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Human plasminogen kringle 3: solution structure, functional insights, phylogenetic landscape.

Martin T Christen1, Pascal Frank, Johann Schaller

  • 1Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.

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Summary
This summary is machine-generated.

Human plasminogen kringle 3 (hPgn K3) lacks lysine-binding affinity, but a Lys57Asp mutation engineered this function. The study determined the structure and binding affinity of the mutated domain, revealing unique properties of hPgn K3 among vertebrate K3-type domains.

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Protein Engineering

Background:

  • Human plasminogen kringle 3 (hPgn K3) possesses elements of a canonical lysine-binding site (LBS) but lacks affinity for lysine.
  • Previous research demonstrated that lysine-binding activity can be engineered through a Lys57Asp mutation.

Purpose of the Study:

  • To determine the three-dimensional solution structure of wild-type hPgn K3 and the engineered K57D mutant.
  • To characterize the lysine-binding affinity and map the binding site of the engineered hPgn K3 domain.
  • To compare the structural and functional properties of hPgn K3 with other vertebrate K3-type domains.

Main Methods:

  • NMR spectroscopy was used to determine the solution structure of wild-type and mutated hPgn K3.
  • Ligand-induced NMR chemical shift perturbations were employed to map the binding site and determine the affinity for AMCHA.
  • Homology modeling and in silico docking (AutoDock 4.0) were utilized to support the engineered LBS functionality.

Main Results:

  • The wild-type and mutated [r(K57D)K3] structures were found to be isomorphous by NMR.
  • The engineered r(K57D)K3 domain exhibited an equilibrium association constant (Ka) of approximately 5.23 mM⁻¹ for AMCHA.
  • The engineered LBS functionality was supported by homology modeling and docking, highlighting electrostatic interactions involving Arg36/Arg71 and Asp55/Asp57.
  • Lys57 was identified as unique to the human variant, suggesting other vertebrate K3-type domains possess lysine-binding capabilities.

Conclusions:

  • Engineering a Lys57Asp mutation successfully introduced lysine-binding activity into the hPgn K3 domain.
  • The structural and binding data provide insights into the molecular basis of lysine recognition.
  • Human plasminogen kringle 3 is unique among vertebrate K3-type domains in its lack of intrinsic lysine-binding affinity, with other homologous domains predicted to bind lysine.