Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : June 15, 2021
Accepted : August 23, 2021
Published online : September 30, 2021
doi:10.24659/gsr.8.3_136

Review article

Conformational changes in the LRP1 receptor related to the amyloid beta
binding on the receptor's II and IV ligand binding domains
Nikola Barić
Presika, Labin, Croatia

Abstract

Related to the great importance of the intra-arterial perivascular drainage (IPAD), the drainage function of the lipoprotein
receptor-related protein-1 (LRP1) that is a huge transmembrane LRP receptor member, is increasingly becoming the subjectof comprehensive researches. Two important questions thus emerged: 1. How does the binding of the whole ligand complex
members, among them amyloid beta (Aβ), the crucial factor in the pathology and pathophysiology of Alzheimer's disease(AD), lead to conformational changes in the extracellular receptor structure? 2. How do these changes reflect in the structureof the receptor intracellular tail, with the binding of PICALM (phosphatidylinositol binding clathrin assembly protein) to the
YXXL tail motif, and how do they reflect in the recruitment of a number of signal proteins, and in the onset of PICALM/
clathrin Aβ endocytosis, transcytosis and Aβ exocytosis into the capillary circulation? Both questions require an exact
explanation. The profound insight into the available literature related to these events does not give a satisfactory explanation tothese questions; consequently, the present knowledge of the AD pathophysiology requires additional comprehensive research.
This study is not concerned with the details of the AD pathology and pathophysiology; it is primarily concerned with the
problem of Aβ binding to the LRP1, with local conformational changes in the tail, and with the activators of Aβ transcytosis
and Aβ exocytosis into the capillary blood.

KEY WORDS: lipoprotein receptor- related protein1 (LRP1), intra-arterial perivascular drainage (IPAD),		

Alzheimer's disease (AD), PICALM, YXXL motif, endocytosis, transcytosis, exocytosis

Introduction

The available literature related to the amyloid beta (Aβ)
drainage from the brain often emphasizes the conformationalchanges on the lipoprotein receptor-related protein-1 (LRP1)
tail after Aβ binding to the receptor. Changes on the tail are
obviously the result of induced events on the extracellular
N-terminal chain. It can be supposed that both, -COOH and-NH2 ends of LRP1, are fixed to the surrounding structuresin the cell (tail elements) and to the elements of lamina
reticularis (extracellular matrix, ECM). Aβ binding to the
LRP1 ligand binding domain, conformational changes on
LRP1 extracellular heavy α-chain (ectodomain) of the receptor(induced by the aligning of the electrostatic forces of bothparticipants) lead to mild LRP1 contraction. This contraction
with the fixed LRP1 ends (ECM and tail elements), probably
induces the discrete pulling of the distal di-leucine towards
the receptor extracellular part, with the widening of theaccessed space to the PICALM (phosphatidylinositol binding
clathrin assembly protein) for the contact with the YXXL
motif. This space in which PICALM is trapped, is borderedby the fixed - COOH on one side and the less fixed di-leucineon the other side. Essentially, the LRP1 activation results inthe zigzag wrinkling of its structure with the accompanying
mild shortening. The result is the LRP1-PICALM/clathrindependent endocytosis of Aβ-LRP1 complex ( Fig. 1 ) 1- 6 ) .
Daly NL et al. 7 ) , by NMR (nuclear magnetic resonance)
spectroscopy, solved the problem of the structure of the first
LDL receptor module (CR1, cysteine-rich1). This module iscomposed of a β-hairpin structure followed by a series of
β-turns. In its structure are clearly observable 6 disulfidebound cysteines and a cluster of negatively charged aminoacids (Asp-15, Asp-26, Glu-30, Asp-33, Asp-36, Glu-37,
and Glu-40), primarily clustered on one face of the module.
Between the fourth and sixth cysteine, there was found
the region for high-affinity binding of positively charged
sequences in LDLR's ligands. The CR module also contains
about 40 amino acids and a coordinated calcium ion
important for the structure stability ( Fig. 1).

Contact address: Nikola Barić MD, PhD
Presika 153, 52220 Labin, Istria, Croatia
TEL: + 385-52-285- 008 e-mail: nikola.baric@pu.t-com.hr

Glycative Stress Research 2021; 8 (3): 136 -147
(c) Society for Glycative Stress Research_ 13 6 _Glycative Stress Research

NH2 fixed-COOH

Cysteine-rich module
Ligand-binding repeats (40 AA in every module)
C I Ligand-binding cluster I
No ligands known to bind

Peptide bond-NH2
EGF repeats
2 cysteine-rich EGF-repeats

YWTD domains����������� ������ (much AA in every domain)�� ��������

.

β2-strand- - +Asp15, Asp26, Glu30, Asp33, Asp36, Glu37, Glu40
pullingC- +���� Ref. 7�
Ligand-binding repeats
Ligand-binding
domains I-IV contain a
The three-dimensional structure of
Ligand-binding cluster II +net
negativechargeimportant forthis module has been determined by
C II+ binding positively charged ligands.
Binding of Aβ
two-dimensional NMR spectroscopy.
- β1-strand+
Extracellular heavy α-chain of LRP1-+
Extracellular N-terminal chain, 515 kDaN
2 cysteine-rich EGF repeats
pulling
Cysteine-rich module
Ligand binding repeat (one, 1)
EGF repeats��� �������
(30-40 AA in every repeat).

