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Replacement of Phe274 with conserved residue Tyr274 for reactive center loop expulsion in antithrombin
1Center for Molecular Biology of Oral Diseases, College of Dentistry, Chicago, IL, USA. uduhan@uicalumni.org
Background:
The reactive center loop (RCL) of native antithrombin is partially inserted in the main serpin body. It must be fully exposed for optimal inhibitory function.
Objective:
To test the hypothesis that P(14)-s2B interaction affects loop insertion in antithrombin. By mutating Phe(274) to Tyr(274), the objective was to introduce P(14)-s2B interaction in antithrombin.
Methods:
Site-directed mutagenesis and affinity chromatography were used to obtain purified recombinant protein. Antithrombin's ability to form sodium dodecyl sulfate (SDS)-stable complex with thrombin, stoichiometry of thrombin inhibition, second-order rate constant for thrombin and factor Xa (fXa) inhibition (M(-1) s(-1)), and heparin dissociation constant (K(D); tryptophan fluorescence emission spectra) were determined.
Results And Conclusion:
A marginal, but inconclusive, difference between the wild type and the mutant was observed. The result highlights the variable effect of P(14)-s2B interaction in different serpins. Alternate hypothesis for achieving loop expulsion is proposed.
Insights
Mutating antithrombin to introduce P(14)-s2B interaction showed a marginal effect on reactive center loop (RCL) exposure. This suggests variable P(14)-s2B interaction effects across different serpins.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Structure-Function Relationships
Background:
- The reactive center loop (RCL) of antithrombin must be fully exposed for optimal inhibitory function.
- In native antithrombin, the RCL is partially inserted within the main serpin body.
Purpose of the Study:
- To investigate if P(14)-s2B interaction influences RCL insertion in antithrombin.
- To engineer a P(14)-s2B interaction by mutating phenylalanine at position 274 to tyrosine.
Main Methods:
- Site-directed mutagenesis and affinity chromatography for protein purification.
- Assays to determine antithrombin's ability to form SDS-stable complexes with thrombin.
- Quantification of thrombin and factor Xa inhibition kinetics and heparin binding affinity.
Main Results:
- A minor, inconclusive difference was observed between the wild-type and mutated antithrombin.
- The study indicates that P(14)-s2B interactions have a variable impact on different serpins.
Conclusions:
- The introduced P(14)-s2B interaction had a limited and inconclusive effect on antithrombin's RCL exposure.
- An alternative hypothesis for achieving RCL expulsion in serpins is proposed.
