From the Department of Biochemistry and Molecular Biology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612-4316
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ABSTRACT |
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The low density lipoprotein receptor-related
protein is a member of the low density lipoprotein receptor family and
contains clusters of cysteine-rich complement-like repeats of about 42 residues that are present in all members of this family of receptors. These clusters are thought to be the principal binding sites for protein ligands. We have expressed one complement-like repeat, CR8,
from the cluster in lipoprotein receptor-related protein that binds
certain proteinase inhibitor-proteinase complexes and used
three-dimensional NMR on the
13C/15N-labeled protein to determine the
structure in solution of the calcium-bound form. The structure is very
similar in overall fold to repeat 5 from the low density lipoprotein
receptor (LB5), with backbone root mean square deviation of 1.5 Å. The
calcium-binding site also appears to be homologous, with four carboxyl
and two backbone carbonyl ligands. However, differences in primary
structure are such that equivalent surfaces that might represent the
binding interfaces are very different from one another, indicating that different domains will have very different ligand specificities.
The low density lipoprotein receptor-related protein
(LRP)1 is a member of the low
density lipoprotein receptor family (1). Members of this family of
receptors contain a small cytoplasmic domain, a single
membrane-spanning helix, and clusters of two types of small
cysteine-rich repeats on the extracellular side, interspersed with
regions that contain a YWTD motif. The two types of cysteine-rich
repeat are an epidermal growth factor-like repeat of about 50 residues
and a complement-like repeat, so named for its presence in complement
components C8 and C9, of about 42 residues. Each of these types of
repeat contains 6 cysteines that are involved in three intradomain
disulfide bonds. The limited evidence on the location of protein
ligand-binding sites indicates that such sites are restricted largely
to the clusters of complement-like repeats. The LDL receptor contains
one cluster of seven such repeats and binds various apolipoproteins,
including apoE and apoB100 (2). LRP contains a total of 31 such repeats
organized into four clusters of 2, 8, 10, and 11 repeats (3). These
clusters have been designated clusters I, II, III, and IV,
respectively. LRP binds a much wider range of protein ligands including
apolipoproteins, serpin-proteinase complexes, and
Although structures of three complement-like repeats have been reported
from the LDL receptor, one by x-ray crystallography (5) and two by NMR
spectroscopy (6, 7), no structure of such a repeat has previously been
reported from LRP. Both because of the sequence differences between
repeats in regions that might form the ligand-binding sites and also
because of the more extensive range of protein ligands that LRP can
bind compared with the LDL receptor, it is important to make structural
comparisons between repeats from different receptors and between
repeats from the same receptor to understand differences in ligand and
calcium specificity (Fig. 1). We report here the first structure of a complement-like repeat from LRP, CR8. CR8 is the eighth complement-like repeat from the N terminus and is the sixth repeat in cluster II. In
overall fold CR8 is very similar to LB5, which in turn is similar to
the folds of two other domains from the LDL receptor for which NMR
structures are known, LB1 (6) and LB2 (7). However, because only 4 of
25 residues are identical between these four domains (excluding
cysteines and calcium coordinating residues), 1) each domain presents very different
charge densities and hydrophobic patches on its surface, suggesting
that each domain will be capable of very different interactions with
protein ligands and thus have its own distinct ligand specificity.
