODS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL METHODS
RESULTS
 DISCUSSION
 REFERENCES

Materials
DMPC was purchased from Avanti Polar Lipids (Pelham, AL), and stock solutions were stored in chloroform;–methanol 2:1 (v/v) under nitrogen at - 20°C; the purity was assayed by TLC on silica gel G plates (Analtech, Newark, DE) in chloroform;–methanol;–water 65:25:4 (v/v/v). Lipids were visualized by spraying developed TLC plates with a 50% sulfuric acid solution and charring them at 200°C for 15 min; 100-µg quantities gave a single spot. 2-Mercaptoethylamine (cysteamine) was purchased from Sigma (St. Louis, MO). D2O (Cambridge Isotope Laboratories, Andover, MA) was routinely deoxygenated and stored under nitrogen. [13C]formaldehyde (99% isotopic enrichment) as a 20% solution in water and [{epsilon}-13C]lysine dihydrochloride (6-13C, 99%) were from Cambridge Isotope Laboratories. [14C]Formaldehyde (55 Ci/mol) in distilled water was purchased from NEN Life Sciences (Boston, MA). Anionic contaminants were removed from the formaldehyde by passage through a small Dowex 1-chloride column (21). NaCNBH3 (Aldrich, Milwaukee, WI) was recrystallized from methylene chloride before use (21). K3Fe(CN)6 was from Aldrich; other salts and reagents were analytical grade.

Bacteriological media were obtained from Difco Laboratories (Detroit, MI). The prokaryotic expression vector pET32a and the competent Escherichia coli strain BL21 (DE3) were from Novagen (Madison, WI). Competent E. coli strain DH5{alpha} and nucleotides were from Life Technologies (Gaithersburg, MD). Pfu DNA polymerase was from Stratagene (La Jolla, CA). Restriction enzymes were purchased from New England BioLabs (Beverly, MA). Isopropyl ß-D-galactopyranoside, 2-mercaptoethanol, aprotinin, and ampicillin were from Sigma. Ultrapure guanidine hydrochloride was from ICN Pharmaceuticals (Costa Mesa, CA). Oligonucleotides were from Oligos Etc. (Wilsonville, OR), and DNA purification kits were from Qiagen (Chatsworth, CA).

Expression and purification of 22-kDa fragments and preparation of 22-kDa apoE·DMPC complexes
The 22-kDa fragments of apoE2, apoE3, and apoE4 were expressed in E. coli and purified as described (22). If further purification (>95%) was needed, the fragments were subjected to gel filtration with a Superdex 75 column.

To obtain 22-kDa apoE3 fragments enriched in [{epsilon}-13C]lysine, the fragment was expressed and purified as described for selenomethionine labeling of apoE (23). Briefly, starter culture (1 ml) was added to 1,000 ml of lysine-free LeMaster medium (24) supplemented with 250 µl of Kao-Mychayluk vitamin solution (25) and 417 mg of [{epsilon}-13C]lysine. Cultures were grown at 37°C to an optical density (at 600 nm) of 0.6 (8 h), induced with 0.5 mM isopropyl-ß-D-thiogalactopyranoside, and harvested after 2 h. Incorporation of [{epsilon}-13C]lysine was confirmed by electrospray mass spectrometry, performed by the Mass Spectrometry Facility at the University of California, San Francisco (San Francisco, CA). A molecular mass increase of 24 mass units indicated 50% incorporation of [{epsilon}-13C]lysine into the eight lysine positions in the apoE3 22-kDa fragment.

The 22-kDa fragments of all three isoforms were complexed with DMPC and isolated by gel-filtration chromatography as described (20). The 22-kDa apoE·DMPC complexes were labeled with 13C as described (26).

Cysteamine modification of 22-kDa apoE·DMPC complexes
The 22-kDa apoE2 fragment was modified with cysteamine as described (27). After reductive methylation, the 22-kDa apoE2·DMPC complexes were further modified by incubating 5 mg of 22-kDa apoE2 in 100 mM NH4HCO3 with 10 µl of cysteamine (100 mg/ml) per 150 µg of protein for 4 h at 37°C. The modified complexes were dialyzed against saline-EDTA followed by D2O-borate buffer before use in NMR experiments.

