Download Comparison of Ligand-Binding Sites of Modeled Apo[a] Kringle

758
Comparison of Ligand-Binding Sites of
Modeled Apo[a] Kringle-like Sequences
in Human Lipoprotein [a]
Juan Guevara Jr., Amy Y. Jan, Roger Knapp, Alexander Tulinsky, and Joel D. Morrisett
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Human lipoprotein [a] contains at least two high-molecular-weight, disulfide-linked apolipoproteins,
apo[a] and apo B-100. Apo[a] is a highly glycosylated, hydrophilic apoprotein that somewhat resembles
plasminogen by containing an extended kringle domain and a carboxyl-terminal serine protease domain.
The apo [a] kringle domain is composed of 11 distinct kringle types. Ten of these display high sequence
homology to plasminogen kringle 4 (PGK4). The crystallographic coordinates for PGK4 were used to
generate three-dimensional molecular models of the apo[a] kringle types, and the lysine-binding region
of PGK4 was used to compare the different potential receptor-ligand and ligand-binding sites contained
in each different PGK4-like kringle of apo [a]. A receptor-ligand site can be proposed for each kringle
type. Potential serine protease cleavage sites, containing arginine-threonine and threonine-arginine, are
located on the surface of the kringles. The ligand-binding site of one apo [a] kringle model is almost
identical to that of PGK4 and may be a lysine-binding site of apo [a]. Four other apo [a] kringle models
appear to have structurally similar lysine-binding sites, but with differences that may influence
ligand-polypeptide specificity. Five apofa] kringle models have ligand-binding sites that probably do not
bind lysine; one of these is the highly repeated kringle in the known apo [a] polymorph. (Arteriosclerosis
and Thrombosis 1993;13:758-770)
KEYWORDS • apolipoprotein[a] • plasminogen kringle 4 • ligand-binding sites
E
levated plasma levels of lipoprotein [a] (Lp[a]) in
humans correlate with the incidence of coronary and cerebral artery disease.1"4 However,
little is known about the mechanism(s) by which Lp[a]
leads to atherosclerosis or whether an elevated level of
plasma Lp[a] is a cause or a result of atherosclerosis.
Lp[a] contains two major and distinctly different highmolecular-weight apoproteins.5-7 Within Lp[a], apoprotein[a], apo[a], a highly glycosylated, hydrophilic protein
with little apparent affinity for lipid, is disulfide-linked
to one molecule of apo B-100,68-13 a highly hydrophobic
apolipoprotein. Apo B-100 is partly embedded in the
lipid-rich, pseudomicellar Lp[a] particle. The primary
structure of a single apo[a] polymorph inferred from
cDNA sequencing14 suggests a polypeptide mass of
about 530 kD. Apo[a] contains two distinct domains: a
kringle-containing part and a serine protease domain.
For the 530-kD polymorph, there are 38 repeated
From the Departments of Medicine (J.G.Jr., A.Y.J., R.K.,
J.D.M.) and Biochemistry (J.D.M.), Baylor College of Medicine,
Houston, Tex., and the Department of Chemistry (A.T.), Michigan State University, East Lansing, Mich.
Supported in part by National Institutes of Health (NIH) grants
HL-32971 to J.D.M., 2S07RR-05425-29 to J.G.Jr, and HL-25942
to A.T. and The W.M. Keck Center for Computational Biology.
Some molecular modeling was performed using facilities of the
Molecular Biology Information Resource, Department of Cell
Biology, and 3DEM Resource Center (NIH grant RR-02250),
Department of Biochemistry, Baylor College of Medicine.
Address for correspondence: Dr. Joel D. Morrisett, The Methodist Hospital, MS A601, 6565 Fannin Street, Houston, TX 77030.
Received September 14, 1992; revision accepted February 12,
1993.
sequences in the kringle domain, and these occur as 11
different kringle types.15 The segments between kringles
have short sequences in common with intercellular
adhesion molecules16-17 and with the class of active sites
of serine protease inhibitors known as serpins.1819
In apo[a], kringle types 1-10 are similar but not
identical in sequence to plasminogen kringle 4 (PGK4);
primary structural homologies range between 78% and
88% (Figure 1). The apo[a] kringle type 2 sequence
(LPaK2) is serially repeated 28 times. A kringle is a
highly conserved, triloop polypeptide structure stabilized by three disulfide bridges; its function is not
catalytic but rather involves recognition and ligand
binding.2021 Other proteins such as prothrombin, tissuetype plasminogen activator (t-PA), urokinase, factor
XII, and hepatocyte growth factor also contain kringlelike sequences.22-23 There has been much speculation
about the potential of apo[a] kringles to confer on Lp[a]
the capability to compete with plasminogen for binding
the lysine residues of fibrin, thereby interfering with the
fibrinolytic process and leading to thrombosis and
atherosclerosis.24-29
We have studied the structure of apo[a] kringle
sequences to gain new insight into the possible positive
physiological role(s) played by the Lp[a] particle and to
help identify the mechanism(s) by which this lipoprotein may express its atherogenicity. The PGK4 and
prothrombin kringle 1 structures elucidated by x-ray
crystallography show conservation of the overall molecular structure,30"36 while kringle 2 of t-PA shows some
small but significant differences.37-38 These observations
provide a sound basis for using the three-dimensional
FIGURE 2. (Left) Comparison of the amino acid residues composing the ligand-bi
of PGK4 and apo[a] kringle sequences (LPaKl-LPaKlO). Conserved amino a
cyan; conservative substitutions are in green; nonconservative substitutions are in
Arg35, shown in purple, is a conservative change that is consistent in all PGK4-like
in apo[a]. Residues 31-35 and 69 define the cationic region of the PGK4 ligand-b
The residue is conserved in position 35. The anionic region in PGK4 is defined
53-57, with Asp56 as the important negatively charged side group. This feature is
in LPaKlO, and a conservative substitution to glutamate maintains the chara
LPaKS, LPaK6, LPaK7, and LPaK8. In PGK4, Trp60 and Trp70 form the h
trough region of the ligand-binding site. LPaKZ is the highly repeated kringle s
apo[a].
