Salt bridge relay triggers defective LDL receptor bindingby a mutant apolipoprotein
Charles Wilsoni t , Ted Mau l , Karl H Weisgraber 2, Mark R Wardell 2t ,Robert W Mahley2 and David A Agard'
1Howard Hughes Medical Institute, Department of Biochemistry and Biophysics, and Graduate Group in Biophysics, University ofCalifornia, San Francisco, CA 94143-0448, USA and 2Gladstone Foundation Laboratories for Cardiovascular Disease, Cardiovascular
Research Institute, Department of Pathology, University of California, San Francisco, CA 94140, USA
Background: Apolipoprotein-E (apo-E), a 34kDa bloodplasma protein, plays a key role in directing cholesteroltransport via its interaction with the low density lipoprotein(LDL) receptor. The amino-terminal domain of apo-Eforms an unusually elongated four-helix bundle arrangedsuch that key basic residues involved in LDL receptorbinding form a cluster at the end of one of the helices. Acommon apo-E variant, apo-E2, corresponding to thesingle-site substitution Argl58-*Cys, displays minimal LDLreceptor binding and is associated with significant changesin plasma cholesterol levels and increased risk of coronaryheart disease. Surprisingly, the site of mutation in thisvariant is physically well removed (>12A) from the clusterof LDL receptor binding residues.Results: We now report the refined crystal structure of the
amino-terminal domain of apo-E2, at a nominal resolutionof 3.00A. This structure reveals significant conformationalchanges relative to the wild-type protein that may accountfor reduced LDL receptor binding. Removal of the Arg158side chain directly disrupts a pair of salt bridges, causing acompensatory reorganization of salt bridge partners thatdramatically alters the charge surface presented by apo-E toits receptor.Conclusions: It is proposed that the observed reorganiza-tion of surface salt bridges is responsible for the decreasedreceptor binding by apo-E2. This reorganization, essentiallyfunctioning as a mutationally induced electrostatic switch toturn off receptor binding, represents a novel mechanism forthe propagation of conformational changes over significantdistances.
IntroductionThe high affinity binding of apolipoprotein-E (apo-E)by cell-surface receptors, including the low densitylipoprotein (LDL) receptor, allows lipoproteins asso-ciated with apo-E [such as very low density lipoproteins(VLDL), high density lipoproteins (HDL), and chylo-micron remnants] to be targeted for endocytosis andintracellular degradation [1]. Interference with suchreceptor-mediated processing can cause lipoproteins toaccumulate in the plasma and can ultimately lead to theformation of atherosclerotic plaques. The apo-E gene isone of the most polymorphic human genes character-ized to date and mutations that alter LDL receptorbinding are known to have significant effects on choles-terol levels and the risk of coronary artery disease [2].
Apo-E2 is a commonly occurring point mutant of apo-E, initially identified by its altered electrophoreticmobility [3]. Relative to apo-E3 (the wild-type protein),the most common apo-E2 isoform is characterized bythe substitution Argl58--4Cys [4]. This mutation(present in approximately 8 % of the population) lowersLDL receptor binding to <2 % of normal levels,although the protein appears to bind to lipoproteinswith the same affinity and specificity as the wild-typeprotein [5]. While plasma cholesterol and LDL concen-trations are generally lowered in people expressing theapo-E2 protein (presumably as a consequence of the
up-regulation of LDL receptors) [6], a subpopulation ofapo-E2 homozygotes are predisposed to type III hyper-lipidemia, a lipoprotein disorder associated withpremature atherosclerosis [7].