NMR measurements
1H-13C heteronuclear single quantum coherence (HSQC) two-dimensional NMR spectra of 13C-labeled 22-kDa apoE·DMPC complexes were obtained with a Bruker DMX400 wide-bore spectrometer equipped with an SGI 02 computer and a 5-mm inverse broad-band probe as previously described (20). The sample temperature for the two-dimensional NMR spectra was set at 310 K. The two-dimensional 1H-13C HSQC spectra were recorded with carbon decoupling during acquisition. The time-proportional phase-increment method (28) was used to obtain phase-sensitive spectra. One-dimensional 13C spectra were obtained with proton composite pulse decoupling. Chemical shifts and line widths for lipid-protein complexes were measured as described elsewhere (20) (26) (29). The pseudocontact shifts observed when K3Fe(CN)6 was added to the aqueous phase were used to explore the exposure of [13C]dimethyl lysines to the aqueous medium (29). The chemical shifts of [{epsilon}-13C]dimethyl lysine and [{epsilon}-13C]dimethyl-terminal amino residues of the complexes were determined as a function of pH as described previously (20). The pKa values of the [13C]dimethyl lysines were obtained by nonlinear regression fitting of the chemical shifts at different pH values to the Henderson-Hasselbalch equation with the Graph Pad Prism computer program (GraphPad Software, San Diego, CA). The sigmoidal equation is

where Y is the chemical shift, U is the lower limit of the shift, W is the upper limit, X is pH, and Xc is pKa.

Analytical procedures
Protein concentrations were determined by the Lowry procedure (30), and phospholipid content was determined by phosphorus analysis (31). 14C radioactivity was assessed by standard liquid scintillation procedures. Polyacrylamide gel electrophoresis (8;–25% gradient) in the presence or absence of SDS was performed with a Pharmacia (Piscataway, NJ) Phast electrophoresis system to monitor the purity of the proteins or to determine the size of the DMPC complexes. Circular dichroism spectra were obtained on a Jasco (Easton, MD) J600 spectropolarimeter equipped with a temperature-controlling device and interfaced with a computer. The {alpha}-helical content of each preparation was derived from the molar ellipticity at 222 nm by established procedures (26) (32).


  RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL METHODS
 RESULTS
DISCUSSION
 REFERENCES

Previously, we used two-dimensional NMR spectra of [13C]dimethyl lysines to study the microenvironments in 22-kDa apoE·DMPC complexes (20). The lysine resonances were poorly resolved when the protein is present in the lipid-free state in the four-helix bundle conformation. Fig 1A shows an NMR spectrum and the sequence-specific assignment of the eight [13C]dimethyl lysine resonances apparent when the 22-kDa apoE3 is associated with DMPC. This enhanced resolution reflects the changes in lysine microenvironments induced by the opening of the four-helix bundle on interaction with DMPC. These changes are accompanied by an ~104-fold increase in the affinity of the 22-kDa apoE3 molecule for the LDLR. Using a fibroblast LDLR competitive binding assay (17), the concentrations of 22-kDa apoE3 required to reduce binding of 125I-labeled LDL to the LDLR by 50% were 7,000 and 0.7 nM for the lipid-free protein and protein complexed to DMPC, respectively. Fig 1B shows a similar two-dimensional NMR spectrum for a 22-kDa apoE2·DMPC complex. The resonances from the eight [13C]dimethyl lysine residues are again resolved. The resonance with a 13C chemical shift ({delta}) of 41.0 ppm is from the N-terminal {alpha}-amino group; the lysine assignments were made by comparison with 13C chemical shifts and pKa values observed for 22-kDa apoE3·DMPC discs (20). As with apoE3 (20), the spectrum of lipid-free 22-kDa apoE2 was poorly resolved (data not shown), suggesting that apoE2 and apoE3 undergo similar conformational rearrangements on interaction with DMPC.



