Deciphering mutational effects on inducible NO synthase conformational dynamics via quantitative cross-linking mass...

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Mammalian nitric oxide synthases (NOSs) are flavo-hemoproteins crucial for nitric oxide biosynthesis.
• Their activity depends on dynamic interdomain interactions, particularly the FMN-heme complex facilitated by Calmodulin (CaM).
• Previous studies used quantitative cross-linking mass spectrometry (qXL MS) to analyze neuronal NOS dynamics.

Purpose of the Study:

• To investigate the impact of the E546N mutation on the interdomain dynamics of human inducible NOS (iNOS) using qXL MS.
• To compare the structural and dynamic differences between wild-type (wt) and E546N mutant iNOS oxygenase/FMN (oxyFMN) constructs.
• To integrate qXL MS data with computational modeling for a comprehensive analysis of mutation-induced protein dynamics.

Main Methods:

• Quantitative cross-linking mass spectrometry (qXL MS) with parallel reaction monitoring.
• AlphaFold2 structural modeling and subsampling.
• Cross-link-guided AlphaLink2 modeling.

Main Results:

• The E546N mutation significantly reduced inter-subunit cross-links between FMN and heme domains in iNOS.
• Mutation-induced changes in CaM-iNOS domain cross-links indicated allosteric propagation of structural alterations.
• Integrated modeling and qXL MS data revealed distinct conformations for wt and E546N iNOS, with the mutant showing a higher population of undocked states.

Conclusions:

• The E546N mutation disrupts the functional dynamics of the iNOS-CaM complex by altering FMN-heme interactions.
• An integrated approach combining qXL MS and AlphaFold2 subsampling provides a quantitative framework for mapping mutation-induced dynamic changes in multidomain proteins.
• This strategy offers insights into the allosteric regulation and functional consequences of mutations in complex enzymes like iNOS.