Apo-E appears to contain a 22 kDa amino-terminaldomain responsible for LDL receptor binding and a10 kDa carboxy-terminal domain involved in lipo-protein binding [8]. Crystallographic studies haveshown that the LDL receptor binding domain isarranged as an extremely elongated four-helix bundle(the helical segments extend up to 36 residues or 54Ain length) [9]. A cluster of key arginine and lysineresidues required for high affinity LDL receptor bindingdecorate the solvent-accessible face of one of thebundle helices. Several lines of evidence suggest thatreceptor binding is driven by electrostatic comple-mentarity between this group of positively chargedamino acids (spanning residues 136-150) and a setof negatively charged aspartates and glutamates in theshort disulfide-rich repeats of the LDL receptor (see [1]for review). Because of its clear role in modulatingreceptor binding, it had been assumed that Arg158 (thesite of the apo-E2 mutation) must be positioned nearthe other basic amino acids known to be directlyinvolved in binding. It was unclear whetherthe mutation functioned by interacting directly withsome complementary residue on the LDL receptor or
*Corresponding author. Present addresses: tDepartment of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA and*Department of Haematology, University of Cambridge, Cambridge, CB2 2QH, UK.
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only indirectly affected receptor binding. Surprisingly,the crystal structure of the LDL receptor bindingdomain did little to elucidate the mechanism of the E2mutation. The structure revealed that position 158 isseparated from the basic cluster by well over 10A [9].
To understand the structural basis for defective receptorbinding by apo-E2, we have crystallized and solved thestructure of the amino-terminal domain of the mutantprotein. Our results indicate that the apo-E2 amino acidsubstitution induces a concerted change in salt bridgeconformation that is propagated down the length of thehelical bundle. This conformational transition induces amajor change in the electrostatic potential surroundingthe cluster of basic residues, potentially explaining thepoor binding of apo-E2 to the LDL receptor.
Results and discussionComparison with the wild-type moleculeTo understand the structural basis for defective LDLreceptor binding by the apo-E2 protein, we haveanalyzed conformational differences between the wild-type and mutant proteins. Although the resolution ofthe apo-E2 data is limited, the analysis is based on theuse of the well-determined structure of the apo-E3wild-type fragment (Table 1) which should provideconfidence for the observed structural changes. Astereoview of the electron density in a 2Fo-F c map sur-rounding this region is shown in Fig. 1. As can be seen,the majority of the density is continuous and the modelis well positioned. The mutation results in rearrange-ments of both the backbone and side chain atoms in alarge zone around the substituted residue, Arg158 (Fig.2). For the sake of clarity, we shall consider structuralchanges in backbone atoms and side chain atoms separ-ately. Direct comparison of the two structures iscomplicated by two factors. First, the extended loopbetween helix 2 and helix 3 of the bundle (residues82-91) is quite poorly defined in the electron densityfor both the wild-type and E2 structures. As a conse-quence, its position is relatively unconstrained duringsimulated annealing refinement. For this reason, con-formational differences in this region are not consideredto be informative and have been omitted from Fig. 3.Second, shrinkage of the apo-E2 unit cell along the c-axis forces a slight change in crystal contacts whichleads to dispersed conformational changes in those sidechains that make protein-protein contacts. Fortunately,the zone surrounding the apo-E2 mutation site issolvent-exposed in the crystal and thus local conforma-tional changes are not influenced by contacts withsymmetry-related protein molecules.
The apo-E2 mutation has no major effect on most ofthe protein backbone (Figs 2 and 3). The root meansquare (rms) deviation between apo-E2 and apo-E3 forthe relatively unaffected residues 27-75 and 111-155is only 0.42A, within the error expected from analysisof the R-factor dependence upon resolution [10].
Certain localized regions of the backbone, however,including residues 92-96 and 156-163 are significantlydistorted (rms deviation 1.06A; Figs 3 and 4). Themost severely affected portion of the molecule is theamino-terminal half of helix 3 (residues 92-104). Thispart of the helix is displaced away from the remainderof the bundle (Fig. 2) as a slight kink near residues105-108 becomes exaggerated in the mutant protein.Surprisingly, the region thought to be in direct contactwith the LDL receptor (136-150) shows essentially nochange in backbone configuration, while most of thebackbone perturbations are on the other side of themolecule.