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  		Figure 1. Phase-sensitive 1H, 13C HSQC spectra of 22-kDa apoE·DMPC (1:3.2, w/w) discoidal complexes showing [13C]dimethyl lysine resonances. The spectra were obtained under conditions similar to those described previously (20). Sequence-specific assignments for (A) 22-kDa apoE3·DMPC (pH 10.0) (20) and (B) 22-kDa apoE2·DMPC (pH 10.0) are shown.

Several control experiments confirmed that dimethylation of the lysines in apoE does not perturb their titration characteristics. Dimethylation does not alter the lipid-binding properties of apoE (26). The {delta} of [{epsilon}-13C]lysine reflects the titration of the {epsilon}-amino group (33). In our hands, the {delta} of [{epsilon}-13C]lysine in solution increased from 39.1 to 40.3 ppm as the pH was raised from 8 to 12, and the pKa was 10.5. Fig 2A shows the [{epsilon}-13C]lysine resonance after incorporation into lipid-free 22-kDa apoE. A single broad resonance was apparent at {delta} = 40.1 ppm. When {delta} was measured as a function of pH, the pKa was 10.3. This result agrees with the values obtained from two-dimensional NMR spectra of [13C]dimethyl lysine 22-kDa apoE3 (20). There was no advantage in using two-dimensional spectra with the [{epsilon}-13C]lysine apoE; the protons attached to the 13C atom resonated as a triplet (because of coupling with protons on the adjacent methylene group), reducing resolution in the 1H dimension. As with [13C]dimethyl lysine 22-kDa apoE3 (20), the interaction with DMPC altered the pKa of the lysines. Three resonances at {delta} = 39.4, 39.8, and 40.1 ppm were resolved in the spectrum of an [{epsilon}-13C]lysine 22-kDa apoE·DMPC complex (Fig 2B). Titration curves ( Fig 3) show that most lysines retained a pKa of ~10.3, but subsets of lysines had pKa values of 9.5 and 11.0, consistent with the low pKa values of K143 and K146 (9.2;–9.5) and the higher pKa of K157 (11.1) previously obtained with [13C]dimethyl lysine 22-kDa apoE3 (20). Overall, our results confirm that valid information about the microenvironments of lysine in apoE can be obtained by using 13C-dimethylated protein (Fig 2 and Fig 3). This is consistent with the fact that dimethylation of lysines does not significantly perturb protein structure or lysine pKa values (21).



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  		Figure 2. Proton-decoupled 13C NMR spectra of [{epsilon}-13C]lysine 22-kDa apoE3 at 100.6 MHz. The degree of incorporation of [{epsilon}-13C]lysine by expression in E. coli was 50%. A: Lipid-free 22-kDa apoE3 (1.9 mg/ml) was dissolved in borate-buffered D2O. B: 22-kDa apoE3 (1.8 mg/ml) was dissolved as a 1:3.2 (w/w) complex with DMPC. The pKa values were derived by measuring the chemical shifts in spectra obtained at different pH values. The spectra were obtained at 37°C, and 2,600 transients were accumulated with CPD decoupling and the MLEV sequence. Chemical shifts are given in parts per million with aqueous 1,4-dioxane (66.55) as external reference.



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  		Figure 3. 13C NMR chemical shifts (ppm) as a function of pH for the [{epsilon}-13C]lysine resonances of [{epsilon}-13C]lysine 22-kDa apoE3. The chemical shifts were obtained from spectra of the type shown in Fig 2. The pH titration curves and pKa values for lysines were obtained by nonlinear regression fitting to the Henderson-Hasselbalch equation, as described in Experimental Methods. A: pH dependence of the [{epsilon}-13C]lysine resonance from lipid-free [{epsilon}-13C]lysine 22-kDa apoE3 dissolved in borate buffer (cf. Fig 2A). B: pH dependence of the [{epsilon}-13C]lysine resonances from an [{epsilon}-13C]lysine 22-kDa apoE3·DMPC complex. The three resonances are shown in Fig 2B at {delta} = 40.1 ppm (solid squares), 39.8 ppm (solid triangles), and 39.4 ppm (solid inverted triangles).