Cloning, Expression, and Folding of CR8--
CR8 was cloned as
described previously (8). 13C/15N-labeled CR8
was expressed as a GST fusion protein in minimal medium containing 0.6% basal medium Eagle vitamin solution (Life Technologies, Inc.), 1 g liter NMR Spectroscopy--
NMR spectra were recorded at the
University of Illinois at Chicago on a four channel Bruker DRX600
equipped with a pulsed field gradient accessory and operating at 600.13 MHz for 1H. NMR data were processed and analyzed using
Triad 6.3 software (Tripos, Inc., St. Louis, MO). Lyophilized CR8 was
dissolved in 20 mM CD3COOD, 10%
D2O, pH 5.5, and the pH was maintained at 5.5 by addition
of NaOH. The final concentration of protein was approximately 2 mM. Spectra were recorded at 298 K. Resonance assignments
and distance constraints were determined using HBHA(CO)NH (11), CBCA(CO)NH (12), TOCSY-HSQC (13), 15N-NOESY-HSQC (13),
HCCH-TOCSY (14), and 13C-NOESY-HSQC and HNHA (15)
experiments. HNCA (16) was used for the backbone sequential assignment
through Structure Determination--
With resonance assignments in hand,
NOEs for backbone-backbone, backbone-side chain, and side chain-side
chain were identified and classified as small, medium, or large. For
backbone-backbone NOEs, upper bounds of 5.0, 4.0, and 3.0 Å,
respectively, were used for these three classes. For backbone-side
chain and side chain-side chain NOEs, upper bounds of 5.5, 4.5, and 3.5 Å, respectively, were used for these classes. These values were also
the base values used to make pseudo-atom corrections where
stereospecific assignments were not available. These distance
constraints, as well as torsion angle constraints derived from HNHA
measurements of 3JHNH
Energy minimization used the package AMBER (20) and was carried out for
steps up to a maximum of 0.5 ps, except for the first step, which was
for 0.01 ps. Force constants of 32 kcal mol Materials--
Plasmid pGEX-2T(XbaI) was a kind gift
from Dr. Robert Costa (University of Illinois at Chicago). Sepharose
4B, Sephadex G-50 F and Mono Q (HR5/5) were from Amersham Pharmacia
Biotech. Terbium peroxide was from Sigma. It was dissolved in
concentrated HCl to give the trichloride. 99.997% CaCl2
was from Johnson Matthey, Ltd.
(15NH4)2SO4 and
13C glucose were from Cambridge Isotope
Laboratories, Inc.
Assignments--
We have previously shown qualitatively that CR8,
the eighth complement-like repeat from LRP, only adopts a well defined
structure in the presence of calcium (8), which is consistent with the requirement for calcium for tight binding of protein ligands by both
LRP and the closely related LDL receptor. The assignments and structure
determination reported here are therefore for the calcium complex,
obtained in the presence of a saturating level of calcium. All NMR
assignments of CR8 were carried out on a uniformly 13C,15N-labeled or 15N-labeled
44-residue construct prepared as described above. This molecule
contains 42 residues (3-44) from LRP (residues 1040-1081) with an
additional N-terminal Gly-Ser that resulted from the restriction site
present in the vector used to create the GST-CR8 fusion protein DNA
construct. The calcium complex was well behaved and gave a single set
of resonances, consistent with a single well defined conformation.
Despite the small size of the molecule, there was good spectral
dispersion, which is illustrated by a 13C-decoupled
[15N-1H]-HSQC NMR spectrum (Fig.
2). Backbone amides had a 1H
chemical shift range of 3.5 ppm, (from 6.73 ppm for Lys39
to 10.23 ppm for Asp31) and a 15N chemical
shift range of 21ppm (from 107.7 ppm for Gly5 to 128.8 ppm
for Asp31). Assignment of backbone atoms (amide
15N and 1H, C
NOE constraints (Fig. 3) were obtained
from the following two experiments: three-dimensional
15N-edited NOESY-HSQC with a mixing time of 120 ms and a
three-dimensional 1H-13C-correlated NOESY-HSQC
in D2O with a mixing time of 120 ms. Only three i, i+3
d Structure Calculation--
Structural calculations for
Ca2+-CR8 were performed using the torsion angle dynamics
annealing simulation program DYANA, with a total of 545 NOE upper
distance constraints, 31 torsion angle constraints, three disulfide
bonds, and 6 hydrogen bonds as input (Table
I). The NOEs consisted of 208 intraresidue, 160 sequential, 74 medium range (1< i-j <5), and 103 long range interactions. The composition of the three disulfides was
based on the previously determined pattern of disulfides in the
homologous LB1 (19) and LB5 (5) domains from the LDL receptor.