Salt bridge rearrangementThe most striking structural change resulting from theArg-4Cys mutation is a concerted conformational tran-sition in the side chains directly surrounding position158. The rms deviation between equivalent atomswithin 10A of the mutated residue is 1.6A, significantlyhigher than that for all atoms (<1.OA). The major effectof the mutation is to disrupt an interconnected networkof salt bridges that runs down one face of the four-helixbundle. This network is made up as a linear chain ofcharged residues, with each amino acid forming bifur-cating salt bridges to oppositely charged neighbors oneither side of it. The salt bridge path alternatelyincludes residues from the amino-terminal end of helix3 and the carboxy-terminal end of helix 4, effectivelybinding them together. In the wild-type protein, the setof salt bridges can be written as:
At the center of this native salt bridge network, theguanidinium group of Argl58 pairs with the side chaincarboxylates of both Glu96 and Asp154. Substitutionof the positively charged Arg158 by a neutral cysteinein the apo-E2 variant forces its two salt bridge partners
Table 1. Statistics for data collection and structure refinement.
Native apo-E2
Space group P212121 P212121Cell dimensions (a,b,c, A) 40.7, 54.0, 85.4 41.1, 53.9, 83.9Resolution (A) 2.25 3.0Diffraction data
Number of unique reflections >2c0 6899 2749<I>/<Cy> 43.5 32.1Completeness (10-3.5 A) 100 95.5Completeness (10-3.0 A) 100 67.9
X-PLOR refinement statisticsNo. of protein atoms (non-hydrogen) 1172 1167No. of water molecules 76 0Rcryst (8-3 A, for all reflections l/l > 2) 0.172 0.195Rms deviations from ideality
Bond lengths (A) 0.017 0.015Bond angles (°) 3.2 3.6
Defective receptor binding by mutant apolipoprotein Wilson et al. 715
Fig. 1. Stereoview of 2Fo-F ¢ electrondensity map in the region around theapo-E2 mutation (Argl 58-Cys). Thedensity is contoured at 1.25a abovethe mean. Cys158 and Arg150 areshown in red. (Figure produced usingthe MidasPlus program [17,181.)
to seek alternative stable conformations, ultimatelydisrupting most of the surrounding native salt bridges(Figs 4 and 5). Glu96 swings up and away from residue158, maintaining a single salt bridge to Arg92. Asp154moves down towards the previously unpaired Arg150.Of the five salt bridges indicated above for the wild-type protein, only one (Arg92E(-Glu96) remains intactin the apo-E2 structure. As a result, the helical interfaceis no longer spanned and stabilized by salt bridges (withthe exception of the pairings farther away from themutation site which are retained in the mutantstructure: Asp107<--Arg147--Asp10 0). In fact, it ismost likely that the observed alterations in localbackbone conformation are also a direct result of thisdramatic salt bridge rearrangement.
Fig. 2. Comparison of the protein backbones for the LDLreceptor binding domain of wild-type (apo-E3, white) andmutant (apo-E2, cyan) amino-terminal 22 kDa fragments. Theapo-E2 mutation (Argl58-Cys) is shown in red and theputative receptor binding residues (136-150) are highlightedin yellow. The structures were superimposed by a least-squares fit using the backbone atoms of residues 27-48,64-75 and 138-1 53 and have an rms deviation of only0.35 A. The loop between helix 2 and helix 3 (82-91) ispoorly determined in both structures and, for apo-E2, theconnection is shown as a discontinuous line to make thismore obvious. The distorted amino-terminal end of helix 3 isfound in the upper right corner. (Figure produced using theMidasPlus program 117,181.)
While lacking a structure of the protein in complexwith the LDL receptor, we can speculate on the mech-anisms by which receptor binding is disrupted for theE2 mutant. Site-directed mutagenesis has shown that anumber of basic residues in the region 136-150 arerequired for full receptor binding [11,12]. Replacementof one of these key residues, Arg150, by alanine reducesLDL receptor binding to one-quarter of normal values[13]. In the apo-E3 structure, Arg150 is solvent-exposed, presumably accessible for interaction withcomplementary acidic residues on the LDL receptor. Inthe E2 structure, however, this residue has moved outof the highly positive region of helix 4 and is pairedwith Asp154. Arg150 thus serves as the replacement saltbridge partner for Asp154 upon mutation of Arg158.The net result of this rearrangement is a significantchange in the electrostatic potential surrounding thereceptor-binding region of helix 4 (Fig. 6). Thus, whilethe charged side chain of Argl58 does not contributedirectly to the positive region surrounding the
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Fig. 3. Rms deviation between wild-type and apo-E2 structures forbackbone atoms (N, Ca, C) plottedagainst residue number. The region ofthe disordered loop between helices 2and 3 (residues 82-91) has beenomitted for clarity.