NMR spectra were obtained at various pH values to generate titration curves ( Fig 4A). The pKa values for K143, K146, and K157, which are located on helix 4 of 22-kDa apoE2 (15), were 9.4, 9.9, and 10.9, respectively. The pKa values for all the lysines in 22-kDa apoE2 and 22-kDa apoE4 are listed in Table 1, together with the values for 22-kDa apoE3 reported previously (20). Most lysine pKa values were not affected uniformly by the polymorphism. In contrast, the pKa values of K143 and K146 in apoE2, apoE3 and apoE4 differed significantly. Both lysines are in the LDLR-binding region, and their pKa values reflect the functionality of the domain with respect to recognition by the receptor. When complexed with DMPC, full-length apoE3 and apoE4 and their 22-kDa fragments have the same affinity for the LDLR (4) (34). In contrast, the affinity of full-length apoE2 is ~1% that of apoE3, and the affinity of the 22-kDa fragment is ~10% (4). This difference reflects the influence of the C-terminal domain on receptor-binding activity (13). Conversion of cysteines in the apoE2 22-kDa fragment to positively charged lysine analogs by treatment with cysteamine increases the binding affinity to near normal levels (4). Consistent with the requirement for an appropriate microenvironment in the LDLR-binding region of apoE, treatment of apoE2 with cysteamine affected the pKa values of K143 and K146 (Fig 4B and Table 1). This chemical modification did not alter the pKa of K157, which is also located in helix 4.



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  		Figure 4. 13C NMR chemical shifts (ppm) as a function of pH for the resonances from selected [13C]dimethyl lysine residues of 22-kDa apoE2·DMPC discoidal complexes. The chemical shifts were obtained from NMR spectra of the type shown in Fig 1. The pKa values were obtained as described in Fig 3. A: 22-kDa apoE2·DMPC. B: Cysteamine-modified 22-kDa apoE2·DMPC. K143 (solid squares), K146 (solid triangles), K157 (solid inverted triangles).


 
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  		Table 1. Lysine pKa values in apoE isoforms

Because binding of apoE to the LDLR is influenced by the degree of exposure of the polar face of helix 4 to the aqueous phase (20), ferricyanide shift reagent experiments were performed to assess the effects of apoE polymorphism on the relative exposure of K143 and K146 to the aqueous phase ( Table 2). In all cases, ferricyanide shifted the resonances of K143 and K146 downfield more than the other lysine resonances. Thus, the cysteine/arginine interchanges at positions 112 and 158 that differentiate the apoE isoforms did not significantly affect the exposure of any of the lysines to the aqueous phase.


 
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  		Table 2. Potassium ferricyanide-induced changes in NMR spectra of the 22-kDa apoE isoforms in DMPC discs


  DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL METHODS
 RESULTS
 DISCUSSION
REFERENCES

The greater affinity of lipid-bound apoE3 for the LDLR, compared with lipid-free apoE3, is a consequence of reorganization of the {alpha}-helical domains. This reorganization involves opening of the N-terminal four-helix bundle structure (18) (19), which leads to changes in interhelical interactions. Activation of the LDLR-binding region (residues 136;–150) involves two changes in the microenvironment. First, removal of interhelical salt bridges increases the positive electrostatic potential. Second, limited penetration of the amphipathic {alpha}-helix into the nonpolar lipid milieu increases the relative exposure to the aqueous phase (20). The same effects confer full LDLR-binding affinity on apoE4 complexed to lipid but not on apoE2 (4). In the current studies, we directly demonstrated that the microenvironments of apoE2 and apoE3 differ in the LDLR-binding region when complexed with lipid and that the positive charge of the receptor-binding region in apoE2 is significantly reduced.

