Structural independence of ligand-binding modules five and six of the LDL receptor

The low-density lipoprotein receptor (LDLR) is the primary mechanism for the uptake of plasma cholesterol into cells and serves as a prototype for a growing family of cell surface receptors. These receptors all utilize tandemly repeated LDL-A modules to bind their ligands. Each LDL-A module is about 40 residues long, has six conserved cysteine residues, and contains a conserved acidic region near the C-terminus which serves as a calcium-binding site. The structure of the interface presented for ligand binding by these modules, and the basis for their specificity and affinity in ligand binding, is not yet known. We have purified recombinant molecules corresponding to LDL-A modules five (LR5), six (LR6), and the module five-six pair (LR5-6) of the LDL receptor. Calcium is required to establish native disulfide bonds and to maintain the structural integrity of LR5, LR6, and the LR5-6 module pair. Folding studies of the I189D and D206Y mutations within LR5 indicate that each change leads to misfolding of the module, explaining the previous observation that each of these changes mimics the functional effect of deletion of the entire module [Russell, D. W., Brown, M. S., and Goldstein, J. L. (1989) J. Biol. Chem. 264, 21682-21688]. By fluorescence, the affinity of LR5 for calcium, which is crucial for folding and function of these modules, remains approximately 40 nM whether LR6 is attached. Comparison of proton and multidimensional heteronuclear NMR spectra of individual modules to those of the module pair indicates that most of the significant spectroscopic changes lie within the linker region between modules and that little structural interaction occurs between the cores of modules five and six in the 5-6 pair. These findings strongly support a model in which each module is essentially structurally independent of the other.     

Evidence that familial hypercholesterolemia mutations of the LDL receptor cause limited local misfolding in an LDL-A module pair

Mutations at conserved sites within the ligand-binding LDL-A modules of the LDL receptor cause the genetic disease familial hypercholesterolemia (FH), and several of these FH mutations in modules five and six prevent the isolated single modules from folding properly to a nativelike three-dimensional structure. Because LDL-A modules occur as a series of contiguous repeats in the LDLR and related proteins, we investigated the impact of two FH mutations in LDL-A module five (D203G and D206E) and two mutations in module six (E219K and D245E) in the context of the covalently connected module five-six pair. HPLC chromatography of the products formed under conditions that efficiently refold the native module five-six pair demonstrate that, for each mutation, a folding defect persists in the module pair. NMR spectroscopy and calcium affinity measurements of the ensemble of misfolded products demonstrate that the unaltered module of each pair can fold to its native structure regardless of the range of misfolded conformations adopted by its mutated neighbor. These findings lend additional support to a model in which individual LDL-A modules of the LDL receptor act as independent structural elements.     

Characterization of the LDL-A module mutants of Tva,the subgroup A Rous sarcoma virus receptor,and the implications in protein folding

Tva is the cellular receptor for subgroup A Rous sarcoma virus (RSV-A), and the viral receptor function is solely determined by a 40-residue motif called the LDL-A module of Tva. In this report, an integral approach of molecular, biochemical, and biophysical techniques was used to examine the role of a well-conserved tryptophan of the LDL-A module of Tva in protein folding and ligand binding. We show that substitution of tryptophan by glycine adversely affected the correct folding of the LDL-A module of Tva, with only a portion giving a calcium-binding conformation. Furthermore, we show that the misfolded LDL-A conformations of Tva could not efficiently bind to its ligand. These results indicate that this conserved tryptophan in the LDL-A module of Tva plays an important role in correct protein folding and ligand recognition. Furthermore, these results suggest that the familial hypercholesterolemia (FH) French Canadian-4 mutation is likely caused by protein misfolding of low-density lipoprotein receptor, thus explaining the defect for this class of FH.     

