LIGAND PREFERENCES OF KRINGLE-2 AND HOMOLOGOUS DOMAINS OF HUMAN PLASMINOGEN - CANVASSING WEAK, INTERMEDIATE, AND HIGH-AFFINITY BINDING-SITES BY H-1-NMR
Dn. Marti et al., LIGAND PREFERENCES OF KRINGLE-2 AND HOMOLOGOUS DOMAINS OF HUMAN PLASMINOGEN - CANVASSING WEAK, INTERMEDIATE, AND HIGH-AFFINITY BINDING-SITES BY H-1-NMR, Biochemistry, 36(39), 1997, pp. 11591-11604
The interaction of various small aliphatic and aromatic ionic ligands
with the human plasminogen (HPg) recombinant kringle 2 (r-K2) domain h
as been investigated by H-1-NMR spectroscopy at 500 MHz. The results a
re compared against ligand-binding properties of the homologous, lysin
e-binding HPg kringle 1 (K1), kringle 4 (K4), and kringle 5 (K5). The
investigated ligands include the omega-aminocarboxylic acids 4-aminobu
tyric acid (4-ABA), 5-aminopentanoic acid (5-APA), 6-aminohexanoic aci
d (6-AHA), 7-aminoheptanoic acid (7-AHA), lysine and arginine derivati
ves with free and blocked alpha-amino and/or carboxylate groups, and a
number of cyclic analogs, zwitterions of similar size such as trans-(
aminomethyl)cyclohexanecarboxylic acid (AMCHA) and p-benzylaminesulfon
ic acid (BASA), and the nonzwitterions benzylamine and benzamidine. Eq
uilibrium association constant (K-a) values were determined from H-1-N
MR ligand titration profiles. Among the aliphatic linear ligands, 5-AP
A (K-a similar to 3.4 mM(-1)) shows the strongest interaction with r-K
2 followed by B-AHA (K-a similar to 2.3 mM(-1)), 7-AHA (K-a similar to
0.45 mM(-1)), and 4-ABA (K-a similar to 0.22 mM(-1)). In contrast, r-
K1, K4, and 1(5 exhibit a preference for 6-AHA (K-a similar to 74.2, 2
1.0, and 10.6 mM(-1), respectively), a ligand similar to 1.14 Angstrom
longer than 5-APA. Mutations R220G and E221D increase the affinity of
r-K2 for these ligands but leave the selectivity profile essentially
unaffected: 5-APA > 6-AHA > 7-AHA > 4-ABA (K-a similar to 6.5, 3.9, 1.
8, and 0.74 mM(-1), respectively). We find that, while r-K2 definitely
interacts with Na-acetyl-L-lysine and L-lysine (K-a similar to 0.96 a
nd 0.68 mM(-1), respectively), the affinity for analogs carrying a blo
cked carboxylate group is relatively weak (K-a similar to 0.1 mM(-1)).
We also investigated the interaction of r-K2 with L-arginine (K-a sim
ilar to 0.31 mM(-1)) and its derivatives Na-acetyl L-arginine (K-a sim
ilar to 0.55 mM(-1)), N-alpha-acetyl-L-arginine methyl ester (K-a simi
lar to 0.07 mM(-1)), and L-arginine methyl ester (K-a similar to 0.03
mM(-1)). Zwitterionic gamma-guanidinobutyric acid, containing one less
methylene group than arginine, exhibits a K-a of similar to 0.28 mM(-
1). The affinity of r-K2 for lysine and arginine derivatives suggests
that K2 could play a role in intermolecular as well as intramolecular
interactions of HPg. As is the case for the HPg K1, K4, and K5, among
the tested ligands, AMCHA is the one which interacts most firmly with
r-K2 (K-a similar to 7.3 mM(-1)) while the aromatic ligands BASA, benz
ylamine, and benzamidine exhibit K-a values of similar to 4.0, similar
to 0.04, and similar to 0.03 mM(-1), respectively. The relative stabi
lity of these interactions indicates a strict requirement for both cat
ionic and anionic polar groups in the ligand, whereas the presence of
a lipophilic aromatic group seems to be of lesser consequence. Ligand-
induced shifts of r-K2 H-1-NMR signals and two-dimensional nuclear Ove
rhauser effect (NOESY) experiments in the presence of 6-AHA reveal dir
ect involvement of residues Tyr(36), Trp(62), Phe(64), and Trp(72) (kr
ingle residue numbering convention) in ligand binding. Starting from t
he X-ray crystallographic structure of HPg K4 and the intermolecular H
-1-NMR NOE data, two models of the K2 lysine binding site complexed to
6-AHA have been derived which differ mainly in the extent of electros
tatic pairing between the K2 Arg(56) and Glu(57) side chains. Competit
ion between these two conformations in equilibrium may account for the
relatively lesser affinity of the K2 domain for zwitterionic lysine-t
ype ligands.