Comparison of the pKa values of lysines in the four-helix bundle (Table 1) showed that the environments in helices 2 and 3 are similar for the three isoforms. The pKa values of K72 and K75 in helix 2 and of K95 in helix 3 were largely unaffected by the cysteine/arginine interchanges at positions 112 and 158. Somewhat surprisingly, the pKa value of K157 in helix 4 was the same in all three isoforms, despite the C158R mutation that distinguishes apoE2 from apoE3 and apoE4 (Table 1). The introduction of positive charges at C112 and C158 in apoE2 by cysteamine modification did not alter the pKa of any lysine outside the LDLR-binding region on helix 4.

The differences in the pKa values of K143 and K146 in lipid-complexed apoE isoforms are consistent with the notion that the mutations at positions 112 and 158 directly influence the LDLR-binding region. The changes in lysine in pKa values in this region reflect variations in the positive electrostatic potential in the microenvironment (20); this potential influences the affinity for the LDLR. Despite differences in the basicity of the LDLR-binding region in the apoE isoforms, the polar face of helix 4 seems to be similarly exposed to the aqueous phase in all three isoforms, as suggested by the ferricyanide shift reagent experiments (Table 2). Changes in {delta} for K143 and K146 were similar for all three isoforms and for cysteamine-treated 22-kDa apoE2. Thus, the differences in the LDLR-binding affinities of apoE2 and apoE3 do not reflect differences in penetration of the amphipathic {alpha}-helix containing residues 136;–150 into the nonpolar lipid environment.

Effect of the R158C mutation
The 22-kDa apoE3·DMPC complex binds with maximal affinity to the LDLR (34), and the pKa values of K143 and K146 are 9.5 and 9.2, respectively (Table 1). These values are unusually low because the increased positive electrostatic potential in the LDLR-binding region reduces the affinity of the {epsilon}-amino groups for protons (20). The local positive charge appears to be greater near the K146 side chain because K146 has a lower pKa than K143. The R158C mutation in apoE2 has little effect on the pKa of K143 but increases the pKa of K146 by 0.7 pH unit to 9.9. This increase reflects a loss of basicity in the immediate environment of the K146 side chain and is associated with reduced LDLR-binding affinity (1% of apoE3) (4). The change in basicity around K146 is consistent with X-ray crystallography of lipid-free 22-kDa fragments of apoE2 and apoE3 (12) (14) (15). In apoE3, there is a salt bridge between D154 and R158; because of the R158C substitution in apoE2, this salt bridge is replaced with a D154;–R150 salt bridge (12) (15). This new interaction reduces the net positive charge on the R150 side chain and shifts it out of the receptor-binding region. The positions of these residues along the polar face of helix 4 in apoE2 and apoE3 are shown in Fig 5.



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  		Figure 5. Organization of selected residues on the amphipathic {alpha}-helices 3 and 4 in lipid-free 22-kDa apoE isoforms. The crystal structure coordinates are from previous publications (14) (15) (16) and the pictures are drawn with RasMol (38). The {alpha}-helix backbones are shown as strands, and the amino acid side chains are drawn as balls and sticks. The juxtaposition of the D154 and R158 side chains in apoE3 and apoE4 is shown in the bottom two panels. The juxtaposition of the D154 and R150 side chains in apoE2 is shown in the top panel.

NMR measurements of the pKa values for K143 and K146 in 22-kDa apoE·DMPC complexes strongly suggest that the intrahelical ionic interactions in helix 4 are similar in the lipid-free and lipidated 22-kDa apoE molecules. The partial neutralization of the R150 positive charge in apoE2 by participation in a salt bridge with D154 (15) would be expected to reduce the net positive electrostatic potential experienced by K146, located one turn along the {alpha}-helix from R150 (Fig 5). This is reflected by an increase in the pKa from 9.2 in apoE3 to 9.9 in apoE2 (Table 1). Restoration of a positive charge at position 158 by treatment of apoE2 with cysteamine decreased the pKa of K146 to 9.5. This reflects partial restoration of the basicity in this microenvironment because D154 interacts not only with R150 but also with the positive charge at position 158 created by the cysteamine modification.