Disulfides were accordingly specified for the cysteine pairs 6-19,
13-32, and 26-41.
The final 40 best conformers produced by DYANA from an input of 100 initial structures were subject to energy minimization using the
all-atom potential program AMBER 5.0 (20, 21). All NMR constraints were
employed in the energy minimization. Energy minimization calculations
were carried out both without and with calcium specifically
coordinated. Because the four carboxylates and two backbone carbonyls
that coordinate to calcium in LB5 are all present in CR8 in equivalent
positions (carboxylates of Asp27, Asp31,
Asp37, and Glu38 and carbonyls of
Trp24 and Asp29 using CR8 numbering), similar
ligation was used for CR8. Evidence concerning the coordination of the
two backbone carbonyls was provided from 13C carbonyl
chemical shifts of the tryptophan and amide 15N chemical
shifts of the residues immediately C-terminal to Trp24 and
Asp29. Thus the tryptophan carbonyl was strongly downfield
shifted relative to the random coil value (177.9 ppm compared with
173.6 ppm, respectively) (22, 23), and the amide nitrogens of
Arg25 and Thr30 were also strongly downfield
shifted about 7 ppm relative to random coil values (observed values of
127.3 and 121.0 ppm compared with random coil values of 120.8 and 114.2 ppm respectively (22)). The chemical shift of the carbonyl of
Asp29 was less definitive, being close to the expected
random coil value. However, the large perturbation of the amide
nitrogen of the adjacent threonine makes it likely that calcium does
coordinate to the Asp29 carbonyl.
Although the number of distance and angle violations did not differ
very much between calculations carried out in the absence or presence
of calcium coordination (Table I), the overall energy of the final
structures obtained in the presence of calcium (mean of Final Structures--
The final set of 20 best structures (Fig. 4)
and mean structure (Fig. 5) can be roughly described as formed from a
Greek
Most of the structure is very well defined, with rmsd for the backbone
of 0.52 Å for residues 6 to 41 (Table I). This increases to 1.34 Å when all heavy atoms are included for this region. The N-terminal 5 residues and C termini 3 residues are only poorly defined and so cause
large increases in rmsd for both backbone (2.10 Å) and heavy atoms
(2.44 Å). This is not surprising because both ends act as linkers to
the next domains in intact LRP and presumably must show some
conformational flexibility. In addition residues 1 and 2 (Gly and Ser,
respectively) are not part of CR8, being present as a consequence of
the means of expressing and purifying the domain.
Detailed inspection of the best low energy structures (Figs. 4 and 5)
shows that residues His7-Glu10 form a
The calcium-binding site is composed entirely of ligands from the
right-hand loop of the structure (Fig.
6). Because the front and back parts of
the loop do not form a Comparison of the Structures of CR8 and LB5--
In the region
between the first and last of the six cysteines, LB5 is one residue
shorter than CR8 (Fig. 1): an arginine inserted at position 14. Despite
this insertion, there is overall a very high degree of structural
similarity between the two proteins (Fig.
7 and Table
II). Thus the rmsd for the backbone of
the whole structure, excluding residues beyond the end cysteines
(residues 6-41) is 1.45 or 1.34 Å, depending on whether comparison is
made with the lowest energy CR8 structure or the mean structure,
respectively. If the left- and right-hand loops are considered
separately, these comparisons further improve substantially. The
left-hand " Comparison of the Surfaces of CR8, LB5, LB1, and LB2--
Two
different proposals have been made to explain the binding of protein
ligands to complement-like repeats of proteins of the LDL receptor
family. One is that clusters of basic residues on the ligand interact
with acidic residues on the receptor (2), recognizing the four acidic
residues that are almost completely conserved in all of these repeats
and that a cluster of basic residues has been shown to be required for
binding of apoE to the LDL receptor (2). The second is that hydrophobic
interactions play a more important role (5), because most of the
conserved acidic residues present in the C-terminal parts of these
domains are involved in calcium binding and would therefore not be
available to bind basic residues on the protein ligands. Both of these
proposals are perhaps too one-sided and assume a common binding mode
for all ligands to receptors from this family. Instead, it should be
recognized that because most residues in these repeats that are neither
cysteines nor putative calcium ligands will be on the surface of these
small domains, the high variability of sequence between different
domains will result in unique surfaces for each domain and
corresponding unique ligand specificities. This is well illustrated by
a comparison of the face of LB5 proposed to be a ligand-binding face
(5) with the equivalent face of CR8 (Fig.
