Human Plasminogen Kringle 3: Solution Structure, Functional Insights, Phylogenetic Landscape,

Abstract

Human plasminogen kringle 3 (hPgn K3) domain contains most elements of the canonical lysine-binding site (LBS) found in other Pgn kringles. However, it does not exhibit affinity for either lysine or structurally related zwitterionic ligands. It has been shown that lysine-binding activity can be engineered via a Lys57 --> Asp mutation [Burgin, J., and Schaller, J. (2009) Cell. Mol. Life Sci. 55, 135]. Using a recombinant construct expressed in Escherichia coli, the three-dimensional solution structure of hPgn K3 was determined via NMR spectroscopy [heavy atom averaged rmsd = 0.35 +/- 0.07 A (backbone) and 0.75 +/- 0.12 A (all)]. The (1)H/(15)N heteronuclear single-quantum correlated (HSQC) spectra for both wild-type K3 and mutated [r(K57D)K3] structures are essentially identical, implying that the two structures are effectively isomorphous. The affinity of r(K57D)K3 for the lysine analogue trans-(aminomethyl)cyclohexanecarboxylic acid (AMCHA) was investigated from ligand-induced NMR chemical shift perturbations, which enabled for mapping the binding site on the mutated domain surface. The equilibrium association constant, K(a), was determined to be approximately 5.23 +/- 0.03 mM(-1). Homology modeling combined with in silico docking of lysine-like zwitterionic ligands via AutoDock 4.0 supports functionality of the engineered (K57D)K3 LBS, whose electrostatic focal centers are defined by the Arg36/Arg71 cationic and Asp55/Asp57 anionic pairs. Comparison of K3-type sequences from different vertebrates, including kringles from hedgehog apolipoprotein(a) [Apo(a)] and Apo(a)-related (Arp) sequences, reveals that Lys57 is confined to the hPgn variant. Based on the likely phylogeny and ligand affinities of the homologous domains, it is suggested that the hPgn K3 is unique in that all other K3-type domains, including hedgehog Apo(a) and all Arp domains, except K3(1), are predicted to variously exhibit lysine-binding capability. In Arp K3(1) an Arg residue fills site 72, replacing the key aromatic residue found in other kringles, thus interfering with a requisite kringle-ligand hydrophobic interaction.

Hereditary angioedema: the plasma contact system out of control

“…This substitution mutation changes a positively charged lysine at position 311 (mature protein numbering) in kringle 3 into a negatively charged glutamic acid. Interestingly, this kringle normally lacks lysine-binding properties in human PLG, but this can be enabled by replacing the lysine at position 57 with a negatively charged aspartic acid (K57D; [82]). Strikingly, this same amino acid (K311) is mutated in full-length PLG, causing PLG-HAE.…”

Plasminogen receptors and their role in the pathogenesis of inflammatory, autoimmune and malignant disease

“…Four out of these five domains (K1, K2, K4 and K5) contain lysine‐binding sites (LBS) that allow plasminogen binding to proteins with carboxyl‐terminal lysines or conformational mimetics of these residues. These four LBS share a common structure, whereas K3 contains a mutation in the LBS sequence [9]; thus K3‐LBS is non‐functional and does not bind lysine. Biochemical analysis demonstrated that, relative to K2 or K5, K1 and K4 have significantly higher affinities for lysine [10].…”

The Biochemistry and Regulation of S100A10: A Multifunctional Plasminogen Receptor Involved in Oncogenesis

“…As discussed, S100A10 is present in mammals, birds, reptiles, amphibians, and fish, but not in insects, nematodes, protozoa, fungi, or plants (Figure 9). Similarly, plasminogen, one of the established ligands of S100A10, is present in vertebrates including fish [96]. In contrast, S100A2, S100A7, S100A12, S100Z, and S100P are present in humans but not in mouse and rat [73].…”

“…Furthermore, S100A10 possesses the requisite carboxy-terminal lysine that has been shown to be essential for plasmin activation at the cell surface. Removal of the carboxy-terminal lysines (carboxy-terminus of S100A10- [85]-Y-F-V-V-H-M-K-Q-K-G-K-K [96]) attenuates plasminogen and tPA binding [63]. Second, binding of plasminogen to the candidate regulatory protein must convert plasminogen into the open, activation-susceptible conformation.…”