In lipid-associated 22-kDa fragments of apoE2 and apoE3, K143 has the same pKa value (~9.5) (Table 1). This indicates that the C158R mutation does not affect basicity in the microenvironment of the K143 side chain (Fig 5). Although the pKa of K143 in apoE4 is 9.9, this isoform binds as well as apoE3 to the LDLR, suggesting that the magnitude of the positive electrostatic potential around K143 is less important for receptor binding than that around K146. This suggestion is consistent with the idea that ligand recognition by repeat modules of the LDLR is mediated by electrostatic complementarity of conserved patches of negative electrostatic potential on the receptor (35) and the positive charge of apoE. The net positive charges of the lysine side chains at positions 143 and 146 are both important for LDLR interaction because replacement of either lysine with a neutral amino acid reduces binding to about 25;–30% of the apoE3 value (36).

Role of the C112R mutation
Although the presence of a positive charge at position 112 in apoE4 does not alter LDLR-binding affinity (4), it increased the pKa of K143 by ~0.4 pH unit (Table 1). It is not obvious from examination of the four-helix bundle structure formed by 22-kDa apoE3 and apoE4 (Fig 5) why this would occur. In the lipid-free four-helix bundle structure, R112 is ~20 Å from K143 on the opposite side of helix 3, so the residues are too far apart to interact. However, the pKa data show that there is an interaction when the four-helix bundle is open and the protein is associated with DMPC in a discoidal complex. These two residues are located at similar positions along helices 3 and 4 but protrude away from each another in the bundle. If interaction with lipid induced rotation in these helices, bringing R112 and K143 into closer proximity, then the observed change in pKa values of K143 in apoE4 would be explained. It is plausible that helix 3 in apoE·DMPC complexes is oriented differently in apoE3 and apoE4. In apoE3, C112 is located on the nonpolar face of the amphipathic {alpha}-helix 3 (37); in apoE4, the introduction of R112 disrupts the nonpolar face, reducing its angle from 180° to 120° and likely inducing a rotation of the {alpha}-helix with respect to the plane of the lipid-water interface. These suggested effects of the R112;–K143 interaction are based on the assumption that intramolecular effects predominate. However, intermolecular interactions between helices 3 and 4 could give rise to similar effects. The interaction apparently involves more than simple juxtaposition of the positively charged R112 and K143 side chains. If that were the case, the pKa of K143 would be expected to decrease rather than increase.

Summary and conclusions
For optimal high-affinity binding of apoE to the LDLR, the microenvironment in the LDLR-binding region must have the appropriate distribution of positive electrostatic potential and degree of exposure to the aqueous phase (20). Interaction with lipid alters helix- helix interactions in the four-helix bundle structure, causing the LDLR-binding region spanning residues 136;–150 on helix 4 to assume a high-affinity binding conformation. Comparison of the pKa values of K143 and K146 in 22-kDa apoE2·DMPC and apoE3·DMPC complexes reveals that the R158C mutation in apoE2 alters the microenvironment in the LDLR-binding region; this is associated with the decrease in receptor-binding affinity. The C112R substitution in apoE4 also affects the microenvironment in the receptor-binding region because the pKa of K143 is increased. Because this change has no effect on receptor binding, it seems that the C-terminal portion of this region plays a more critical role than the N-terminal portion in the interaction with the LDLR. These insights into the molecular features required for high affinity binding provide a basis for designing ways to enhance the binding of apoE to the LDLR.


  ACKNOWLEDGMENTS

We thank Faye Baldwin and Padmaja Dhanasekaran for expert technical assistance, and Gary Howard and Stephen Ordway for excellent editorial assistance. Mass spectra were provided by the UCSF Mass Spectrometry Facility (A. L. Burlingame, Director) supported by the Biomedical Research Technology Program of the National Center for Research Resources, NIH NCRR BRTP RR01614. This work was supported by NIH grants HL56083 and HL41633.

Manuscript received December 12, 2000; and in revised form February 22, 2001

Abbreviations: apo, apolipoprotein; {delta}, chemical shift; DMPC, dimyristoyl phosphatidylcholine; HSQC-NMR, heteronuclear single quantum coherence-nuclear magnetic resonance; LDLR, LDL receptor


  REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL METHODS
 RESULTS
 DISCUSSION
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