Fi. 4. Stereoview of the wild-type(white) and apo-E2 (cyan) structures inthe zone surrounding the mutatedresidue 158. (Figure produced usingthe MidasPlus program 117,18].)
receptor-binding helix, its presence is required tomaintain the important Arg150 in an unpaired,receptor-accessible conformation.
Previous chemical modification experiments withapo-E2 are consistent with the hypothesis that a salt-bridge-mediated conformational change accounts foraltered receptor binding [14]. The thiol-specific reagentcysteamine reacts with Cys158 to form a lysine analogwhich is capable of forming a single salt bridge. Thischemical modification raises LDL receptor binding toapproximately 10% that of the wild-type protein. Thefact that LDL receptor binding is not completelyrestored may be explained by the observation that themodified cysteine residue is incapable of forming bifur-cating salt bridges such as those formed by the
wild-type arginine residue. As such, the native saltbridge network can only be partially restored.
While the E2 mutation has had little or no effecton main chain conformation in the LDL receptorbinding region of apo-E, alterations of side chain con-formations have been dramatic. The spatially distantsubstitution of cysteine for arginine at 158 has causedan extended network of salt bridges to be disrupted. Asa consequence, the structural alteration that began at158 spreads in a 'domino effect' to encompass key sidechains in the receptor binding region. The net result isa significant alteration in the electrostatic field and side-chain conformation in the LDL receptor bindingsegment. These changes would seem sufficient toaccount for the reduced binding of the apo-E2 mutant.
2.0-
1.5-
<i:C0
> 1.0_
0.5
UA.
20 40 60 80 100residue number
A I I I I I
120 140 160 ISO
Defective receptor binding by mutant apolipoprotein Wilson et al. 717
Fig. 5. Comparison of the salt bridgenetworks in the apo-E2 (left) and wild-type apo-E3 (right) structures. Saltbridges dashed lines) are indicatedtogether with the distance betweencharged atoms (in A). (Figureproduced using the MidasPlusprogram 117,181.)
Fig. 6. Electrostatic potential map cal-culated for (a) the wild-type proteinand (b) the apo-E2 mutant protein.The DELPHI program (Biosym, SanDiego, CA) was used to calculate anapproximate solution to the linearizedPoisson-Boltzmann equation. Theprotein and solvent dielectrics wereset to 2 and 80, respectively. The ionicstrength was set to 150mM(mimicking blood plasma). Onlyformal protein charges were includedin the calculation. Positive (cyan) andnegative (red) contours in the potentialare evaluated at +2 and -2 kT/e-,respectively.
The observed conformational change suggests that thereceptor may directly interact with only a subset ofthose residues that appear to be important for binding,while the remainder may be required to maintain theintegrity of the native salt-bridge network.
Biological implicationsApolipoprotein-E (apo-E) is a major componentin most of the lipoprotein classes includingchylomicrons, very low density, low density andhigh density lipoproteins (VLDL, LDL andHDL). Functioning as a specific, high-affinity
ligand for cell-surface receptors, apo-E plays afundamental role in mammalian lipid andcholesterol metabolism.
There are three major apo-E isoforms (apo-E2,apo-E3, and apo-E4 [3]) which result from singleamino acid mutations within the structural gene[4]. The most common form, apo-E3, is consid-ered normal for LDL receptor binding andparticle specificity. The commonly occurringapo-E2 isoform (Argl58-4Cys) retains <2 % ofnormal binding to the LDL receptor but showsthe same specificity as the wild-type protein [5].In people homozygous for the apo-E2 allele
718 Structure 1994, Vol 2 No 8
(~1 % of the population), remnant lipoproteinsaccumulate in the plasma, a condition known asdysbetalipoproteinemia [15]. As a consequenceof environmental or genetic factors, a subpopu-lation of apo-E2 homozygotes will develop typeIII hyperlipidemia which is characterized byseverely elevated cholesterol and triglyceridelevels and premature atherosclerosis [7].