Implications for familial hypercholesterolemia from the structure of the LDL receptor YWTD-EGF domain pair

The low-density lipoprotein receptor (LDLR) is the primary mechanism for uptake of cholesterol-carrying particles into cells. The region of the LDLR implicated in receptor recycling and lipoprotein release at low pH contains a pair of calcium-binding EGF-like modules, followed by a series of six YWTD repeats and a third EGF-like module. The crystal structure at 1.5 A resolution of a receptor fragment spanning the YWTD repeats and its two flanking EGF modules reveals that the YWTD repeats form a six-bladed beta-propeller that packs tightly against the C-terminal EGF module, whereas the EGF module that precedes the propeller is disordered in the crystal. Numerous point mutations of the LDLR that result in the genetic disease familial hypercholesterolemia (FH) alter side chains that form conserved packing and hydrogen bonding interactions in the interior and between propeller blades. A second subset of FH mutations are located at the interface between the propeller and the C-terminal EGF module, suggesting a structural requirement for maintaining the integrity of the interdomain interface.     

The structure, dynamics, and binding of the LA45 module pair of the low-density lipoprotein receptor suggest an important role for LA4 in ligand release

The low-density lipoprotein receptor (LDLR), the primary receptor for cholesterol uptake, binds ligands through its seven LDL-A modules (LAs). We present nuclear magnetic resonance (NMR) and ligand binding measurements on the fourth and fifth modules of the LDLR (LA45), the modules critical for ApoE binding, at physiological pH. Unlike LA5 and all other modules in LDLR, LA4 has a very weak calcium affinity, which probably plays a critical role in endosomal ligand release. The NMR solution structure of each module in the LA45 pair only showed minor differences compared to the analogous domains in previously determined crystal structures. The 12-residue linker connecting the modules, though slightly structured through an interaction with LA4, is highly flexible. Although no intermodule nuclear Overhauser effects were detected, chemical shift perturbations and backbone dynamics suggest cross talk between the two modules. The ligand affinity of both modules is enhanced when the two are linked. LA4 is more flexible than LA5 and remains so even in the module pair, which likely is related to its weaker calcium binding affinity.     

Folding determinants of LDL receptor type A modules.

To investigate how three disulfide bonds and coordination of a calcium ion cooperate to specify the structure of an LDL-A module, we studied the interdependence of disulfide bond formation and calcium coordination in the folding of ligand-binding module 5 of the LDL receptor (LR5). In variants of LR5 containing only a single pair of cysteines normally disulfide-bonded in the native polypeptide, the addition of calcium does not alter the effective concentration of one cysteine for the other. LR5 only exhibits a calcium-dependent preference for formation of native disulfide bonds and detectable calcium-induced changes in structure when the two C-terminal disulfide bonds are present. Furthermore, when the conformation of this two-disulfide variant of LR5 is probed by NMR in the presence of calcium, only the C-terminal lobe of the module, which contains the calcium coordination site, acquires a near-native conformation; the N-terminal lobe appears to be disordered. These findings contrast with studies of other model proteins, like BPTI, in which formation of a single disulfide bond is sufficient to drive the entire domain to acquire a stable, nativelike fold.     

Backbone dynamics of a module pair from the ligand-binding domain of the LDL receptor

The ligand-binding domain of the LDL receptor consists of seven contiguous LDL-A modules. The fifth of these ligand-binding modules is absolutely required for recognition of both LDL and beta-VLDL particles. A four-residue linker of variable sequence connects each pair of modules, except for modules four and five, which are connected by a 12-residue linker. To provide a more detailed understanding of the structural relationship in a typical pair of functionally important LDL-A repeats of the LDLR, we investigated the backbone dynamics of repeats five (LR5) and six (LR6) alone and in the context of the covalently connected LR5-6 pair. Our results reveal substantial flexibility in the four-residue linker connecting the two repeats in the LR5-6 pair. The intrinsic dynamic behavior of each repeat is essentially unchanged when the repeats are covalently connected. These observations indicate that the relative orientation of repeats in LR5-6 is not fixed. Modeled in an extended conformation, the linker can separate LR5 and LR6 by up to 15 A, a distance that would allow substantial freedom of motion of each repeat with respect to the other in the pair.     

