The LDLR (low-density lipoprotein receptor) is a modular protein built from several distinct structural units: LA (LDLR type-A), epidermal growth factor-like and β-propeller modules. The low pH X-ray structure of the LDLR revealed long-range intramolecular contacts between the propeller domain and the central LA repeats of the ligand-binding domain, suggesting that the receptor changes its overall shape from extended to closed, in response to pH. Here we discuss how the LDLR uses flexibility and rigidity of linkers between modules to facilitate ligand binding and low-pH ligand release.

Introduction

The LDLR (low-density lipoprotein receptor) mediates cellular uptake of cholesterol-laden lipoprotein particles from the circulation. The receptor binds cholesterol-carrying LDL particles at the cell surface at neutral pH, transporting them to endosomes where the low-pH environment induces ligand release. The two activities of the receptor, ligand binding and pH-triggered release, are attributed to distinct domains, referred to as the ligand-binding domain and the EGFP (epidermal growth factor-precursor) homology domain, respectively [1].

The two functional domains of the LDLR consist of several distinct structural units connected by short linkers (Figure 1). Seven LA (LDLR type-A) modules constitute the ligand-binding domain, and the EGFP region consists of two EGF-like modules followed by a β-propeller domain and a third EGF-like module. Electron microscopy studies suggest that the receptor adopts an elongated conformation at neutral pH, presenting the tandemly repeated LA modules for ligand binding [2]. The 3.7 Å X-ray structure revealed that at endosomal pH the receptor closes by forming intramolecular long-range contacts between the β-propeller domain and the central LA repeats of the ligand-binding domain [3]. Here, we discuss how the LDL receptor combines flexibility and rigidity of linkers between modules to make efficient ligand binding and ligand release possible.

Intermodule relationships in the LDL receptor

Figure 1
Intermodule relationships in the LDL receptor

Arrows identify intermodule connections where direct (red) or indirect (black) evidence is available regarding the relative mobility of the adjacent modules. Wavy and straight lines indicate interdomain flexibility and rigidity, respectively. Double-headed blue arrows denote interfaces putatively induced by low pH.

Figure 1
Intermodule relationships in the LDL receptor

Arrows identify intermodule connections where direct (red) or indirect (black) evidence is available regarding the relative mobility of the adjacent modules. Wavy and straight lines indicate interdomain flexibility and rigidity, respectively. Double-headed blue arrows denote interfaces putatively induced by low pH.

Flexible linkers in the ligand-binding domain allow the LDLR to accommodate heterogeneous ligands

The N-terminal LA modules (LA1–LA7) constitute the ligand-binding domain of the LDLR. The receptor binds two different classes of lipoprotein particles, LDL particles, which contain a single copy of apoB (apolipoprotein B) and β-VLDL (β-migrating very-low-density lipoprotein) particles, which contain multiple copies of apoE. It was shown previously that repeats LA3–LA7 all contribute to the binding of LDL, and LA5 is crucial for high-affinity binding of β-VLDL [4]. Further in vitro studies have shown that the LA4-LA5 pair is sufficient to bind apoE•dimyristoylphosphatidylcholine particles, which are believed to mimic β-VLDL [5].

The LA modules share a similar tertiary fold stabilized by three disulphide bonds and a bound Ca2+ ion that is required to maintain the structural integrity of the module [3,6,7]. A cluster of highly conserved acidic residues that form an acidic patch on the surface of the LA modules participates in Ca2+ ion coordination. The presence of a complementary group of basic residues on the surface of the receptor-binding domain of apoE [8], which are important for LDLR binding [9], suggests that this acidic region participates in binding of ligands as well.

The LA modules of the ligand-binding domain are connected by short four- or five-residue linkers, except for the linker between LA4 and LA5 of 12 residues. Solution NMR studies of the tandem repeats LA1-LA2 and LA5-LA6 have demonstrated that the linkers in both module pairs are flexible, permitting essentially unrestricted relative motion of each module with respect to its partner in the pair [10,11]. The absence of contacts between adjacent ligand-binding modules in the X-ray structure suggests that the interdomain flexibility directly observed in these two cases applies generally to the linkers connecting all of the LA repeats in the ligand-binding domain. Such flexible intermodule connections allow the LDLR to bind a variety of heterogeneous lipoprotein particles different in size, shape and curvature.