8). Very different charge densities are
found in these two domains. These arise from substantial differences in
residues that occur on this face. The most notable differences are as
follows, using the numbering for each domain given in Fig. 1:
Phe8 in LB5 is Asp9 in CR8; Arg14
in CR8 is an insertion; Ser21 of LB5 is Arg23
in CR8; Glu37 of LB5 is Lys39 in CR8; and
Ala40 in LB5 is Glu42 in CR8. These represent
changes from acidic to basic, from polar to positively charged, from
hydrophobic to negatively charged, and, for Arg14 of CR8,
an addition of a positive residue. CR8 is not atypical in regard to
these differences between pairs of complement-like domains. There are
thus major differences in composition of equivalent residues in two
other domains of known structure from the LDL receptor, LB1 and LB2
(Table III), and differences in the
resulting contour surface and charge density (Fig. 8), again despite
very similar backbone folds. This further emphasizes that from the point of view of understanding receptor-ligand interactions involving this family, the structure of each interacting domain must be specifically considered and therefore be available by x-ray and/or NMR
means.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
2-macroglobulin-proteinase complexes (1). Studies on
truncated LRP species suggest that cluster II is a major locus for
protein ligand binding (4).
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Fig. 1.
Primary structure of CR8 and alignment with
primary structure of LB5 from the LDL receptor, for which an x-ray
structure of the calcium complex has been determined (5). The
first two residues of the CR8 construct (GS) are not present in LRP and
were introduced as part of the restriction site in the GST-CR8 fusion
protein. Alignment was made at the six conserved cysteines. The 6 residues shown in bold are those that coordinate to the
calcium in LB5 either through backbone carbonyls (W and G) or through
carboxyl side chains. The numbering for LB5 is that used by Fass
et al. (5).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1
(15NH4)2SO4, and 2 g liter
1 13C-labeled glucose. Optimal yield was achieved
by harvesting the cells 6 h after induction. GST-complement repeat
fusion protein was purified according to Ref. 9, cleaved with thrombin
(1/1000 w/w, 5-10 min at 20 °C) and rechromatographed on
GSH-Sepharose. Refolding was performed as described. Correct folding
was indicated by the ability of the protein to bind calcium tightly, as
shown by tryptophan fluorescence change, and to give sensitized terbium fluorescence emission when terbium was bound (8). Protein
concentrations were determined spectrophotometrically using an
extinction coefficient of 5500 M
1
cm
1 estimated from the amino acid composition (10).
-carbon. Some ambiguities in sequential connectivities of
backbone C
atoms were resolved on the basis of H
and H
chemical shift comparisons between HBHA(CO)NH and TOCSY-HSQC. From
these three triple resonance experiments, it was possible to complete
the sequential assignments. TOCSY-HSQC and HCCH-TOCSY were used for
side chain proton chemical shift assignment. HBHA(CO)NH was used to
distinguish
-protons and
-protons when their proton chemical
shifts were very close. The ambiguities in H
and H
assignment
(threonine and serine) were resolved by their connected carbon using
HCCH-TOCSY. HCCH-TOCSY and 13C-NOESY-HSQC were also used
for side chain carbon assignments from known proton assignment. NOE
constraints were obtained from 15N-NOESY-HSQC and
13C-NOESY-HSQC. Dihedral angle constraints were obtained
from an HNHA experiment. HSQC spectra of a sample of CR8 freshly
prepared in D2O were recorded to identify slowly exchanging
amide protons and hydrogen bonds. Identification of backbone carbonyls
involved in coordination to the calcium was made using a
two-dimensional HACACO to permit assignment of backbone and side chain
carbonyl chemical shifts. A two-dimensional
[15N-1H] HSQC experiment was also carried out
on the doubly labeled sample, with 13C decoupling at CO and
C
frequencies. Sensitivity enhancement gradient pulse
sequences were employed for all experiments in which magnetization was
detected on the amide proton. Center frequencies were 4.70 ppm for
1H, 118 ppm for 15N, 55 ppm for
13C
, 43 ppm for
13C
, and 177 ppm for carbonyl.