Solution structure of the viral receptor domain of Tva and its implications in viral entry

Tva is the cellular receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A). The viral receptor function of Tva is determined by a 40-residue, cysteine-rich motif called the LDL-A module. Here we report the solution structure of the LDL-A module of Tva, determined by nuclear magnetic resonance (NMR) spectroscopy. Although the carboxyl terminus of the Tva LDL-A module has a structure similar to those of other reported LDL-A modules, the amino terminus adopts a different conformation. The LDL-A module of Tva does not contain the signature antiparallel beta-sheet observed in other LDL-A modules, and it is more flexible than other reported LDL-A modules. The LDL-A structure of Tva provides mechanistic insights into how a simple viral receptor functions in retrovirus entry. The side chains of H38 and W48 of Tva, which have been identified as viral contact residues by mutational analysis, are solvent exposed, suggesting that they are directly involved in EnvA binding. However, the side chain of L34, another potential viral contact residue identified previously, is buried inside of the module and forms the hydrophobic core with other residues. Thus L34 likely stabilizes the Tva structure but is not a viral interaction determinant. In addition, we propose that the flexible amino-terminal region of Tva plays an important role in determining specificity in the Tva-EnvA interaction.     

A two-module region of the low-density lipoprotein receptor sufficient for formation of complexes with apolipoprotein E ligands

The low-density lipoprotein (LDL) receptor transports two different classes of cholesterol-carrying lipoprotein particles into cells: LDL particles, which contain a single copy of apolipoprotein B-100 (apoB-100), and beta-migrating very low-density lipoprotein (beta-VLDL) particles, which contain multiple copies of apolipoprotein E (apoE). The ligand-binding domain of the receptor lies at its amino-terminal end within seven adjacent LDL-A repeats (LA1-LA7). Although prior work clearly establishes that LA5 is required for high-affinity binding of particles containing apolipoprotein E (apoE), the number of ligand-binding repeats sufficient to bind apoE ligands has not yet been determined. Similarly, uncertainty exists as to whether a single lipid-activated apoE receptor-binding site within a particle is capable of binding to the LDLR with high affinity or whether more than one is required. Here, we establish that the LA4-5 two-repeat pair is sufficient to bind apoE-containing ligands, on the basis of binding studies performed with a series of LDLR-derived "minireceptors" containing up to four repeats. Using single chain multimers of the apoE receptor-binding domain (N-apoE), we also show that more than one receptor-binding site in its lipid-activated conformation is required to bind to the LDLR with high affinity. Thus, in addition to inducing a conformational change in the structure of N-apoE, lipid association enhances the affinity of apoE for the LDLR in part by creating a multivalent ligand.     

Structure of an LDLR-RAP complex reveals a general mode for ligand recognition by lipoprotein receptors

Proteins of the low-density lipoprotein receptor (LDLR) family are remarkable in their ability to bind an extremely diverse range of protein and lipoprotein ligands, yet the basis for ligand recognition is poorly understood. Here, we report the 1.26 A X-ray structure of a complex between a two-module region of the ligand binding domain of the LDLR and the third domain of RAP, an escort protein for LDLR family members. The RAP domain forms a three-helix bundle with two docking sites, one for each LDLR module. The mode of recognition at each site is virtually identical: three conserved, calcium-coordinating acidic residues from each LDLR module encircle a lysine side chain protruding from the second helix of RAP. This metal-dependent mode of electrostatic recognition, together with avidity effects resulting from the use of multiple sites, represents a general binding strategy likely to apply in the binding of other basic ligands to LDLR family proteins.     

Thermodynamics of protein‐cation interaction: Ca+2 and Mg+2 binding to the fifth binding module of the LDL receptor

The ligand binding domain of the LDL receptor (LDLR) contains seven structurally homologous repeats. The fifth repeat (LR5) is considered to be the main module responsible for the binding of lipoproteins LDL and β‐VLDL. LR5, like the other homologous repeats, is around 40‐residue long and contains three disulfide bonds and a conserved cluster of negatively charged residues surrounding a hexacoordinated calcium ion. The calcium coordinating cage is formed by the backbone oxygens of W193 and D198, and side‐chain atoms of D196, D200, D206, and E207. The functionality of LDLR is closely associated with the presence of calcium. Magnesium ions are to some extent similar to calcium ions. However, they appear to be involved in different physiological events and their concentrations in extracellular and intracellular compartments are regulated by different mechanisms. Whether magnesium ions can play a role in the complex cycle of LDLR internalization and recycling is not known. We report here a detailed study of the interaction between LR5 and these two cations combining ITC, emission fluorescence, high resolution NMR, and MD simulations, at extracellular and endosomal pHs. Our results indicate that the conformational stability and internal dynamics of LR5 are strongly modulated by the specific bound cation. It appears that the difference in binding affinity for these cations is somewhat compensated by their different concentrations in late LDL‐associated endosomes. While the mildly acidic and calcium‐depleted environment in late endosomes has been proposed to contribute significantly to LDL release, the presence of magnesium might assist in efficient LDLR recycling. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Characterization of Determinants for Envelope Binding and Infection in Tva, the Subgroup A Avian Sarcoma and Leukosis Virus Receptor