..

��� ������������� ������

��������� �� ���� � ���� ������� �� �� �������� ��������������� �� ��� �����
���� ����� ��������� �����
������ �� ��� ��������
���������

Ligand binding cluster III
C III

YWTD domainβ-propeller domain
Transmembrane domain
NPXY motif

Peptide bondDileucine motif

2 cysteine-rich EGF repeats

Aβ
Length of one Aβ monomer(120 Å = 12 nm) (β1-strand)

..

-

-

pulling

YXXL motif��� ������������� ������ �������� ������������ ������������� �� ��� ������������ ��������� ���������� �������

.+- Ligand-binding cluster IV
β2-strand.+.- + -- - BindingCofIVAβ.+.+ C.+ 2 cysteine-rich EGF repeats
β1-strand

Lys28

C R26

Width of one Aβ monomer
30Å = 3 nm (β1-strand)

C R27

Lys16

His14

LRP1 length 50-70 nm, 500-7�� Å

His13

N

-

His6

YWTD domain

Arg5

β-propeller domain
EGF repeats

extracellularpulling
Transmembrane domain
Abluminal cell membrane

Serve as dominantServe as the endocytosis signal for receptor
NPXY motifs
endocytosis signals for
intracellular
the rapid endocytosis
Di-leucine motifs Tail�������� �������� ���������� ��������� �� ���
LRP1 β-chain���� �����
PICALM
S76
COOH fixed ������ ������� ����.

Fig.1 of LRP1 (CD91) receptor molecule.
Fig. 1. Presentation

Second ligand-binding cluster contains eight cysteine-rich ligand-binding repeats (blue circles); it also contains eight cysteine-rich EGF
repeats and six YWTD domains. In the tail there are visible two NPXY m motifs, two dileucine motifs, and one YXXL motif; every
ligand binding domain consists of a β-hairpin structure followed by a series of β-turns. LRP1, lipoprotein receptor-related protein-1; EGF,
epidermal growth factor; Aβ, amyloid beta; AA, amino acids; NMR, nuclear magnetic resonance; PICALM, phosphatidylinositol binding
clathrin assembly protein.

_ 137 _

Amyloid beta (Aβ) binding on the LRP1 receptor

RAP role in LRP1 biosynthesis
Before further analysis of the above mentioned events,
it is important to explain the structure and function of
the receptor-associated protein (RAP), the LRP receptor
chaperone (protein that assists the conformational changesof other macromolecules). Its artificially synthethysedform is the crucial factor in all experiments related to theligand binding to LRP receptor. On account of this, it isnecessary to primarily emphasize some facts about the
LRP1 generation. LRP1 gene is located on the long armof chromosome 12 ( long arm chr. 12Q13.3). Biosynthesisof the LRP1 protein (location ER, endoplasmic reticulum)
proceeds by the 39 kDa receptor-associated protein (RAP
gene, short arm, 4 p16. 3 gene), normally the resident in theendoplasmic reticulum (ER). Naturally, RAP is a chaperone
which is the key factor for the conformational folding andunfolding, as well as assembly and disassembly of many
macromolecules, among them LRP1. Its crucial role is toprevent the binding of superfluous ligands on the LRP1
during its formation, and to prevent the aggregation offormed structure in the nonfunctional shape. RAP, which
is bound with LRP1, after entering into Golgi apparatus
dissociates from LRP1 by effects of low pH. After this, RAP
returns back to the ER and continues its function as a blockerof the superfluous ligands and aggregation. A dissociated
LRP1 exits from Golgi, enters the transport vesicle, andcontinues its way to the cell membrane, primarily on theabluminal side. On account of its molecular structure andaffinity for binding to LRP1, RAP artificial synthetic form is
optimal for the mentioned experiments (Fig. 2) 7, 8 -10 ).

The importance of the CR34 distanceshortening in the structure of LRP1
Prasad JM et al. 8 ) in their study present experimental
results related to the distance between LRP1 CR2 and CR7
in the endoplasmic reticulum (ER), which ranges from20- 43 Å. The distance between CR3 and CR4, measured
in conditions of the neutral endosomal, is 32 Å, which isquite different from the distance of 21 Å between the same
modules in the RAPD3/CR34 structure. The difference
between these two conditions indicates obvious narrowing of
the CR34 distance after the adequate electrostatic attraction
and binding (H-bonds, salt bridge, isopeptide bonds) Hys13,
Hys14, Gin15, Lys16 (positive charge cluster - e.g., on theAβ monomer) and Asp residue (on CR34 LRP1 receptor).
It is evident that the decrease of this distance has a direct
effect on the pulling of the extracellular LRP1 part, on the
structural change on the tail, and on the widening of thepassage for PICALM towards the YXXL motif. All thisindicates the marked CR repeat flexibility in the LRP family.
Generally, the study presents the probable events afterAβ binding to LRP1, which, however, require a detailed andcritical examination. The electrostatic forces that attract
Aβ and LRP1 residues induce a certain movement (about
5.5 Å) on both sides of the contact region (interaction).