coupling
constants and chemical shift index-based assignment of secondary
structure, were used as input for the torsion angle dynamics annealing
simulation program DYANA (17). Three disulfide bonds between cysteines
were used as additional constraints before annealing. Six hydrogen
bonds derived from proton exchange HSQC experiment were also included
in the calculation.
1
A
2 for distance constraints and 50 kcal
mol
1 radian
2 for dihedral angle constraints
were employed. The constraint energy given in Table I is a measure of
the mean energy violation, based on these force constants and using the
formula: constraint energy = force constant - (current interatom separation
target separation)2. Comparisons of the structures of CR8 and
LB5 and graphics representations of structures of CR8, LB5, LB1, and
LB2 were made with MOLMOL (18).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
13C
and 1H) was made with the aid of triple resonance
experiments. Starting from distinctive residues such as glycine, small
sequence elements were determined through sequential connectivities
using triple resonance experiments. Experiments used in this work
include HNCA, HBHA(CO)NH and N-15 edited TOCSY-HSQC. From these
experiments, the identities of individual amino acids were tentatively
determined from their C
, H
, and H
chemical shifts, and these
small sequence elements were then further assigned to areas in the
protein by comparison with the known sequence. The C-terminal amino
acid triplet Glu-Gly-Val C
sequential assignment was easily made
from an HNCA experiment, because Gly-Val is a unique pair in the CR8 sequence. This assignment was independently confirmed by comparison of
H
and H
between HBHA(CO)NH and TOCSY-HSQC experiments. The sequential connectivities from the C-terminal valine through to Pro21 were determined by HNCA and, where C
was degenerate, by HBHA(CO)NH and TOCSY-HSQC. Cys19 H
has the highest H
chemical shift (5.24ppm). This was also used as a
starting point for sequential assignment by comparison of HBHA(CO)NH
and TOCSY-HSQC spectra. The same strategy as described above for the
C-terminal half of the molecule was used for the sequential assignment
from Ile20 through to Pro3. Sequential
assignments were checked against both amide-amide and side chain-amide
NOE cross-peaks. Three-dimensional 15N-edited TOCSY-HSQC
and three-dimensional 13C-edited HCCH-TOCSY were used for
other proton and carbon side chain assignments. Some ambiguities of
proton assignment were resolved by HBHA(CO)NH connectivities or by
their attached carbon in HCCH-TOCSY (e.g. H
and H
of
threonine). Carbon side chain assignment ambiguities were resolved by
13C-HCCH-NOESY. Some aromatic proton assignment were made
from two NOESY experiments. A two-dimensional experiment, HA(CA)CO, was used for side chain and backbone carbonyl assignments to unambiguously assign seven backbone carbonyls, including the backbone carbonyls of
residues 24 (Trp in both CR8 and LB5) and 29 (Asp in CR8 but Gly in
LB5), which are the two residues that have been shown by x-ray
crystallography to contribute carbonyl ligands to the bound calcium in
a repeat from the LDL receptor (5). A total of 419 assignments were
made, consisting of all backbone amide and C
atoms,
except for the C
13C for residues
Gly1 and Ser2 and the two proline
15N resonances, 7 backbone carbonyl carbons, 73 side chain
carbons, and 158 side chain hydrogen atoms.
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Fig. 2.