Tva is the cellular receptor for subgroup A avian leukosis and sarcoma virus (ALSV-A). The viral interaction domain of Tva is determined by a 40-residue, cysteine-rich module closely related to the ligand binding domain of the human low-density lipoprotein receptor (LDLR). In this report, we examined the role of the LDLR-like module of Tva in envelope binding and viral infection by mutational analysis. We found that the entire LDLR module in Tva is essential for efficient binding to the viral envelope protein. However, the 17 N-terminal residues of this module can be deleted without affecting receptor function, suggesting that the major determinants for viral entry are located at the C terminus of the module. The effect on viral infection of many amino acid substitutions and deletions in the LDLR module is context dependent, suggesting that the residues important for viral entry are dispersed throughout the LDLR module. In addition, we found that all 27 mutations at residues D46, E47, and W48 greatly reduced envelope binding. These results are discussed in relation to a recently elucidated structure for an LDLR module.     

Gene Structure and Molecular Phylogeny of the Linker Chains from the Giant Annelid Hexagonal Bilayer Hemoglobins

Giant extracellular hexagonal bilayer hemoglobin (HBL-Hb), found only in annelids, is an ∼3500-kDa heteropolymeric structure involved in oxygen transport. The HBL-Hbs are comprised of globin and linker chains, the latter being required for the assembly of the quaternary structure. The linker chains, varying in size from 225 to 283 amino acids, have a conserved cysteine-rich domain within their N-terminal moiety that is homologous to the cysteine-rich modules constituting the ligand binding domain of the low-density lipoprotein receptor (LDLR) protein family found in many metazoans. We have investigated the gene structure of linkers from Arenicola marina, Alvinella pompejana, Nereis diversicolor, Lumbricus terrestris, and Riftia pachyptila. We found, contrary to the results obtained earlier with linker genes from N. diversicolor and L. terrestris, that in all of the foregoing cases, the linker LDL-A module is flanked by two phase 1 introns, as in the human LDLR gene, with two more introns in the 3′ side whose positions varied with the species. In addition, we obtained 13 linker cDNAs that have been determined experimentally or found in the EST database LumbriBASE. A molecular phylogenetic analysis of the linker primary sequences demonstrated that they cluster into two distinct families of linker proteins. We propose that the common gene ancestor to annelid linker genes exhibited a four-intron and five-exon structure and gave rise to the two families subsequent to a duplication event.     

Domain Swapping Reveals That Low Density Lipoprotein (LDL) Type A Repeat Order Affects Ligand Binding to the LDL Receptor