These movements induce mutual successive pulling of thefollowing protein segments toward the contact place (onsetof a certain chemical bond), while the energy packet, or thesignal, travels in the direction opposite to the LRP1 segment
movement. It is supposed that the energy released by thechemical reaction (between Aβ and LRP1) with the probablesmaller resistance of LRP1 structure to this spreading ofenergy towards the tail, leads to the transmision of thesignal (packet of energy) through the membrane and to theembracing the tail (enclosing). The result is the movement of
two NPXY and two di-leucine motifs toward the abluminal
cell membrane, with the spreading of space necessary forthe contact of PICALM with YXXL motif. The -COOH
part of the receptor is most probably firmly fixed to theneighboring structures, so that the intracellular part distends
and enables the above mentioned widening. The shifted
PICALM activates the YXXL motif, with the activation of
the adaptor proteins, which leads to the PICALM/clathrin
dependent endocytosis, transcytosis, and exocytosis. The
analysis of the ligand binding cluster IV (C IV) contributes
to the understanding of these events. The chemical bindingof Aβ and LRP1 residues most probably happens between
Lys16 on Aβ and three Asp on CR26, and His13 on Aβ andthree Asp on CR27 (tripatite salt bridge). With the beginningof the electrostatic balance between the involved residues,
the earlier distance CR2627 with 31 Å decreases to 20 Å.
This induces the above mentioned mutual shift of 5.5 Å. The
pulling of two NPXY and two di-leucine motifs, due to theclose contact with α-unit of the G protein-coupled receptor,
leads to the distension and separation of α-unit from theother two members of the receptor (β, γ ) and its activation.
Following is the activation of adenyl cyclase, transformationof adenosine triphosphate (ATP) to the cyclic adenosine
monophosphate (cAMP), and the activation of protein kinase
A (PKA) and serine 76 (Ser 76) on the receptor tail (Fig. 3, 4,5 ) 1- 3 ) .
The analysis of Aβ and LRP1 binding indicates the mostprobable participation of CR26 and CR27 that with His13
and Lys16 contact with three Asp residues from each CR.
The retrograde pulling signal (energy packet) that travels to
the tail, must pass through four CR modules, one YWTD
motif, and nine EGF (epidermal growth factor) repeats. If
the signal travelled through peptide links only for four CR, it
would have to pass through 160 peptide bonds. The passageof the signal through one β-propeller domain built of sixbladed β-propellers, each composed of four antiparallel
β-strands, is also an extremely long way. Each of nine EGFlike domains is composed of 30-40 amino acid residues andsix cysteine residues (three disulfide bonds), which all together
also create a great resistance to the signal propagation. The
signal transmission through the total length of this route
would surely markedly decline the signal strength up tocomplete loss of the signal. This type of signal transmission
obviously is not possible. All this indicates that the signal
is transmitted through the complete module structure,abundantly using side chains, and not exclusively the central
direction. The peptide bonds would have the transmission role
only at the entrance and at the exit module point 8 -10).

_ 13 8 _Glycative Stress Research

Lrp1 gene (12q13.3) activation

Transcription factors

Nucleus
Coding for the precursor protein (transcription, mRNA);mRNA exits from the nucleus and enters into the endoplasmic reticulum (ER);mRNA location on ribosomes; mRNA translation; production of specific AA;
Precursor protein generation;
Binding of the precursor protein to the receptor associated protein RAP;
(Endoplasmic reticulum resident chaperone, 39-kDa; composed of D1, D2, andD3 domains); negatively charged CR repeats and positively charged residues
on RAP (ligand);ER
RAP (chaperone) localize on the precursor ligand binding domains (clusters)
preventing the binding of other ligands on these domains;

RAP/LRP1 precursor complex exits from ER and enters into the Golgi
apparatus (GA), (important role of NPXY proximal motif on the precursor);

GA

Protonation (addition of proton to the atom, molecule or ion) of the histidine
residues in RAP 3 domain;

RAP dissociation from LRP1 precursor protein; low pH in Golgi;

Cleaving of the LRP1 precursor by Furin protease (it is a protease that
proteolytically cleaves many protein substrates);

Formation of large extracellular heavy α-chain and a shorter β-chain whichbecome noncovalently linked;

The two chains travel together in a transport vesicle to the cell membrane andthere are embeded in its structure;

Fig. 2. Coding for the LRP1 precursor protein and its transformation into the mature LRP1.

According to a number of recent investigations, transcription of the Lrp1 gene can be activated, among the number of different
transcription factors, particularly by sterol regulatory element binding protein 2, hypoxia-induced factor 1, and nitric oxideFig.2.dependent transcription factors; Lrp1 gene activation, coding for the LRP1 precursor protein, and mRNA generation occurs in
the cell nucleus. Transcribed mRNA exits from the nucleus and immediately enters into ER and contacts with ribosomes. Now,
on the ribosome occurs the translation during which the mRNA is decoded with the production of a specific amino acid (AA)chain, or polypeptide. LRP1, lipoprotein receptor-related protein-1; Rap, Ras proximate; RAP protein, chaperone; protonation,
RAP dissociation; CR, cysteine-rich repeat on LRP1; ER, endoplasmic reticulum.