13C-decoupled
[1H-15N] HSQC of Ca2+-CR8, with
assignments shown for all of the protonated backbone amides, as well as
the indole NH of tryptophan 24 (labeled W24NE1).
The horizontal line at 112.5 ppm connects the two peaks from
the side chain amide of Gln12. The cross-peak for
Ser2 is weak and below the contour level used. Its
location, at 8.46ppm for 1H, is indicated by an empty
box.
N NOEs were observed, between Pro21 and
Trp24, between Leu22 and Arg25, and
between Ser36 and Lys39. Dihedral angle
constraints were obtained from HNHA measurements of
3JHNH
coupling constants and
chemical shift index-based assignment of secondary structure. Six
slowly exchanging amide protons were identified from HSQC spectra of a
freshly prepared sample in D2O (Fig. 3).
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Fig. 3.
Short and medium range NOEs and slowly
exchanging amide protons indicative of stable hydrogen bonds used in
structure determination of CR8. Thin,
medium, and thick lines represent weak, medium,
and strong NOEs, respectively.
Structure statistics for energy minimized 20 best structures
362 kcal
mol
1) was very much lower than in the absence of calcium
(mean of
262 kcal mol
1) (Table I). The best 20 structures reported (Fig. 4) and the mean
structure (Fig. 5) are therefore for
structures calculated with calcium coordinated. For the 20 best
structures only 1.3 distance violations >0.2 Å were found per
structure, with no angle violations >10 ° (Table I).
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Fig. 4.
Relaxed stereo view of the 20 best structures
of CR8 obtained after energy minimization by AMBER. Statistics for
these structures are given in Table I. The structures were displayed
using MOLMOL.
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Fig. 5.
Ribbon representation of the mean structure
of CR8, based on the 20 best structures depicted in Fig. 4. The N
terminus is at left, and the C terminus is at
right. The two-stranded anti-parallel mini- -sheet is
shown in blue, and the one and a quarter turn
-helix is
shown in yellow/ochre. The calcium-binding loop is at
bottom right. The three disulfides are shown as
straight lines.
in which the top of the loop has been folded backwards and
down. This gives two loops, one on the left and one one the right. Each loop is held together by a disulfide between front and back, with a
third disulfide holding the two loops together at the top. The left-hand loop contains a small two-stranded anti-parallel
-sheet, and the left- and right-hand loops are linked at the bottom through a
short
-helix.
-turn
structure. The amide proton of residue Glu10 exchanges
slowly and thus appears to be involved in a hydrogen bond. This residue
is followed by a short two-strand anti-parallel
-sheet formed by
residues Phe11-Cys13 and residues
Leu18-Ile20. Strong sequential
d
N and cross-strand dNN, d
N,
and d
a NOEs could be observed in this
-sheet. The
amide protons of Phe11 and Ile20 also exchange
slowly and thus appear to form hydrogen bonds with each other. This
mini-sheet is followed by a one and a quarter turn
-helix from
residues Pro21-Arg25. NOE cross-peaks were
observed in 15N NOESY-HSQC between the Pro21
-proton and the Trp24 amide proton and between the
Leu22
-proton and the Arg25 amide proton.
The amide protons of residues Trp24 and Arg25
exchanged slowly, consistent with hydrogen bond formation to the
carbonyls of Pro21 and Leu22, respectively.
Residues Phe11 and Ile20, which are strongly
conserved in LRP repeats and also present in LB5 from LDL receptor,
form a small hydrophobic core together with Leu18 and
Leu22.
