Solution structure of the complex of VEK-30 and plasminogen kringle 2

Abstract

The solution structure of the complex containing the isolated kringle 2 domain of human plasminogen (K2(Pg)) and VEK-30, a 30-amino acid residue internal peptide from a streptococcal M-like plasminogen (Pg) binding protein (PAM), has been determined by multinuclear high-resolution NMR. Complete backbone and side-chain assignments were obtained from triple-resonance experiments, after which structure calculations were performed and ultimately refined by restrained molecular simulation in water. We find that, in contrast with the dimer of complexes observed in the asymmetric unit of the crystal, global correlation times and buoyant molecular weight determinations of the complex and its individual components showed the monomeric nature of all species in solution. The NMR-derived structure of K2(Pg) in complex with VEK-30 presents a folding pattern typical of other kringle domains, while bound VEK-30 forms an end-to-end alpha-helix (residues 6-27) in the complex. Most of the VEK-30/K2(Pg) interactions in solution occur between a single face of the alpha-helix of VEK-30 and the lysine binding site (LBS) of K2(Pg). The canonical LBS of K2(Pg), consisting of Asp54, Asp56, Trp60, Arg69, and Trp70 (kringle numbering), interacts with an internal pseudo-lysine of VEK-30, comprising side-chains of Arg17, His18, and Glu20. Site-specific mutagenesis analysis confirmed that the electrostatic field formed by the N-terminal anionic residues of the VEK-30 alpha-helix, viz., Asp7, and the non-conserved cationic residues of K2(Pg), viz., Lys43 and Arg55, play additional important roles in the docking of VEK-30 to K2(Pg). Structural analysis and kringle sequence alignments revealed several important features related to exosite binding that provide a structural rationale for the high specificity and affinity of VEK-30 for K2(Pg).

Fig. 1

Stereo view of the solution structure of the VEK-30/K2_Pg complex. (A). Backbone trace of the ensemble of 20 superimposed lowest energy structures. Color coding: Cys1-Cys78 of K2_Pg and Ala6-Lys27 of VEK-30 are shown in blue and orange, respectively. The N- and C-termini of each chain are shown in gray (K2_Pg) and cyan (VEK-30), respectively. (B). Ribbon representation of the lowest-energy structure. Helices are shown in red, β strands are shown in blue, coil and turn regions are shown in gray.

Fig. 2

NMR spectral analysis of the K2_Pg/VEK-30 complex. (A). 2D 1H-15N HSQC spectra of ¹⁵N-labeled K2_Pg bound to unlabeled VEK-30. (B). ¹⁵N-labeled VEK-30 bound to unlabeled K2_Pg. Sequence-specific assignments are indicated and peaks corresponding to the NH₂ groups of the side-chain amides of Gln and Asn residues are connected by lines.

Fig. 3

Surface representation of the binding interface between VEK-30 and K2_Pg. The exposed hydrophobic groove (formed by the aromatic residues Tyr35, Phe40, Trp60, Phe62, Trp70 and Tyr72) on the surface of the K2_Pg domain is highlighted red. The hydrophobic residues and Arg17 of VEK-30 involved are labeled and depicted as sticks (dark blue).

Fig. 4

Electrostatic interactions between K2_Pg and VEK-30. The K2_Pg domain is shown in surface representation with color coding according to electrostatic potential (blue = positively charged, red = negatively charged). The side-chains of charged residues in VEK-30 interacting with K2_Pg are represented as magenta and green sticks for positive- and negative-charged residues, respectively.

Fig. 5

Superimposition of X-ray crystallographic and NMR solution structures of the VEK-30/K2_Pg. (A). The backbone trace of two molecules (molecule-1 and molecule-2) in the unit cell of the crystal structure (PDB entry 115K), and the lowest energy NMR structure of VEK-30/K2_Pg are overlaid and represented as blue, green and red lines, respectively. (B). Overlay of side-chain residues involved in the binding interactions. The intermolecular side-chain interactions of K2_Pg and VEK-30 of molecule-1 (blue) and molecule-2 (green) of the X-ray crystal structure and the lowest energy NMR structure (red) are compared with side-chain orientations (represented as sticks). The main-chain of VEK-30 in the three structures is depicted as a ribbon diagram.

Fig. 6

Close-up views of the intermolecular interactions comparing VEK-30/K2_Pg and K2_Pg/AMCHA interactions as derived from their solution structures. (A). Side-chain atoms in the hydrophobic groove (green-formed by Y35, F40, W60, W70 and Y72) and anionic center (formed by D54 and D56) of K2_pg that interact with side-chain atoms from three critical amino acids of VEK-30 (blue-K14, R17, and H18) are shown. A portion of the VEK-30 helix is shown in yellow. An overlay of the K2_Pg/AMCHA structure on the K2_Pg/VEK-30 structure was performed and all atoms undisplayed, except for AMCHA (magenta), thus providing the relationships between the pseudo-lysine of VEK-30 and AMCHA in the K2_Pg lysine binding site. (B). The binding of K2_Pg to VEK-30 and K2_Pg to AMCHA are overlaid for comparison and selected residues are displayed. The side-chains of K2_Pg residues in the VEK-30/K2_Pg complex and in the AMCHA/K2_Pg complex are shown as dark blue and red sticks, respectively. The ligand, AMCHA, is colored cyan and the ribbon of VEK-30 and side-chains in VEK-30 are both colored green.

Fig. 7

Sequence alignment of the 5 human Pg kringles. Strictly conserved residues are designated by *. Several important non-conserved residues presented in K2_Pg and are boxed in the sequences. In K2_Pg, C4, E56, and L72 were mutated to C, D, and Y (mK2_Pg), respectively to bring the canonical LBS of K2_Pg into conformity with other lysine binding kringles.