_ 13 9 _Amyloid beta (Aβ) binding on the LRP1 receptor

Amyloid beta (Aβ) binding with the LRP1 ligand binding clusters II and IV;electrostatic attraction between Aβ positively charged residues (Arg5, His6, His13,
His14, Lys16, Lys28), and negativaly charged residues on the LRP1 cluster (Asp15,
Asp26, Glu30, Asp33, Asp36, Glu37, Glu40);
Aβ Binding (ligand) binding to LRP1 ectodomain
Conformational changes in the LRP1 extracellular heavy α-chain (515 kDa);(structural extension of LRP1 with the transmission of tension to the tail)Conformational changes in the LRP1 intracellular tail; tail extension; morefavorable approach of PICALM to the YXXL motif;

Accelerated recruitment of PICALM on the tail and binding with YXXL motif(dominant determinant for internalisation);
Local recruitment of clathrin and AP-2 protein to the cell membrane with the
beginning of PICALM/clathrin-dependent endocytosis of Aβ-LRP1 complex;
On the place of PICALM, clathrin, and AP-2 recruitment, occurs the formationof special cell membrane patches called clathrin coated pits;

By the interaction of PICALM, clathrin, and AP-2, continues the invagination of
the cell membrane, and the formation of the completely embeded vesicle
surrounded by the clathrin scaffold;
Additional effects by Hsc70, auxillin, and Rab5, induce the uncoating ofclathrin coated vesicle and moving of AP-2, clathrin molecules, Hsc70, andauxillin toward the cell membrane; PICALM remains bound around the
evolved early endosome;By coordinated effects of PICALM, Rab5, Rab11, syntaxin, synaptobrevin, and
SNAP-25, late �ndosom� becomes the exocytic vesicle, approaches the cell
membrane, fuses with it, dissolves the membrane structure, and discharges thecontent in the blood; LRP1 remains bound with the straightened cell membrane;

Fig. 3. Events typical for LRP1/clathrin coated vesicles endocytosis and transcytosis.

������ to the LRP1 cluster II and IV induces conformational changes into the LRP1 extracellular and intracellularAβbinding
segments. Changes in the intracellular segment induce the receptor slight elongation and concomitant pulling forces which
propagate through its structure, pass across the cell abluminal membrane, and enter into the receptor's tail segment. Now,
it follows the structural change of the tail's structure, which results in PICALM/ clathrin induced Aβ endocytosis andtranscytosis. The crucial importance in these events has the protein PICALM, whose binding with YXXL motif is the first stepin all events. LRP1, lipoprotein receptor-related protein-1; Aβ, amyloid beta; PICALM, phosphatidylinositol binding clathrin
assembly protein; AP-2, adaptor protein 2; HSC70, 71 kDa heat shock cognate protein; Rab5, Ras analog in brain 5; SNAP25,
synaptosome associated protein 25.

_ 14 0 _Glycative Stress Research

EGF repeat
Ab.l.c.m.

GPCR
inac tive
α

β

NPXY
Di-leucine

LRP1

PICALM
S76
-COOH
fixed

.

B/

γ

Adenyl cyclase
adaptorinactiveATP
adaptor�������

Tail

Fe-65

YXXL
PKA
inactivecAMP
EGF repeat

PSD95

Disabled 1
inactive���� ���� �������

C III
Ligand binding cluster III
Peptide bondA/

2 cysteine-rich EGF repeats

Aβ
Lys28
β2-strand
Lys16

-

.

His14

Tendency for electrostatic equilibrium.+- +.. + F= k*Q *Q /r- C. ++ K=9*10 Nm /C
C R27
Asp
Electrostatic attractionAspm ove m e nt

Binding of Aβ

O nset of pulling
Tendency for electrostatic equilibrium

2

1

His13

-

Onset of pulling- + Distance CR2627:32Å
Ligand-binding cluster IV- -C R26
β1-strand
C IV

20Å

Distan ce Lys16-His13: 20Å

One cysteine-rich repeat (module) is
composed of approximately 40 AA, andβ-hairpin structure followed by β-turns

His6
Arg5

.

9

2

2

2 cysteine-rich EGF repeats

2

YWTD domain

Coulomb's law

β-propeller domain
EGF repeats

N
LRP1 extracellular segment
Cell membrane Transmembrane domain
intracellularServe as the endocytosis signal for receptor

NPXY motifs
Di-leucine
motifsLRP1 β-chain
Membrane spanning C-terminal fragment, 85 kDa
PICALM
S76
-COOH

.

fixed

Ab.l.c.m.
Tail
Serve as dominantendocytosis signals forthe rapid endocytosis
YXXL motif��������
PICALM binding site
PKA

Fig. 4. Partial presentation of LRP1 receptor without complete activation.
A and B: The difference compared to Fig. 5 is clearly visible; Aβ is free; LRP1 cluster IV is also free; the space for PICALM

Fig. 4.
movingto the YXXL motif is narrowed, and the PICALM approach to the YXXL is blocked; PKA is inactive; there is no sign of S76
phosphorylation; Ab.l.c.m., abluminal cell membrane; adaptor proteins, Disabled1, Fe-65, PSD95; NPXY motifs, Di-leucine motifs,
LRP1 tail; B/Aβ positive residues, Lys28, Lys16, His14, His13, His6, membrane spanning C-terminal fragment, 85 kDa; EGF repeats;
YWTD domains, Tyr-Trp-Thr-Asp (YWTD)-repeat; LRP1, lipoprotein receptor-related protein-1; EGF, epidermal growth factor; Aβ,amyloid beta; AA, amino acids; PICALM, phosphatidylinositol binding clathrin assembly protein; PKA, protein kinase A; YXXL, Tyrx-x-Lue; NPXY, Asn-Pro-x-Tyr, a sequence often found in cytoplasmic tails; PSD95, postsynaptic density protein 95; GPCR, G proteincoupled receptor; CR, cysteine-rich repeat on LRP1.