The low density lipoprotein receptor (LDLR) plays a key role in plasma cholesterol homeostasis by binding and internalizing lipoprotein ligands. Studies have revealed that one or more of the seven LDL type A repeats (LA1–LA7) in the receptor are responsible for apolipoprotein binding. In the present study, protein engineering was performed to swap or replace key LA repeats in a recombinant soluble LDLR (sLDLR). Although wild type sLDLR showed strong ligand binding activity, an sLDLR variant in which LA repeat 5 was replaced by a second copy of LA repeat 2 showed low binding activity. Likewise, a variant wherein LA repeats 2 and 5 were swapped displayed low binding activity. At the same time, substitution of LA repeat 2 with a second a copy of repeat 5 resulted in a receptor with ligand binding activity similar to wild type LDLR. When binding assays were conducted with human low density lipoprotein as ligand, LA repeat order was a less important determinant of binding activity. Taken together, the data indicate that the sequential order of LA repeats plays a key role in ligand binding properties of LDLR.The low density lipoprotein receptor (LDLR)³ plays an important role in plasma cholesterol homeostasis (1). A fundamental function of LDLR is transport of cholesterol-rich lipoproteins into cells via receptor-mediated endocytosis (2). Human LDLR is 839 amino acids in length and is comprised of five distinct modules that arose from gene duplication. At the N terminus of LDLR, there exists a series of seven imperfect, disulfide bond-rich, LDL type A (LA) repeats, each ∼40 amino acids in length. Calcium binding induces LA repeats to fold into a ligand binding-competent conformation (3). Adjacent to the ligand binding module is a ∼400-residue module that bears homology to epidermal growth factor (EGF) precursor. This module consists of two disulfide bond-rich EGF-like repeats (A and B) and a YWTD β-propeller motif followed by a third EGF-like repeat C (4). The third module of LDLR is distinguished by an abundance of O-linked sugars, whereas the fourth module is comprised of a single membrane-spanning sequence. Finally, a short intracellular C-terminal cytoplasmic domain, required for receptor internalization, is present (5).LDLR binds two apolipoprotein ligands, apolipoprotein (apo) E and apoB (6). Although these proteins do not share structural similarity, sequence elements rich in positively charged amino acid side chains are present in each that are required for binding. Deletion studies have demonstrated that specific LA repeats are required for apolipoprotein binding to LDLR (7, 8).Recently, another LDLR ligand, termed proprotein convertase subtilisin-like kexin type 9 (PCSK9), has emerged (9): PCSK9 serves to regulate cholesterol homeostasis by modulating LDLR processing. Unlike lipoprotein ligands, PCSK9 binds EGF repeat A and, apparently, is not released from the receptor at endosomal pH.LA1–LA7 are ∼40–50% identical in primary sequence. Each repeat contains a Ca²⁺ binding site and three disulfide bonds. The importance of these structural features for ligand binding is widely recognized. For example, it is known that LA5 is essential for optimal binding of apoB- and apoE-containing ligands (7, 8). On the other hand, deletion of LA2 had no effect on binding of apoE-containing lipoproteins. X-ray crystal structure information is available for isolated LA5 at pH 5.0 (10) as well as the entire ectodomain of LDLR (residues 1–699) at endosomal pH (11). Based on this structural information and complementary data on apolipoprotein ligands, it has been postulated that electrostatic interactions modulate LDLR conformation and ligand binding. Given this, it remains unclear whether the precise order of LA repeats within the ligand binding module may impact ligand binding.In the present study, protein engineering of a soluble LDLR (sLDLR) was performed to swap or replace specific LA repeats within the ligand binding module of sLDLR. Ligand binding to wild type (WT) and engineered sLDLR was then determined. The results show that LA repeat 5 must not only be present, it must exist in the correct context with respect to other LA repeats within the ligand binding module.     

The role of a conserved acidic residue in calcium-dependent protein folding for a low density lipoprotein (LDL)-A module: implications in structure and function for the LDL receptor superfamily

One common feature of the more than 1,000 complement-type repeats (or low density lipoprotein (LDL)-A modules) found in LDL receptor and the other members of the LDL receptor superfamily is a cluster of five highly conserved acidic residues in the C-terminal region, DXXXDXXDXXDE. However, the role of the third conserved aspartate of these LDL-A modules in protein folding and ligand recognition has not been elucidated. In this report, using a model LDL-A module and several experimental approaches, we demonstrate that this acidic residue, like the other four conserved acidic residues, is involved in calcium-dependent protein folding. These results suggest an alternative calcium coordination conformation for the LDL-A modules. The proposed model provides a plausible explanation for the conservation of this acidic residue among the LDL-A modules. Furthermore, the model can explain why mutations of this residue in human LDL receptor cause familial hypercholesterolemia.     

