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Related Experiment Videos

A novel soluble tissue factor variant with an altered factor VIIa binding interface

G F Lee1, R F Kelley

  • 1Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080, USA.

The Journal of Biological Chemistry
|March 28, 1998
PubMed
Summary

Researchers engineered tissue factor (TF) variants to enhance binding to factor VIIa (FVIIa). A novel variant, K20A,D58W, binds FVIIa comparably to wild-type but is defective in cofactor activity, suggesting altered binding mechanisms.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Engineering

Background:

  • Tissue factor (TF) and factor VIIa (FVIIa) interaction is crucial for blood coagulation.
  • Specific residues Lys20 and Asp58 in TF are critical for high-affinity FVIIa binding.
  • Developing TF-based antagonists requires understanding and modifying these binding interactions.

Purpose of the Study:

  • To engineer novel tissue factor (TF) variants with enhanced affinity for factor VIIa (FVIIa).
  • To investigate the impact of specific residue modifications at the TF-FVIIa binding interface.
  • To elucidate the binding mechanism of engineered TF variants.

Main Methods:

  • Construction and display of TF variant libraries on bacteriophage M13.
  • Phage display selection and sorting based on FVIIa binding affinity.

Related Experiment Videos

  • Biochemical characterization of soluble TF (sTF) variants, including binding assays and cofactor activity measurements.

    • Main Results:

    • Initial library screening yielded predominantly wild-type TF sequences.
    • A targeted strategy fixing Lys20 to alanine and randomizing nearby residues identified a consensus variant with Tryptophan at position 58 (K20A,D58W).
    • The K20A,D58W TF variant exhibited FVIIa binding affinity comparable to wild-type sTF but was cofactor-defective for factor X activation.

    Conclusions:

    • Modifying the TF-FVIIa binding interface can yield variants with altered functional properties.
    • The K20A,D58W variant's binding mechanism appears distinct from wild-type TF, involving more than simple hydrophobic interactions.
    • Further research is needed to fully understand the structural and mechanistic basis of the enhanced binding and defective cofactor activity.