_ 141 _

Amyloid beta (Aβ) binding on the LRP1 receptor

Importance of the tripartite salt bridges
formation
In the case of Aβ and LRP1 binding, the appearanceof electrostatic equlibrium between their opposite charges
creates the tripartite salt bridge (combination of two noncovalent interactions: H-bonding and ionic bonding) between
the carboxilate oxygen atom of three aspartates, and eitherof His13 imidazole ring two side chain nitrogens, Nδ or
Nε. At the same time, the three aspartates (located on theneighboring RC) surround the ε-amino group of the lysine16
residue, also in the form of the tripartite salt bridge (theaverage value of covalent bond energy, 415 kJ/mol; hydrogenbond energy, 4 - 40 kJ/mol; ionic bond energy, 170-1,500 kJ/
mol). As in the presented experiments with RAP in the studyof Prasad JM et al. 8), the binding is linked with the narrowingof the distance between two included neighboring CRs andthe occurence of pulling forces i.e., pulling signals. It is
important to emphasize that the distance between His13 andLys16 on the Aβ-strand, in the case of binding, is about 20-21
Å, as is in the case of RAPD3/CR34 complex 9, 10).
Aβ approaches LRP1 by the influence of the electrostatic
attractive force. This force is explained by the Coulomb's
law: F = K*Q1*Q2/r 2 , where F is the electric force, k is the
Coulomb's constant ( k = 8.9875*10 9 N*m 2 / C 2 ), Q1 and Q2
are the charges on objects, and r is the distance between the
charges. Other members of the equation: F, electric force
in newtons (N); C, coulomb; m, distance r in meters (m).
The equation clearly shows that the closer two charges are,the stronger is the force between them. So, by the increased
mutual approaching, F rises. The pulling of the two
maximally exposed CRs (ligand binding cluster IV, CR26
and CR27, Fig. 4, 5 ) progressively rises up to the occurrenceof tripartite salt bridges. The critical distance for theoccurrence of these bridges is ≈ 2.7-3.3 Å, with 3.0 Å being
the most common value for protein. The force F has the
maximal value just before the occurence of the bridges. In
this moment, parallel to the pulling of both CRs, evolves thestrong reversed energy (signal) propagation to the receptor
tail ( Fig. 4, 5 ) 11).
Fisher C et al. 9) present the 1.26 Å X-ray structureof the complex composed of two module regions of the
LDLR ligand binding domain (D), and the third RAP
domain (escort protein for LDLR family members). RAP
has three domains: D1, D2, and D3. The RAPD3 (positively
charged) domain has three-helix bundles (α7, α 8, α 9),and two docking sites, one for each LDLR module. Three
calcium-coordinated Asp residues (negatively charged) fromeach LDLR module encircle a lysine side chain (positively
charged) protruding from the RAPD3 α 8 helix. The first
explored LDLR module region CR3, with its aspartates,
surrounds Lys 270 (K270), and module region CR4 alsowith its aspartates surrounds Lys256 (K256). In this way,
the three Asp at each contact position surround the ε-aminogroup ( NH 3 + attached to the fourth C atom from the
α-carbon) of the lysine side chain (CH2)4 NH2 . LDLR
aromatic residues, F105 (Phe-105, Phenylalanine, essentialaa) of CR3 and W14 4 (Trp-14 4, Triptofan, essential aa)of CR4, complete the pocket surrounding. The surroundingof the ε-amino groups is in fact a tripartite salt bridge. The
authors mark the LRP module as LA module. There areelectrostatic interactions between complementary charged