FIGURE 1. (Above) Comparison of apo[a] kringle-like sequences (LPaKl-LPa
the 78-amino acid sequence of PGK4. This comparison is based on overal
homology between the apo[a] sequences and PGK4. LPaKlO has a high degr
sequence similarity (88%) and a high degree of amino acid homology at i
ligand-binding site. Residues depicted in green are conservative substitutions, while t
in white are nonconservative amino acid changes. Six changes are shown in pur
which are conserved in all apo[a] kringle-like sequences, i.e., Vall7,Arg20, Thr21,A
Thr76. Twenty-eight copies ofLPaK2 are present in the single apo[a] polymorph inf
cDNA sequencing.u This apo[a] polymorph contains 37 PGK4-like repeat sequenc
PGK5-like sequence.
CVH6HGQSYRGTFSTTVTGRTCQSUSSMTPHRHQRTPENYPNDGLTMNVCRNPDADTGPUCFTHDPS1RUEVCNL
CYHQDGRSYRGISSTTyTGRTCQSUSSHXPHUHQRTPENYPNAGLTENYCRNPDSaKOPUCVTTDPCVRUEVCNL
CVHQDGQSYRG FSTTWTG RTCQSUSSMTPHUHQRTTEVYPN8GLTRNYCRNPDA EIRPUC VTHDPSVRUE YC
CVgggGQSVRGTLSTT I TGRTCQSUSSMTPHUHRRIPLVYPNAGLTRNYCRNPDA EIRPUC THDPSVRWEYC
CYH8DGQSVRG FSTTWTG RTCQSUSSHTPHUHQRTTEVVPN8GLTRNVCRNPDA »"ISPUC - TMDPHVRUEYC
CVVHVGQSVRGTVSTTWTGRTCQAUSSMTPHQHSRTPENYPNAGLTRNYCRNPDt
IR UC THDPSVRUEYCN
CYM6HGQSVRGTVSTTWTGRTCQAUSSMTPHSHSRTPEVYPNAGLIMNYCRNPDI VAA VC TRDP8VRUEVCN
CYH8MGQSYRGTVSTTWTGRTCQAUSSHTPHQHNRTTENYPNAGLIMNVCRNPD1 VAA VCVTRDP8VRUEVCN
CYH6NGQSVRGTVSTTVTGRTCQAUSSMTPHSHSRTPEVVPNAGLIMNVCRNPDPVAA VC "TRDPSURUEVC
CYH6NGQSVOGTVFITVTGRTCQAUSSMTPHSHSRTPAVYPNA6LIKNYCRNPDPVAA
UCVTTDPSVRUEVCH
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Arteriosclerosis and Thrombosis
TABLE 1.
Kringle
model
PGK4
LPaKl
LPaK2
LPaK3
LPaK4
LPaK5
LPaK6
LPaK7
LPaK8
LPaK9
LPaKlO
Vol 13, No 5 May 1993
Summary of Potential Energy Values (kcal) for Models of Apoprotein[a] Kringles
Bond
Angle
Dihedral
Improper
24
25
27
29
29
29
29
27
28
32
29
124
122
125
135
121
128
121
123
127
125
132
226
240
236
249
246
248
225
236
230
236
251
23
23
22
23
23
24
22
23
25
22
25
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crystallographic structure of PGK4 in evaluating the
putative ligand-binding sites of the apo[a] kringle-like
sequences. Thus, the coordinates of PGK435 were used
to generate molecular graphic models of the apo[a]
PGK4-like kringles. In PGK4, the ligand-binding site,
which has both dipolar and hydrophobic characteristics,
appears to accommodate lysine and analogous molecules such as e-aminocaproic acid.3133'36'39"41 X-ray crystallographic data show that in PGK4, Asp54 and Asp56
form the anionic region and interact with the e-amino
group of the ligand. The cationic region is composed of
Lys35 and Arg69, which are located at the opposite side
of the ligand-binding site. Wu et al3S have reported a
crystallographic structure of PGK4 complexed with
e-aminocaproic acid, which shows that in the cationic
region Lys35 and Arg69 are involved in the interaction
with the carboxyl moiety of e-aminocaproic acid and
hence may also be important in the interaction between
the kringle-binding site and the carbonyl oxygens of the
polypeptide main chain of the ligand protein. The
intermediate region, or hydrophobic trough of the ligand-binding site, which interacts with the methylene
backbone of the lysine residue, is composed of Trp60
and Trp70. Therefore, segments of PGK4 that are
important in forming the ligand-binding site that binds
to lysine are located within segments 31-35, 52-57,
59-62, and 69-72 (Figure 2). Subtle amino acid substitutions in these regions of the apo[a] PGK4-like sequences suggest possible differences in the specificity of
the ligand-binding sites and their adjacent microdomains. The different ligand-binding sites of modeled
apo[a] kringle types 1-10 are described in this report.