The structural data described here provide amolecular model for the decrease in LDL re-ceptor binding activity associated with theapo-E2 variant. The Argl58-4Cys mutationdisrupts a salt bridge network, thereby dramati-cally altering the surface charge presented byapo-E to its receptor. In the future, it may bepossible to design drugs directed against type IIIhyperlipoproteinemia by engineering compoundsthat bind specifically to the Cys158 site of apo-E2 and introduce positively charged groupscapable of fully restoring the native salt bridges.
Materials and methodsProtein production and crystallizationThe 22kDa thrombolytic fragment of apo-E2 was isolated asdescribed previously using blood plasma from a single humandonor [16]. Crystals of the E2 mutant were obtained via vapordiffusion by the hanging drop method. Conditions developed forthe native apo-E3 protein [9] [using 15% polyethylene glycol400 (BDH), 20mM sodium acetate, pH 5.3, 0.2% A-n-octylglu-copyranoside (Calbiochem), and 0.1% A3-mercaptoethanol] alsoyielded crystals of the E2 variant. Unfortunately, for severalreasons including limited protein availability, it was only possibleto produce two quite small crystals. Both wild-type and mutantproteins crystallized in the P21 21 21 space group with nearlyidentical a and b unit cell dimensions (see Table 1). The c-dimension of the E2 mutant (c = 83.9 A) is somewhat smallerthan that of the wild-type protein (c=85.4A).
Data collectionData were collected using a Rigaku automated four circle diffrac-tometer (AFC5R), equipped with an MSC cryo-cooling device.Wild-type and mutant apo-E crystals are extremely sensitive toradiation damage. To minimize radiation-induced decay, crystalswere quick-frozen in a stream of boiling liquid nitrogen and alldata were collected at, or below, -1500C. Crystals of the apo-E2variant were significantly smaller than those obtained for thewild-type protein (smaller than 0.1 mm in each dimension), andusable data could be only collected from a single crystal to anominal resolution of 3.0A (as opposed to 2.25A for the wild-type apo-E3). As is typical of diffractometry, only a singleasymmetric unit of data was collected. Lack of sufficient crystalsprecluded the extension of the data to higher resolution.
Molecular replacement and refinementThe 2.25A refined structure of the wild-type 22kDa fragmentprovided starting phases and coordinates for the structuralanalysis of the apo-E2 mutant. Following an initial rigid-bodyminimization to compensate for displacement along theshrunken c-axis, several cycles of simulated annealing moleculardynamics, B-factor and position refinement, and manual rebuild-ing of the structure were performed. Data collection andrefinement statistics are reported in Table 1. The final R-factorfor the apo-E2 variant structure is 19.5% (8-3.0A data). With
the exception of the large disordered loop between helices 2 and3 (residues 82-91) and glycine residues, the 4,-s backbone con-formational angles were all within or very close to allowedlimits. Thus, despite the rather low resolution of the structuralanalysis, the final structure appears to be reasonably well refined.Rigid-body and fiull-atom refinement were performed using X-PLOR, version 2.0 [17].
The coordinates have been deposited with the BrookhavenProtein Data Bank as entry 1LE2.
Acknowledgements: We thank J Newdoll and the UCSFComputer Graphics Laboratory (supported by NIH RR-1081)for help in preparing figures. This work was supported by theHoward Hughes Medical Institute (CW, TM, DAA), the Fannieand John Hertz Foundation (CW) and NIH Program ProjectGrant HL41633 (KHW,MRW,RWM).
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Received: 13 Apr 1994; revisions requested: 5 May 1994;revisions received: 8 Jun 1994. Accepted: 15 Jun 1994.