surfaces at each docking site. The authors emphasize that Aβ
peptide corresponds to RAPD3. Strongly positive RAPD3
potential is concentrated around K256 (Lysine) and K270
(Lysine). These two docking sites are separated by four
helical turns. The positively charged side chains of RAP
K253 and R285 correspond to Aβ peptide His13, His14 andLys16.
Lillis AP et al. 10) in their research strongly confirm theresults of Fisher C et al. 9). They present in detail the RAPD3
structure in the complex with two CRs from the LRP
receptor, and emphasize the importance of further analysis
of ligand interaction with LRP1 receptor. They also confirm
that the YWTD repeat (β-propeller domain) forms a sixbladed β-propeller that packs tightly against C-terminal
EGF module. This observation leads to the conclusion thatthe β-propeller domain functions as an alternate ligand forCR4 and CR5, which bind in a calcium-dependent mannerpromoting ligand release.
The analysis of the distance between the middle ofresidue His13 and the middle of residue Lys16 shows thevalue of ~ 20 Å. This strikingly corresponds to the distance
between CR3 and CR4 in the structure RAPD3/CR34,
which is 21 Å. The distance between Lys16 and Lys28 issignificantly larger than 21 Å. This indicates the purposefuladaptation of LRP1 extracellular N-terminal chain (515kDa), i.e., heavy α-chain, to electropositive residues of Aβmonomer. The resulting link is in the form of a tripartite salt
bridge.
De Nardis C et al. 2) emphasize that the overall
conformation of LRP1 remains largely unchanged after RAP
binding, as shown by the similarity of the SAXS curves andby the negative-stain EM class averages. Conformationaltransitions upon RAP binding might happen at a local level,
perhaps influencing the relations of the neighboring domains(e.g., Cysteine-rich repeats) without producing large structural
rearrangements visible at low resolution, as supposed by
Migliorini MM et al. 12). According to the authors, the lowresolution electron spectroscopic microscopy images of fulllength LRP1 purified from human placenta, suggested anelongated "zigzaged" shape ranging 50-70 nm in length.
Detailed structural studies of the full LRP1 molecule havebeen hindered by the difficulties in generating LRP1. The
authors frequently emphasize the presence of 515 kDa
extracellular right handed α-chain containing the ligand
binding regions. It is non-covalently associated with the
β-chain (85 kDa) composed of the trans-membrane region
and the small intracellular domain (Fig. 1).
Gonias SL et al. 13) present the molecular models of
the organization of structural domains in LRP1. They alsoemphasize the helical shape of the extracellular N-terminal
chain (515 kDa).
As it was mentioned in the introduction, the bindingof Aβ to the LRP1 ligand binding domains (especially II
and IV) induces a certain conformational effect on the
LRP1 cytoplasmic C-terminus with subsequent expressive
binding of PICALM on the YXXL motif, and inductionof the PICALM/clathrin-dependent endocytosis of the
Aβ-LRP1 complex. There are no data about the exact_ 14 2 _Glycative Stress Research

EGF repeat
EGF repeat
Ab.l.c.m.

GPC R

α
NPXY

β
activeDi-leucine.

fixed S76
-COOH

PSD95

Adenyl cyclase

2 cysteine-rich EGF repeats

adaptoradaptor

PICALM
spreading tail

B/

C III
Ligand binding cluster III
Peptide bondγ����acti������ adaptor

phosphorylation
PKA
Fe-65 [P��]3activation
Disabled 1

activation

LRP1 tail segment

Aβpulling- Distance CR2627: 32Å to 21Å
- Ligand-binding cluster IV+
C R26- C IV

.

Lys28
cAMP

One cysteine-rich repeat (module) is
composed of approximately 40 AA
and β-hairpin structure followed by β-turns

pulling

ATP.+.+ - Binding of Aβ.
pulling+.
+C.+ - - 2 cysteine-rich EGF repeats
β1-strand

β2-strand
Lys16

C R27

21Å

His14
His13

A/

-

N

His6

YWTD domainβ-propeller domain

Arg5

EGF repeats

extracellularpulling

LRP1 extracellular segment

Transmembrane domain

Ab.l.c.m.
Serve as the endocytosis signal for receptor
Membrane spanning C-terminal fragment, 85 kDa

intracellular

LRP1 β-chain

Serve as dominantendocytosis signals forthe rapid endocytosis

NPXY motifs
Di-leucine motifs

COOH

.

S76

fixed

PICALMYXXL motif
Tail
PICALM binding

Psite PKA

Fig. 5. Partial
presentation of LRP1 (CD91) receptor molecule (CIII and C IV).
.

Fourth ligand-binding cluster contains 11 cysteine-rich ligand-binding repeats (blue circles); in the direction to the abluminal cell membrane
there are the positions of nine EGF repeats and one YWTD domain (β-propeller domain); in the tail there are visible two NPXY motifs,
two d-ileucine motifs, and one YXXL motif; every ligand binding domain (cysteine rich domain) consists of a β-hairpin structure followed
by a series of β-turns; one cysteine-rich repeat (module) is like a ball of wool; it is composed of approximately 40 AA, among them three
disulfide bonds formed by six cysteine residues; PICALM; cell membrane; endocytosis; transcytosis; length of one Aβ monomer is~120Å (12 nm); width of one Aβ monomer is ~ 30 Å (3 nm); LRP1 length ~ 50-70 nm (500-700 Å); S76, serine residue 76 on LRP1 tail;phosphorylation, attachment of the phosphoryl group to the molecule; phosphate ion, [ P O 4] 3-, P; Ab.l.c.m., abluminal cell membrane;
adaptor proteins, Fe-65, Disabled1, PSD95; GPCR, G-protein-coupled receptor; His6, membrane spanning C-terminal fragment, 85 kDa;
EGF repeats; YWTD domains. Tyr-Trp -Thr-Asp (YWTD)-repeat; LRP1, lipoprotein receptor -related protein-1; EGF, epidermal growth
factor; Aβ, amyloid beta; PICALM, phosphatidylinositol binding clathrin assembly protein; AA, amino acids; PKA, protein-kinase A;
NPXY, Asn-Pro-x-Tyr;
YXX, Tyr-x-x-Lue; PSD95, postsynaptic density protein 95.
Fig. 5nature of this conformational effect. It is evident that theapproach of positively charged Aβ to the negatively charged
ligand-binding LRP1 repeats, their mutual contact, andthe formation of tripartite salt bridges, is all the result ofelectrostatic interactions. As mentioned earlier, the optimal
electrostatic connection is possible by the changes in the
LRP1 backbone structure, which include its total shortening(32Å to 21 Å, distance between CR3 and CR4). It is evident
that this conformational change has induced the occurrenceof tension forces in the backbone structure, which propagate
along its molecular complex in both directions, especially
important for reactions in the LRP1 tail. Here it is also
important to emphasize that hydrogen bond energy varies
between 1- 40 kcal/mole, (4.184 -167.54 kJ/mole), covalentbond energy is 415 kJ (99.2 KCAL; 111 kcal/mole, 464
kJ/mole), and ionic bond 700-4,000 k J (167.30 -956.02
kcal/mole). (Energy in kilojouls is equal to the kilocalories
multiplied by 4.184) 1, 8, 14).