Molecular Modeling Systems and Methods
Modeling was performed on molecular graphics
workstations from Silicon Graphics Inc., using the crystallographic modeling programs CHAIN,42 Pplygen
Corp.'s QUANTA 3.0, the Adopted Basis Newton-Raphson energy minimization procedure in CHARMm (version
21), and Molecular Simulations, Inc., BIOGRAF 3.0 program. There are amino acid substitutions in critical
regions of Lp[a] kringles that distinguish them from
PGK4 (Figure 1). The effect of these changes on the
kringle three-dimensional structure was studied by using two approaches to the molecular modeling of each
apo[a] kringle type. One method built each amino acid
onto the appropriate a-carbon coordinates from the
van der
Waals
Electrostatic
Total
-391
-3,692
-3,686
-379
-361
-367
-3,544
-3,493
-3,549
-3,513
-3,444
-366
-3,419
-3,480
-3,366
-399
-3,477
-3,447
-375
-3,611
-3,622
-3,589
-3,420
-370
-3,435
-3,770
-390
-3,501
-3,454
-377
-395
-3,590
-3,725
x-ray crystallographic structure of PGK4 by adding the
amino acid side groups without positional bias. The
a-carbon atoms were then held fixed as the model was
subjected to energy minimization. Then all the atoms
were allowed to move and the molecule was refined
further. Although total energy values for these models
were similar to those of PGK4, Ramachandran plots
showed the dihedral angles of many nonglycine amino
acids to be in energetically unfavorable regions. Therefore, these models were abandoned and were not used.
The second approach was to use the complete PGK4
crystallographic structure and to make the amino acid
substitutions required to convert the molecule to the
appropriate apo[a] kringle type. Each model structure
was refined as described above, except that amino acids
such as tryptophan, tyrosine, and phenylalanine were
also regularized to maintain strict aromatic ring planarity within CHAIN42 before the final energy minimization
was applied.
Results
Amino Acid Differences Between
Non-Ligand-Binding Regions ofApo[a]
Kringle Sequences and PGK4
Sequence similarities between apo[a] kringles and
PGK4 are shown in Figure 1. Homologies range
between 78% (LPaK4) and 88% (LPaKlO). Five
amino acid replacements, Vall7, Arg20, Thr21, Arg35,
and Thr76, are conserved in the 10 apo[a] kringle
types. Interestingly, Lys20 and Lys21 of PGK4 are
replaced with arginine and threonine in all apo[a]
kringles. A second analogous occurrence of this type
of change is the replacement of Lys76 and Lys77 of
PGK4 with threonine and arginine in LPaK4, LPaK8,
and LPaKlO. (In Glu-plasminogen, Lys76-Lys77 is the
cleavage site for fibrinolytic serine proteases in the
plasmin activation process43-47.) In the remaining
apo[a] kringles this Lys76-Lys77 sequence is replaced
with threonine-glutamine. Another site where a similar change occurs is PGK4 Lys35-Thr36, which becomes Arg35-Thr36 in all apo[a] PGK4-like kringles
except LPaK8. Thus, in all apo[a] PGK4-like kringles
except LPaK8, there are two copies of argininethreonine. Furthermore, all apo[a] PGK4-like kringles
except LPaK9 have one copy of threonine-arginine,
although at different positions in the kringle. All
apo[a] kringles except LPaK4 have one copy of argi-
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FIGURE 3. Stereoview of the ligand-binding site of modeled LPaKlO. LPaKlO is about 88% homologous with PGK4, including
high sequence homology and structural fidelity with respect to the ligand-binding site ofPGK4.
FIGURE 4. Stereoview of the ligand-binding site of modeled LPaK5. The molecular model of LPaKS shows a ligand-binding site
that is similar to that of LPaKlO and PGK4. There are five amino acid changes that may affect the ligand and ligand-polypeptide
specificity in this apoprotein[a] kringle.
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FIGURE 5. The ligand-binding sites of modeled LPaK6 and LPaK7. The ligand-binding sites of LPaK6 (panel A) and LPaK7
(panel B) are almost identical.
FIGURE 6. Stereoview of the ligand-binding site of modeled LPaK8. The structural arrangement of the LPaK8 ligand-binding site
suggests that it would bind lysine.
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FIGURE 7. Stereoview of the ligand-binding site of modeled LPaK9. Significant amino acid differences occur in the anionic
region.
FIGURE 8. Stereoview of the ligand-binding site of modeled LPaKA. LPaK4 has the least sequence homology with PGK4 and has
several important amino acid substitutions at its putative ligand-binding site.
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FIGURE 9. Stereoviews of the ligand-binding sites of modeled LPaKl (panel A), LPaK2 (panel B), and LPaK3 (panel C). The
ligand-binding sites of these apoprotein[a] kringles are very similar, quite hydrophobic, and different from that of PGK4 and the
other apoproteinfa] kringle models.