Fixed LA7–EGF_A interface facilitates intramolecular closure on exposure to acidic pH

The ligand-binding domain is followed by a region of the receptor homologous to the EGFP, which includes two EGF-like modules, followed by a β-propeller domain and a third EGF-like module. The ligand-binding domain is connected to the EGFP region by a four-residue linker joining LA7 to EGF_A. In the low-pH structure, these domains share a hydrophobic interface and it has been hypothesized that the linker between LA7 and EGF_A might be a pivot allowing closure at acidic pH, with Gly-293, a conserved residue in the linker, contributing to flexibility [12].

To resolve this issue, we examined, by solution NMR, the flexibility of the linker connecting the LA7 and EGF_A modules across the physiologic-pH range, and assessed the structural integrity of this interface at neutral pH. The mobility of the linker residues was evaluated by measuring heteronuclear {1H}-15N-NOEs, which are sensitive to fast internal motions and are used to identify flexible residues and regions in the molecule. In contrast with the linker connecting LA5 and LA6, which is flexible in solution, the LA7-EGF_A linker is highly constrained across values of pH ranging from 5.3 to 7.0.

Preliminary structure calculations indicate that the intermodule interface between LA7 and EGF_A at neutral pH is not significantly altered from that seen in the X-ray structure at endosomal pH. The interface appears to be stabilized by a cluster of hydrophobic residues that includes Phe-261, Val-274 and Ile-313. Two familial hypercholesterolaemia mutations, Phe-261/Ser and Ile-313/Val, alter side-chains of residues in the interface emphasizing its importance in normal LDLR function.

Previous structural data show that the EGF_A–EGF_B interface is also constrained at neutral and endosomal pH [3,13,14]. Together, LA7, EGF_A and EGF_B constitute a stiff handle that works in conjunction with the flexible intermodule linkers of the ligand-binding domain. The rigid trio of repeats serves as an anchor, setting a favourable overall topology that permits the ligand-binding repeats to sample effectively a smaller conformational space. This constraint facilitates intramolecular closure of LA4 and LA5 on to the β-propeller domain when the ligand is released.

Role of the EGF_B-β-propeller interface

Similar to LA7, EGF_A and EGF_B, which maintain their rigid relative arrangement independent of pH, the last EGF-like module, EGF_C, packs tightly against the β-propeller at both neutral and acidic pH [3,15]. Thus, all the modules in the EGFP domain, besides the EGF_B-β-propeller pair, are rigidly connected. Available experimental results suggest that the interface between the EGF_B and β-propeller modules, however, might be pH-regulated: although an extensive interface, composed of hydrophobic and charged residues, exists between EGF_B and the propeller at low pH [3], it is not observed in the neutral pH X-ray structure of the EGF_B-propeller-EGF_C fragment, where the EGF_B module is present but disordered. It remains to be clarified, however, whether the interface between EGF_B and the propeller seen at low pH is an intrinsic feature of the two modules (i.e. would the interface form spontaneously at low pH if the two modules were excised from the rest of the receptor?) or is it a secondary consequence of the long-range closure of the receptor at low pH?

Concluding remarks

Studies of the LDLR structure emphasize how different relationships between adjacent domains are tailored for function. The ability of the ligand-binding modules to move independently of one another helps the receptor to capture heterogeneous ligands at the cell surface. On the other hand, a fixed junction between the ligand-binding domain and the EGFP region imposes a topological constraint that positions the ligand-binding domain in an appropriate orientation for long-range intramolecular closure at low pH to release ligands.

Structure Related to Function: Molecules and Cells: A Focus Topic at BioScience2004, held at SECC Glasgow, U.K., 18–22 July 2004. Edited by D. Alessi (Dundee, U.K.), T. Cass (Imperial College London, U.K.), T. Corfield (Bristol, U.K.), M. Cousin (Edinburgh, U.K.), A. Entwistle (Ludwig Institute for Cancer Research, London, U.K.), I. Fearnley (Cambridge, U.K.), P. Haris (De Montfort, Leicester, U.K.), J. Mayer (Nottingham, U.K.) and M. Tuite (Canterbury, U.K.).

Abbreviations

     
  • apoB

    apolipoprotein B

  •  
  • β-VLDL

    β-migrating very-low-density lipoprotein

  •  
  • EGF

    epidermal growth factor

  •  
  • EGFP

    EGF precursor

  •  
  • LDL

    low-density lipoprotein

  •  
  • LDLR

    LDL receptor

  •  
  • LA

    LDLR type-A repeat

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