_ 143 _

Amyloid beta (Aβ) binding on the LRP1 receptor

Mechanical force propagation throughproteinYoung HT et al. 15) in their study emphasize thatmechanical forces are the major factors among regulatorsof biological functions even at the microscopic level,such as proteins. These forces are connected with protein
conformational changes, which on the level of ligand-binding
repeats, are the source of bidirectional signal propagation,
especially in the form of molecular movements.
Like Young HT et al. 15), a number of other researchers
have presented the results of their investigations of themechanical force propagation through biomolecularcomplexes. By experiments, simulations and theory, they
have obtained the fundamental characteristics of the dynamic
response of a protein complex molecular system to mechanicalforces, among them pulling and stretching. All the researchersemphasize the great importance of steered molecular dynamics(SMD) simulations for revealing force propagation pathways
through a mechanically ultrastable multi-domain cellulosome
protein complex. They also add the great importance to theatomistic molecular dynamics (MD) simulations which isnecessary for the understanding of protein dynamics 15-20).
The mentioned distance shortening between neighboring
CR repeats practically induces the movement of moleculeson both sides of the CR2627 (5.5 Å on both sides), which ismanifested on the abluminal cell membrane side, with theevident pulling of LRP1 transmembrane portion and also of
the tail region (5.5 Å). Based the assumption that the receptor
COOH region is tightly fixed to the local tissue structures(ECM), and YXXL motif is practically only connectedwith the distal dileucine motif (which is movable), it isevident that, the until now blocked PICALM, has now a
lot of free space to come in contact with YXXL motif,
and therefore induces the adaptor protein complex (AP-2)
and scaffold recruitment, and enables the clathrin-coatedtranscytosis. It is possible that the NH 2 end of the LRP1 isalso fixed. It is evident that for the explanation of these events
there are no other possible mechanisms. It can be supposedthat during the pulling energy transmission from the pointof its origin to the tail, there is no evident loss of energy(Fig. 4, 5 ) 1, 3, 20-25).
Schoeler C et al. 16), by the employment of single-molecule
spectroscopy with an atomic force microscope (AFM) andsteered molecular dynamics (SMD) simulations, have revealed
force propagation pathways along a mechanically ultrastable
multi-domain cellulosome protein complex. Cellulosomes
are multi-enzyme extracellular complexes, associated withthe cell surface and they mediate cell attachment to insoluble
substrates. According to the authors, mechanical forces havea crucial role in biological systems. Mechanically active
proteins can respond to these forces by conformationalchanges. The AFM and single-molecule force spectroscopy(SMFS) makes it possible to measure directly the molecularmechanical properties. The mentioned methods can exactly
define the pulling geometry of these molecular events. In
this situation, the conventional bioinformatics approachis completely insufficient. For the exact explanation, themolecular dynamics (MD) is very important as well. SMD
simulations are also essential in studying the force propagation
through protein structures, and in this case, the force
distribution analysis ( FDA) is very useful.

Once again, the adequate re-establishment of theequilibrium between Aβ charges and LRP1 CR charges
induces the decrease (from 32 Å to 21 Å) of the interspace
between the two neighboring CR repeats. The consequenceof this decrease is the onset of intermolecular pulling in the
LRP1 backbone structure directed on both sides to the centerof the mentioned interspace. Pulling, on the side of abluminal
cell membrane, as a signal, also propagates in the direction
to the abluminal cell membrane, and after passing acrossit, arrives to the distal Di-leucine motif and YXXL motif.
The structural consequences are explained above. It is clearthat contrary to the direction of the pulling propagation as asignal, forces induced by pulling have an inverse direction to
the CR/CR interspace. It is evident that in these events theintermolecular attractive forces have a crucial role 15-17).