Guevara et al
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FIGURE 9.
Modeling of Apo[a] Kringle Sequences
765
Continued
nine-glycine in positions 10-11. These may be potential cleavage sites for both plasmin and a-thrombin.47-49 Except for ArglO, all other arginine residues
mentioned are located on the surface of the apo[a]
kringle models. In LPaK8, Arg3 is part of the only
"RGD" adhesion sequence present in this polymorph
of apo[a]. This sequence is a /3-turn on the surface of
the kringle in the LPaK8 model.
Amino Acid Differences in the Ligand-Binding-Site
Regions ofApofaJ Kringle Sequences
General. The effects of amino acid differences in
critical sequence positions of the ligand-binding sites of
apo[a] kringles were evaluated by molecular modeling.
The dipolar and hydrophobic ligand-binding region of
PGK4 is composed of kringle segments 31-35, 54-57,
59-62, and 69-72 (Figure 2).31-33-36 Lysine and analogous molecules such as e-aminocaproic acid occupy
most of the available space in the ligand-binding site of
PGK4.36 This can be described as a three-sided pocket,
with segments 31-35, 54-57, and 69-72 forming the
sides and segment 59-62 forming the base of the
pocket. The anionic region, segment 54-57, is formed
by Asp54 and Asp56, which interact with the e-amino
moiety of the ligand. The cationic region is composed of
Lys35 and Arg69, which are located opposite to the
anionic region of the ligand-binding site. The guanidino
group of Arg69 and the e-amino group of Lys35 have
been shown to interact with the carboxyl terminus of
e-aminocaproic acid ligand.36 This suggests that either
or both cationic moieties may interact with the appropriate carbonyls on a ligand polypeptide main chain.
The hydrophobic region, segment 69-72, is formed by
Trp70, which forms the trough, or cradle, of the ligandbinding site together with Trp60 of segment 59-62. This
region interacts with the methylene backbone of the
lysine residue. One residue, Trp70, is partially exposed
to the aqueous solvent on the surface of the kringle. Its
indole ring forms about a 90° angle with the ring of
Trp60. Also integral to the binding site are Phe62 and
Tyr72, which are located at opposite ends of the trough
and perhaps enhance its hydrophobicity. The ligandbinding environment of PGK4 is therefore created by
Lys35, Asp54, Asp56, Trp60, Arg69, Trp70, and perhaps
Phe62 and Tyr72. Amino acid changes in any of these
critical positions will most likely alter the binding environment as well as ligand specificity. The models of
PGK4-like kringle sequences in apo[a] were examined
to determine how amino acid replacements in these
positions would influence the ligand-binding environment and to evaluate their lysine-binding capacities. A
comparison of the total potential energies for these
models and of PGK4 is given in Table 1. Ramachandran
plots for each LPaK model showed that the dihedral
angles of only a few nonglycine amino acids were just
outside energetically favorable regions.
Ligand-binding site ofLPaKlO. The ligand-binding site
of LPaKlO (Figure 3) appears to retain many of the
electrostatic and hydrophobic characteristics of the lysine-binding site present in PGK4. Amino acid differences occur in positions 35 and 57: Lys35 to arginine
and Lys57 to threonine, respectively. The Arg35 is a
mild substitution that still maintains the electropositive
center of the lysine-binding site. The replacement of
Lys57 with threonine could influence the positioning of
Tyr72, but such an effect is not evident in the model of
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LPaKlO. Of all apo[a] kringle types, LPaKlO would
appear to have the highest probability of binding lysine
or a lysine-type ligand by a mechanism similar to the
one used by PGK4. The relatively nonpolar, cradle- or
trough-like environment formed by Trp60 and Trp70 is
conserved between the anionic region formed by the
carboxylic moieties of Asp54 and Asp56 and the cationic region formed by the guanidino groups of Arg35 and
Arg69. The Asp54, Arg35, and Arg69 residues, conserved in all apo[a] kringle sequences, are prominent
features of LPaKlO. The guanidino group of Arg35
forms a hydrogen bond with the carboxyl group of
Asp54, and the guanidino moiety of Arg69 may be
important in the interaction of the apo[a] kringle with
the carbonyl moieties of ligand polypeptides.
Ligand-binding site of LPaK5. Although amino acid
differences between PGK4 and LPaK5 occur at positions 32, 34, 35, 56, 57, 58, and 62 (Figure 2), the
electrostatic and hydrophobic characteristics of the ligand-binding site environment of PGK4 are retained in
LPaK5 (Figure 4). In the anionic region, Asp56 is
replaced with glutamate, but the carboxyl moiety is
positioned at the top of the Trp60/Trp70 hydrophobic
trough. At the base of the PGK4 trough near the
cationic region formed by Arg69, Phe62 has been replaced by a tyrosine in LPaK5. The hydroxyl of Tyr62
appears to be in a polar interaction with the guanidino
group of Arg69, while the Tyr62 phenolic ring interacts
with the indole ring of Trp60. The cationic region is
unchanged, since Lys35 of PGK4 is replaced with
arginine. Lysine in position 57 of PGK4 is replaced with
isoleucine in LPaK5, and its alkyl side group is oriented
away from the putative binding site in the model of this
kringle (Figure 4). Major residue changes are seen at
positions 32 and 34, where PGK4 Arg32 and Gln34 are
replaced with glutamine and serine, respectively. Since
positions 32 and 34 do not appear to be part of the
cationic site, this apo[a] kringle appears to retain the
necessary environment for binding of lysine or lysinelike molecules.
