The structural principles underlying molybdenum insertase complex assembly

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Trace element molybdenum (Mo) is essential for biological activity, primarily within the nitrogenase-specific FeMo cofactor or the molybdenum cofactor (Moco).
• Moco comprises an organic molybdopterin (MPT) component and an inorganic Mo-center.
• Molybdenum insertases (Mo-insertases) catalyze the critical Mo-center formation, featuring distinct G- and E-domains with partially understood functions.

Purpose of the Study:

• To elucidate the structural and functional relationship between the G- and E-domains of the eukaryotic Mo-insertase Cnx1 complex.
• To investigate the overall architecture of the Cnx1 complex and its implications for catalytic activity.

Main Methods:

• Cross-linking mass spectrometry was employed to capture protein-protein interactions within the Cnx1 complex.
• Computational modeling was utilized to generate a structural model based on the cross-linking data.
• Analysis focused on the assembly and domain orientation within the eukaryotic Mo-insertase.

Main Results:

• A structural model of the eukaryotic Cnx1 complex was successfully generated.
• Cnx1 was revealed to form an asymmetric hexameric complex.
• The hexameric structure facilitates the alignment of the G- and E-domain active sites into a catalytically productive orientation.

Conclusions:

• The asymmetric hexameric structure of Cnx1 is crucial for coordinating the sequential steps of molybdenum cofactor synthesis.
• This structural arrangement optimizes the interaction between the G-domain (MPT adenylation) and E-domain (molybdate insertion) active sites.
• The findings provide key insights into the mechanism of molybdenum insertion in eukaryotes.