LRP1 phosphorylation by protein kinase
A (PKA)
L i Y et al. 26) point out that the serine 76 is the major
phosphorylation site within the LRP tail. Serine 73 and serine
79 most probably function as the docking site of the kinase
and are the part of the phosphorylation motif of LRP tail. The
results of their experiments indicate that LRP phosphorylation
is predominantly mediated by the cAMP-dependent protein
kinase A (PKA) on the serine 76 on its tail. They alsoemphasize that LRP/ligand endocytosis is primarily inducedby the YXXL and the distal di-leucine motifs, and thatthe two NPXY motifs and the proximal di-leucine motif
are not included in the initial endocytosis. The binding ofcertain LRP ligands (possibly Aβ) induces dissociation of
the stimulatory heterotrimeric G-protein subunit (Gsα) withconsequent activation of the adenylate cyclase and a rise in
intracellular cAMP. The result is an enhanced PKA activity
and LRP phosphorylation (Fig. 5, 6 ) 27).
Goretzki L et al. 28), at the beginning of their exposition,
specify that LRP-mediated ligand internalization is dependent
on cAMP-dependent protein kinase (PKA) activity. Ligand
binding to the LRP1 cluster II and IV induces conformationalchanges on its cytoplasmic tail characterized by the YXXL
motif activation and the pulling of two NPXY and two dileucine motifs in the direction to the clusters. These changes
induce the dissociation of heterotrimeric G-protein Gs alphasubunit (one of three heterotrimeric G-protein units), which
promptly activates enzyme adenyl cyclase (transmembraneprotein), and consequent conversion of adenosine triphosphate(ATP) to 3, 5-cyclic adenosine monophosphate (cAMP). This
reaction raises the intracellular cAMP concentration. PKA
becomes activated and phosphorylates the LRP1 tail on Ser76.
Phosphorylation of Ser76 elevates the interaction between
the tail and adaptor proteins such as Dab-1 (Fig. 5, 6 ).
It is possible to suppose that the pulling of the LRP1 tailchanges the direction of di-leucine – YXXL motif connection.
This change enables the PICALM approach to the YXXL
motif and its activation. Activated YXXL motif activates
PKA. The mentioned change at the same time induces thebreaking of the connection of the Gα subunit from GPCR,
and its dissociation and activation. This activation induces
the activation of adenyl cyclase, the transition of ATP in
cAMP, and PKA activation (Fig. 5, 6 ) 29) .

_ 14 4 _Glycative Stress Research

A/ G protein-coupled receptor (GPCR)
activation by agonists;

B/ Aβ binding with LRP1; conformationalchanges, both, in ligand-binding
cluster II and IV and tail of LRP1;
contact of the LRP1 tail withheterotrimeric G-protein;

Conformational changes in heterotrimeric G protein complex;

Gs alpha subunit: exchange of GDP to GTP and separation from the βγ subunits;Gs alpha subunit in turn activates adenyl cyclase, which quickly converts ATP intocAMP (enzymatic catalysis); ATP

3,5-cyclic AMP (cAMP);

cAMP concentration in the intracellular space rises;

protein kinase A (PKA) activation;

Serine 76 (Ser76) on the LRP1 tail phosphorylation;

Phosphorylation of adaptor proteins (PSD-95, Fe-65, Disabled 1);

Activation of LRP1 endocytosis and transcytosis;

Fig. 6. The role of GPCR receptor, conversion of ATP into cAMP, and PKA activation.

LRP1; adaptor proteins (PSD-95, Fe-65, Disabled 1); ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate,second messenger included in many biological processes; GPCR, G-protein coupled receptor, transmembrane receptor, has
three intracellular units (α, β, γ); PKA, protein kinase A, a family of enzymes, its activity depends on cellular levels of)LJ
cAMP, cAMP dependent protein kinase, its α-subunit binds to adenyl cyclase which converts ATP into cAMP, importantfunction in LRP1 endocytosis and transcytosis. LRP1, lipoprotein receptor-related protein-1; PSD-95, postsynaptic density
protein 95.

_ 145 _

Amyloid beta (Aβ) binding on the LRP1 receptor

LRP1 Ser 76 by PKA phosphorylation is crucial for
adaptor protein activation and consequent Aβ endothelialtranscytosis and clearance 30- 33).
This study emphasizes the model that implies the relation
between the Aβ/LRP1 binding and LRP1 conformationalchanges on the LRP1 tail, and clathrin dependent Aβ
endocytosis and transcytosis. It is necessary that appropriate
experiments and expert analyses confirm or reject thisconcept. Regardless of the concept evaluation, this study
has certainly contributed to the better understanding of
the Alzeheimer's disease pathophysiology, and presents avaluable attempt in the solving of this contemporary society
serious problem.

Conclusion
The importance of Alzheimer's disease, this severe,chronic, and lethal neurodegenerative disease, is continually
becoming an increasing medical and social problem. Thisis due to the relative and absolute growth of the worldwide
elderly population and the alarming aspect of its incidence
and prevalence. The exact explanation of its complex
polygenetic etiology and effective therapy has not yet beenobtained. However, a lot of researchers emphasize the crucial
importance of intracerebral amyloid beta (Aβ) accumulationand its damaged drainage from the brain. They especiallyemphasize the important role of lipoprotein receptorrelated protein1 (LRP 1) in these disturbed events. A numberof complex experiments confirm that Aβ binding to the
LRP1 ligand-binding cluster II and IV are the trigger for
conformational changes in the receptor's extracellular heavy
α-chain which induce the activation of its tail structures
and the present molecules. This activation is the inductorof the PICALM/clathrin Aβ endocytosis, transcytosis, andexocytosis into the capillary circulation and to the points ofAβ degradation. The important actual problem is how thebinding of Aβ to LRP1 induces the conformational changes
in the tail and the consequent beginning of the drainage.
Experiments emphasize that in these events the mechanicalforces, especially pulling forces, have the crucial role in theenergy transmission through the protein structure, and in theactivation of the tail complex essential for Aβ drainage.

Conflict of interest
The author declares no conflict of interest in this study.

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