Ligand-binding sites ofLPaK6 and LPaK7. The models
of LPaK6 and LPaK7 show that many of the ligandbinding site characteristics of LPaK5 are again conserved. The putative ligand-binding site of these apo[a]
kringles is shown in Figure 5. The anionic and hydrophobic characteristics are retained, since the required
orientations of the critical amino acid side groups are
conserved in both models. The major changes occur in
positions 32 and 34, where tryptophan and glutamine of
LPaK6 replace glutamine and serine, respectively, of
LPaK5, and Arg32 and Lys35 in PGK4 are replaced
with tryptophan and arginine, respectively. These two
kringles differ at positions 58 and 67, where LPaK6 has
serine and asparagine, respectively, while LPaK7 has
arginine and serine. These two positions are located
peripheral to the putative ligand-binding site and do not
appear to influence its polarity or geometry in the
models. Therefore, both LPaK6 and LPaK7 should have
the potential for binding lysine or lysine-like molecules.
Ligand-binding site of LPaK8. The model for the
ligand-binding site of LPaK8 (Figure 6) is similar to
those sites described above for LPaK5, LPaK6, and
LPaK7 with one notable exception. The cationic region
of the LPaK8 model is composed of Arg34, Arg35, and
Arg69. Significant differences between the models of
LPaK8 and PGK4 occur at six positions (Arg32, Gln34,
Lys35, Asp56, Lys57, and Phe62 of PGK4 are replaced
with tryptophan, arginine, arginine, glutamate, isoleucine, and tyrosine, respectively). Notwithstanding these
amino acid differences, the LPaK8 model also appears
to have the potential for binding lysine or lysine-like
ligands.
Ligand-binding site of LPaK9. Unlike other apo[a]
kringle types, which contain six cysteine residues,
LPaK9 contains a seventh cysteine that occurs at
position 67. This position is peripheral to the ligandbinding site in the LPaK9 kringle model. The kringle is
a prime candidate for the site of disulfide linkage to
apo B-100 because of this extra cysteine residue.
Covalent and noncovalent interactions between LPaK9
and apo B-100 have been discussed elsewhere.50 In the
model of LPaK9, the ligand-binding site appears to
have remained intact (Figure 7) but is different from
that of PGK4 and the other apo[a] kringle models
described above. Major differences occur in the anionic region, where Ala55 and Asp56 of PGK4 are
altered to serine and glycine, respectively, in LPaK9,
which not only reduce the polarity of the anionic
center of the ligand-binding site but also increase its
binding volume capacity as well. The hydrophobic
trough formed by Trp60 and Trp70 remains intact.
Therefore, the ligand-binding site of LPaK9 appears
to be less specific for lysine analogues but capable of
binding larger, hydrophobic ligands, such as tyrosine,
phenylalanine, and methionine.
Ligand-binding site ofLPaK4. Although the hydrophobic trough formed by Trp60 and Trp70 is retained by
this apo[a] kringle model, amino acid substitutions at
positions 32, 34, 35, 55, 56, 57, and 62 significantly
change the characteristics of the ligand-binding environment (Figure 8). In the anionic region of LPaK4, Pro55,
Val56, and Ala57 replace alanine, aspartate, and lysine
of PGK4, thus changing the electrostatic properties of
the trough and thereby making it more hydrophobic. In
the LPaK4 model there is a slight shift in the position of
Trp70 and Tyr72, which also changes the orientation of
the trough somewhat. Other amino acid substitutions
that modify the nature of the ligand-binding site for
LPaK4 occur at positions 32 and 34, which are both
serine instead of the arginine and glutamine that are
present in the cationic region of PGK4. Lysine in
position 35 of PGK4 is altered to arginine in LPaK4, as
with the other apo[a] kringles. The extensive primary
and tertiary structural differences between PGK4 and
LPaK4 do not favor the latter kringle for binding
lysine-type ligands. However, an interaction with the
ligand polypeptide main-chain carbonyl groups is suggested by the conservation of the cationic nature of the
binding site with Arg69. The LPaK4 model is compatible with the binding of bulkier hydrophobic ligands.
Ligand-binding sites of LPaK3, LPaK2, and LPaKl.
The models of LPaKl, LPaK2, and LPaK3 suggest that
alteration of Trp60 in PGK4 to Tyr60 in the apo[a]
kringles alters their ligand-binding sites substantially
(Figures 9A-9C). The ligand-binding site now contains
three tyrosines, Tyr60, Tyr62, and Tyr72. In these
kringle models, Trp70 assumes a different orientation
from that in PGK4 or in the other apo[a] kringles. The
hydrophobic trough is not formed, and the anionic
region is likewise changed by the presence of Val56 in
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FIGURE 10. The ligand-binding region of LPaKlO (yellow,
cylindrical) superimposed on the analogous region of PGK4
(green). Two replacements occur at positions 35 and 57, where
arginine in LPaKlO replaces lysine in PGK4 and threonine
replaces lysine, respectively. An e-aminocaproate molecule
(blue) is located in the ligand-binding site.
place of Asp56 in PGK4. Although a ligand-binding site
appears to be partially formed by residues Arg35,
Asp54, Arg69, and Trp70, its size is now considerably
reduced. Side-group differences at positions 32, 34, and
55 are peripheral to the ligand-binding site and do not
appear to alter its geometry or polarity. Based on these
models, it would appear that kringles LPaKl, LPaK2,
and LPaK3 will not bind lysine or lysine-like ligands.
