Human Plasminogen Kringle 3: Solution Structure, Functional Insights, Phylogenetic Landscape,
- 9 July 2010
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 49 (33), 7131-7150
- https://doi.org/10.1021/bi100687f
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 [Bürgin, 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 Å (backbone) and 0.75 ± 0.12 Å (all)]. The 1H/15N 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, Ka, was determined to be ∼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.Keywords
This publication has 94 references indexed in Scilit:
- Structure-oriented methods for protein NMR data analysisProgress in Nuclear Magnetic Resonance Spectroscopy, 2010
- Characterization of the kringle fold and identification of a ubiquitous new class of disulfide rotamersJournal of Structural Biology, 2009
- Clustal W and Clustal X version 2.0Bioinformatics, 2007
- Structural Basis of Interaction between Urokinase-type Plasminogen Activator and its ReceptorJournal of Molecular Biology, 2006
- PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculationsNucleic Acids Research, 2004
- The iProClass integrated database for protein functional analysisComputational Biology and Chemistry, 2004
- SWISS-MODEL: an automated protein homology-modeling serverNucleic Acids Research, 2003
- Electrostatics of nanosystems: Application to microtubules and the ribosomeProceedings of the National Academy of Sciences of the United States of America, 2001
- SWISS‐MODEL and the Swiss‐Pdb Viewer: An environment for comparative protein modelingElectrophoresis, 1997
- AQUA and PROCHECK-NMR: Programs for checking the quality of protein structures solved by NMRJournal of Biomolecular NMR, 1996