Mechanism of low density lipoprotein (LDL) release in the endosome: implications of the stability and Ca2+ affinity of the fifth binding module of the LDL receptor

Uptake of low density lipoproteins (LDL) by their receptor, LDLR, is the primary mechanism by which cells incorporate cholesterol from plasma. Mutations in LDLR lead to familial hypercholesterolemia, a common disease affecting 1 in 500 of the human population. LDLR is a modular protein that uses several small repeats to bind LDL. The repeats contain around 40 residues, including three disulfide bonds and a calcium ion. Repeat 5 (LR5) is critical for LDL and beta-migrating very low density lipoprotein binding. Based on the crystal structure of LDLR at endosomal pH (but close to extracellular calcium concentration), LR5 has been proposed to bind to the epidermal growth factor (EGF) precursor domain of LDLR in the endosome, thus releasing the LDL particles previously bound in extracellular conditions. We report here the conformational stability of LR5 as a function of temperature and calcium concentration under both extracellular and endosomal pH conditions. The repeat was very stable when it bore a bound calcium ion but was severely destabilized in the absence of calcium and even further destabilized at acidic versus neutral pH. The temperature and calcium concentration dependence of LR5 stability clearly indicate that under endosomal conditions the unfolded conformation of the repeat is largely dominant. We thus propose a new mechanism for LDL release in the endosome in which calcium depletion and decreased stability at acidic pH drives LR5 unfolding, which triggers LDL release from the receptor. Subsequent binding of LR5 to the EGF precursor domain, if it takes place at low calcium concentrations, would contribute to a further shifting of the equilibrium toward dissociation.     

Role of calcium in protein folding and function of Tva, the receptor of subgroup A avian sarcoma and leukosis virus

Tva is the cellular receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A). The viral receptor function of Tva is determined by a 40-residue cysteine-rich motif called the LDL-A module. In this study, we expressed and purified the wild-type (wt) Tva LDL-A module as well as several mutants and examined their in vitro folding properties. We found that, as for other LDL-A modules, correct folding and structure of the Tva LDL-A module is Ca2+ dependent. When calcium was present during in vitro protein folding, the wt module was eluted as a single peak by reverse-phase high-pressure liquid chromatography. Furthermore, two-dimensional nuclear magnetic resonance (NMR) spectroscopy gave well-dispersed spectra in the presence of calcium. In contrast, the same protein folded in vitro in the absence of calcium was eluted as multiple broad peaks and gave a poorly dispersed NMR spectrum in the presence of calcium. The calcium affinity (Kd) of the Tva LDL-A module, determined by isothermal titration calorimetry, is approximately 40 microM. Characterization of several Tva mutants provided further evidence that calcium is important in protein folding and function of Tva. Mutations of the Ca2+-binding residues (D46A and E47A) completely abrogated the Ca2+-binding ability of Tva, and the proteins were not correctly folded. Interestingly, mutations of two non-calcium-binding residues (W48A and L34A) also exerted adverse effect on Ca2+-dependent folding, albeit to a much less extent. Our results provide new insights regarding the structure and function of Tva in ASLV-A entry.     

Identification of the human rhinovirus serotype 1A binding site on the murine low-density lipoprotein receptor by using human-mouse receptor chimeras

Human rhinovirus serotype 1A (HRV1A) binds more strongly to the mouse low-density lipoprotein receptor (LDLR) than to the human homologue (M. Reithmayer, A. Reischl, L. Snyers, and D. Blaas, J. Virol. 76:6957-6965, 2002). Here, we used this fact to determine the binding site of HRV1A by replacing selected ligand binding modules of the human receptor with the corresponding ligand binding modules of the mouse receptor. The chimeric proteins were expressed in mouse fibroblasts deficient in endogenous LDLR and LDLR-related protein, both used by minor group HRVs for cell entry. Binding was assessed by virus overlay blots, by immunofluorescence microscopy, and by measuring cell attachment of radiolabeled virus. Replacement of ligand binding repeat 5 of the human LDLR with the corresponding mouse sequence resulted in a substantial increase in HRV1A binding, whereas substitution of repeats 3 and 4 was without effect. Replacement of human receptor repeats 1 and 2 with the murine homologues also increased virus binding. Finally, murine receptor modules 1, 2, and 5 simultaneously introduced into the human receptor resulted in HRV1A binding indistinguishable from mouse wild-type receptor. Thus, repeats 1 and/or 2 and repeat 5 are involved in HRV1A attachment. Changing CDGGPD in the acidic cluster of module 5 in the human receptor to CDGEAD present in the mouse receptor led to substantially increased binding of HRV1A, indicating an important role of the glutamate residue in HRV1A recognition.