Discussion
Several assumptions were made in developing the
molecular graphic models of the apo[a] kringle-like
sequences that are based on the amino acid sequence of
apo[a] inferred from the cDNA sequence.14 This sequence indicates that the apo[a] polymorph contains 11
different types of kringle-like repeats and that 10 of
them are similar but not identical to PGK4.15 Therefore,
because there exists a high degree of sequence homology among all kringles, especially between apo[a] kringle-like sequences and PGK4, we have assumed that the
apo[a] sequences also exist in a kringle conformation.
However, there is no direct structural evidence at
present to indicate that these repeated sequences do in
fact occur in the typical tri-loop kringle conformation.
Sequences such as those of prothrombin kringle 1,
plasminogen kringles 1 and 4, and t-PA kringle 2, with
which sequence homology is about 55%, have been
shown to exist in the kringle structure. To gain a clearer
understanding of the apo[a] protein and how its kringle
domain may influence the physiological role of the Lp[a]
particle, we have developed molecular models of the
apo[a] kringle-like sequences. These sequences share
>78% sequence homology with PGK4. Another consideration supporting the view that these kringle structures
Modeling of Apo[a] Kringle Sequences
767
may also be conserved in apo[a] is that the side-chain
structures of corresponding amino acid residues appear
to be the same for conserved residues in PGK1, PGK4,
t-PA kringle 2, and prothrombin kringle I.35 Other
important structural aspects of PGK4 that appear to be
retained in the apo[a] PGK4-like sequences are the two
hydrophobic cores. In PGK4, these cores are formed by
Trp25, His33, Pro37, Tyr40, Leu45, Pro53, Trp60, and
Phe62 and by Tyr2, Tyr9, Pro59, and Leu75. Interestingly, except for LPaKl, LPaK6, and LPaK7, apo[a]
kringle models contain all residues of the hydrophobic
cores of PGK4. The latter hydrophobic cluster is conserved and is evident in all apo[a] kringle models. In the
model of LPaKl, substitutions in positions 37 and 60 at
which threonine and tyrosine replace proline and tryptophan, respectively, the cluster is partially but not
significantly modified. In LPaK6 and LPaK7, the replacement of Pro37 by threonine does not appear to
alter the cluster. These observations are corroborated
theoretically by the low potential energies for van der
Waals forces shown in Table 1. Nevertheless, it is
understood that although refinement of molecular
structures by nonhydrated, static energy minimization
can yield secondary and tertiary structures that are
energetically favorable (Table 1), the modeled structures may be different from the true structure(s). Suffice
it to add that computer molecular modeling has been
shown to be a reliable method for evaluating structures
that have not been elucidated by x-ray crystallographic
or multidimensional nuclear magnetic resonance methods, especially when the homology is high.31 All of the
apo[a] kringle models are based on coordinates of the
crystallographic structure for PGK4 and are presented
here as predicted structures. We then used these models to evaluate and compare their putative ligandbinding sites with that of PGK4.
In human plasma, plasminogen is present at a concentration range of 1.3-2.3 /xM29 and exists in at least
four molecular forms.51 Two forms are considered to be
the complete molecules and have glutamate at the
amino terminus of 790 amino acids in identical sequences but have different glycosylation levels. These
are referred to as the Glu-I and Glu-II forms of
plasminogen. The other two forms are identical but lack
the first 76 residues and have lysine as the amino
terminus. These are termed Lys-I and Lys-II forms of
plasminogen. All forms can be readily purified from
crude human plasma containing Lp[a] by affinity chromatography by a single pass through a lysine-Sepharose
column in high-phosphate buffers.52-53 Plasma contains
potentially 2.0-4.0 FIM plasminogen lysine-binding
kringles in PGK1 and PGK4. Typically, few other
proteins are retained by lysine-Sepharose, and they
represent probably less than 5% of the total protein
bound. Lp[a] is not one of the species that binds or
competes with plasminogen in this form of affinity
chromatography. Theoretically, if the PGK4-like apo[a]
kringles have a binding affinity for lysine similar to that
of either PGK4 or PGK1, then a small fraction of Lp[a]
from crude plasma, along with plasminogen, should
bind to a lysine-Sepharose affinity gel.
Potential Lysine-Binding Apofa] Kringles
The plasma levels of Lp[a] vary from 10 mg/L to 2,000
mg/L.29-54 Approximately 17% of these values yield an
768
TABLE 2.