-sheet with one another, which might
stabilize the structure (in contrast to the situation in the left-hand
loop of the domain (Fig. 5)), it is understandable why calcium binding
serves to rigidify the conformation of this and other complement-like
domains (5, 8, 24). Thus, the calcium bridges the front and back parts
of the loop (Asp37 and Glu38 in front and
Asp27, Asp29, and Asp31 in the
back) and holds them together and, at the same time, tethers the loop
to the
-helix that links the left and right loops, through the
carbonyl of Trp24. The simultaneous neutralization by the
positively charged calcium of multiple negatively charged residues,
which would otherwise repel one another, would further help to
stabilize this single structure. A potentially significant difference
between LB5 and CR8 is that the glycine that coordinates the calcium in
LB5, through its carbonyl, is an aspartate in CR8. Although chemical
shift evidence strongly suggests that Asp29 also
coordinates calcium through its carbonyl, it is also possible that the
aspartate carboxyl could act as a calcium ligand, either in addition to
the other four carboxyls or in place of one of them. Energy
minimizations carried out with this side chain as a seventh ligand
resulted in no significant improvement in the energy of the structure
(data not shown), so this must remain speculative.
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Fig. 6.
Comparison of the calcium coordination sites
of CR8 and LB5. The views are the same as that in Fig. 5. The
coordinates for the LB5 representation were obtained from Brookhaven
Protein Data Bank, from deposition 1ajj. Because the coordinates of
Ca2+ are not provided in 1ajj, the position was inferred
from the published ligand-calcium separations.
-sheet loop" (residues 6-23) shows 1.20 Å rmsd
between the two structures, and the calcium-binding loop (residues
24-41) shows even smaller rmsd of 0.90 Å. This high degree of
similarity is despite very high variability of the primary structure,
when cysteines and calcium coordinating ligands are excluded (Fig. 1).
It is therefore likely that in overall fold, all of the complement-like
domains in LRP and the LDL receptor will be very similar. Given the
major differences in ligand specificity of LRP and the LDL receptor, the basis for these differences must therefore lie in the different residues that are presented on the surface.
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Fig. 7.
Structural comparison of the backbones of CR8
(dark line) and LB5 (light line)
using deposited coordinates of LB5 and the lowest energy NMR structure
of CR8 from those depicted in Fig. 4. Statistics for the
comparison are presented in Table II. A, best overlay of the
whole structures. B, best overlay of the N-terminal
-sheet-containing region. C, best overlay of the
calcium-binding region.
Comparison of the structures of CR8 and LB5
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Fig. 8.
Comparison of the proposed hydrophobic
ligand-binding face of LB5 with the structurally equivalent faces of
CR8, LB1, and LB2, showing surface contours and charge density.
The orientation of LB5 is the same as that used in Fig. 4B
of Fass et al. (5). The same orientations of CR8, LB1, and
LB2 are presented to facilitate comparison. This orientation for CR8 is
obtained from that in Figs. 4 and 5 by 180 ° rotation about the axis
perpendicular to the plane of the paper. The coordinates for LB1, LB2,
and LB5 were obtained from the Brookhaven Protein Data Bank from
depositions 1ldl, 1ldr, and 1ajj, respectively. Both to provide
landmarks and to highlight two of the prominent differences between CR8
and LB5, two residues are indicated in LB5 and the structurally
equivalent residues in CR8. Red indicates a negative charge,
and blue indicates a positive charge.
Comparison of selected residues on the hydrophobic face of LB5 with
residues in CR8, LB1, and LB2 that appear on the same face and are
structurally equivalent
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ACKNOWLEDGEMENT |
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We thank Nat Gordon for maintaining the DRX600 NMR spectrometer and for help with some NMR experiments.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM54414. The Bruker DRX600 was purchased with National Science Foundation Academic Research Infrastructure Program Grant BIR-9601705 and funds from the University of Illinois at Chicago.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and structure factors (code 1cr8) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
Supported by a postdoctoral fellowship from the Danish Natural
Science Research Council.
§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, M/C 536, University of Illinois at Chicago, 1853 West Polk St., Chicago, IL 60612-4316. Tel.: 312-996-5534; Fax: 312-413-8769; E-mail: pgettins{at}tigger.cc.uic.edu.
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ABBREVIATIONS |
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The abbreviations used are: LRP, low density lipoprotein receptor-related protein; LDL, low density lipoprotein; GST, glutathione S-transferase; HSQC, heteronuclear single quantum coherence; rmsd, root mean square deviation; TOCSY, total correlation spectroscopy; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy.
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