Arteriosclerosis and Thrombosis
Comparison of Lysine-Binding Kringles, Plasminogen Kringles 1 and 5, to LPaKll
1
PGK1
LPaKll
PGKS
Vol 13, No 5 May 1993
30
70
20
40
50
80
10
60
CKTGDOKNYROTMSKTKNOITCQKWSSTSPHRPR-FSPATHPSEQLBENYCRNPDNDPQOPWCYITDPEKRYDYCDILBC
CMFQNQKGYRaKKATTVTGTPCQEWAAQEPHRHSTPIPGTNJCWAGLBKNYCRNPDGDINGPWCYTMNPRKLFDYCDIPLC
CMFGHGKGYRGiaUTTVTGTPCQDWAAQBPHRHSIFTPETNPRAGLBKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQC
Downloaded from http://atvb.ahajournals.org/ by guest on August 3, 2017
estimate of the concentration of apo[a] of approximately 1.7-350 mg/L. The plasma concentration of
apo[a] for the 530-kD polymorph would then range
between 3.0 nM and 660 nM. At the high end of these
plasma levels of Lp[a], the concentration of a 280-kD
apo[a] polymorph could be about 1.25 /xM. If only one
of the PGK4-like apo[a] kringles binds to lysine in each
apo[a] polymorph, then at 1.25 /AM apo[a], a significant
fraction of the protein retained by the lysine-Sepharose
affinity column would be expected to be Lpjaj. Comparison of the molecular model of LPaKlO and PGK4
with e-aminocaproic acid in the ligand pocket of the
kringle reveals that this is the only apo[a] kringle type
with a ligand-binding site capable of binding lysine
(Figure 10). This apo[a] kringle would, therefore, be
expected to bind lysine moieties in a protein such as
fibrinogen or to lysine-Sepharose. Typically, plasminogen represents the bulk of crude plasma protein that
binds to lysine-Sepharose under the conditions reported by others,51-53 which would suggest that LPaKlO
has a different, much lower affinity than PGK4 and
PGK1 for lysine-Sepharose under the conditions used
for plasminogen binding. This change in specificity may
be dependent on changes that occur at locations outside
the ligand-binding site, perhaps at positions 35 and 68.
The Lys35-to-Arg35 change seems innocuous, but the
side group of Ile68 in LPaKlO may interact with amino
acids that precede the lysine ligand. Alternatively, and
more probable, the ligand-binding sites of apo[a] kringles may already be occupied with lysine residues of apo
B-100 and would not recognize other lysine moieties.55"57 Precedence for an extraordinary attraction of
the lysine-binding site for lysine residues has been set by
the intermolecular interactions observed in the crystal
structure of PGK43335 and the trimer formation of t-PA
kringle 2, in which three intermolecular lysyl side chains
bind in respective binding sites.37
Molecular models of LPaK5, LPaK6, LPaK7, and
LPaK8 also show ligand-binding sites that differ slightly
from those of LPaKlO and PGK4 with respect to both
electrostatic and hydrophobic properties. Although the
hydrophobic trough formed by Trp60 and Trp70 remains intact in these kringles, glutamate at position 56
and Tyr62 bring a negatively charged characteristic to
the surface of the ligand trough. Amino acid substitutions in the cationic region, residues 31-35, may also
influence the ligand-binding site formed in the apo[a]
kringles, and it is possible that the residues lend specificity to each kringle type for the residues that precede
lysine. These results suggest a difference in ligandaffinity constants for each kringle, perhaps as different
as those reported for PGK1 and PGK4. 58 ^
Non-Lysine-Binding Apo [a] Kringles
The ligand-binding sites in LPaKl, LPaK2, LPaK3,
LPaK4, and LPaK9 differ significantly from the sites of
apo[a] kringles with respect to their potential for bind-
ing lysine. The ligand specifically bound by LPaK9 may
be related to the association of this apo[a] kringle with
a site on apo B-100. The Cys4,057 residue in apo[a]
(Cys67 in the LPaK9 sequence) is believed to form an
intermolecular disulfide bond with a cysteine of apo
B-100. Molecular models of LPaK9 suggest that Cys67
is located on the surface of the kringle near the ligandbinding site. Ligands such as phenylalanine and methionine appear to fit well into the ligand-binding site of
the modeled LPaK9. LPaK9 is probably an important
region of apo[a] for understanding the interaction of
apo[a] with apo B-100 and perhaps for identifying other
regions of the kringle that are involved in the binding
interaction. LPaK9 is approximately 80% homologous
to PGK4 and contains a modified ligand-binding site
with some nonhomologous replacements in and adjacent to the ligand-binding region. For example, Asp56,
an essential amino acid for the binding of lysine to
PGK4, is replaced with glycine in LPaK9. In the cationic
region, Trp32 and Arg35 replace Arg32 and Lys35,
respectively, in PGK4, thereby changing the surface
characteristics peripheral to the ligand-binding region.
These changes appear to influence the interaction of
apo B-100 segments with this apo[a] kringle. In a
separate study, selected segments of the low density
lipoprotein apo B-100 sequence, which contain free
sulfhydryl cysteines, have been subjected to energy
minimization and docking with the ligand-binding site
and adjacent regions of the LPaK9 model.50 In the
docking experiments, apo B-100 segment 3,732-3,745
(PSCKLDFREIQIYK) displayed the best fit and the
largest number of van der Waals contacts with models of
LPaK9. The models showed that Phe3,738 fit and
minimized well in the ligand-binding site of LPaK9 with
numerous atomic contacts with Trp60 and Trp70 of the
kringle. Other interactions between apo[a] and apo
B-100 are likely and may include noncovalent binding
through one or more of the apo[a] kringles.55-56 These
interactions may involve ligands such as L-proline and
L-lysine, but with lysine having a lesser role and/or
affinity.57
Proline, an amino acid that is thought to be more
important than lysine in the binding of apo[a] and Lp[a]
to biotinylated low density lipoprotein,57 may bind to
LPaKl, LPaK2, LPaK3, and/or LPaK4. The models of
these apo[a] kringles demonstrate ligand-binding pockets that are significantly different from the lysinebinding site of PGK4. Proline may be a specific ligand
for these apo[a] kringles. Previously, proline was modeled with LPaK9 alone and as part of an apo B-100
fragment. In the models, proline occupies only a fraction of the apparent space in the binding site of LPaK9.
As part of apo B-100 fragment 1,476-1,490, proline
appeared to be minimally compatible with LPaK9. Our
experience with modeling the interaction of small ligands, such as single amino acids with kringles, has
produced inconclusive results, suggesting that addi-
Guevara et al Modeling of Apo [a] Kringle Sequences
tional amino acids (e.g., those preceding and succeeding
the residue of interest) most likely play a role in the
overall interaction. However, it is possible that proline
alone, when added in excess, can interfere with the
interactions between apo[a] and biotinylated low density lipoprotein by competing with one or more proline
residues on apo B-100 that bind to the apo[a] kringles,
especially the highly repeated LPaK2. The critical anionic residue, Asp56 in PGK4, is replaced by valine in
LPaK2, a change that influences the polarity and size of
the kringle lysine-binding site. The trypsin-releasable
fractions of apo B-100 from low density lipoprotein
contain 78 proline residues60; 56% of these prolines are
located in the ^/-terminal region. Perhaps some of these
prolines might occur in exposed structural /3-turns and
are sterically accessible to interact with the many repeats of LPaK2.
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LPaKll as a Lysine-Binding Kringle
Little attention has been devoted to the PGK5-like
sequence contained in this apo[a] polymorph, which we
have designated as LPaKll (Table 2). LPaKll comprises
80 residues and shares high homology with the non-lysinebinding kringle of plasminogen, PGK5 (about 93%), and
the high-affinity, lysine-binding kringle PGK1 (about
69%). A molecular model for LPaKll was not developed,
since we considered this highly speculative. However,
several laboratories have shown that Lp[a] and recombinant apo[a] bind to intact fibrin as well as to digested
fibrin.27'28'61 Those results suggest that LPaKlO recognizes
lysines located within a polypeptide chain as well as at a
carboxyl terminus, a functional feature not generally attributed to PGK4. An alternative explanation would be
that LPaKll is sufficiently similar to the high-affinity,
lysine-binding kringle PGK1 to also bind to lysine residues
within a polypeptide chain.
Other Potential Apo[a] Ligand Sites
There are other sites on the apo[a] kringle models that
may be ligands that interact with specific receptors. The
structure of these sites was suggested by molecular modeling of the kringle sequences. For example, there is a
single copy of the RGD adhesion sequence that occurs at
positions 3-5 (Figure 1) in LPaK8 or positions 3,8923,894 in the complete apo[a] polymorph sequence. This
motif occupies the first J3-turn on the surface of LPaK8.
The sequence STTVTG at positions 14-19 in most apo[a]
kringle sequences shares some homology with the receptor-ligand site of thrombospondin that is contained in the
sequence SCSVTC.6263 Binding of plasminogen to specific
receptors may also involve these short sequences, three
copies of which are similar but not identical to this
sequence and are present in plasminogen at positions
16-18, 270-272, and 476-478. Whether these sites on
both apo[a] and plasminogen are receptor-binding sites
remains to be shown.
A third ligand type present in the apo[a] kringle sequence is the arginine-threonine and threonine-arginine
sequence; it is repeated at least twice in every kringle
sequence and, therefore, occurs many times throughout
the entire apo[a] molecule. These sequences may be
cleavage sites for other serine proteases, providing a
mechanism for release of the Lp[a] particle by altering the
kringle structure. Molecular modeling has shown these
769
short motifs to be located on the surface of the kringles,
facilitating steric access to the motifs.
Conclusions
The kringle-like sequences in apo[a] readily minimize
into the kringle conformation of PGK4 with very similar
potential energy values and dihedral angle characteristics.
The models reveal subtle amino acid differences between
LPaK models and PGK4 that influence the microenvironments of the kringle structure, which could suggest differences in specificity for the ligand moiety alone and also for
the ligand within a polypeptide. The model of the highly
repeated apo[a] kringle type LPaK2 displays a ligandbinding site that is very different from that of PGK4 and
most likely does not bind lysine, lysine analogues, or lysine
adducts. Our models of apo[a] kringles and the complete
primary structure of apo[a] suggest that apo[a] imparts to
Lp[a] the capacity to recognize and bind a wide variety of
ligand-receptor proteins. The varied ligand-binding sites
contained by the apo[a] kringles as well as the adhesionlike regions of the kringle and interkringle domains
strongly suggest that Lp[a] may bind a wide variety of
different ligands that involve the lipoprotein in different
physiological roles.
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