Structure-Function Studies of the Bacillus subtilis Ric Proteins Identify the Fe-S Cluster-Ligating Residues and...

Area of Science:

• Bacterial protein complexes
• Structural biology
• Gene regulation

Background:

• RicA, RicF, and RicT proteins in Bacillus subtilis regulate Spo0A phosphorylation, impacting genetic competence, sporulation, and biofilm formation.
• These proteins are essential for the maturation of protein-encoding and riboswitch RNAs.
• The RicAFT complex contains two [4Fe-4S]+2 clusters, but its structure and the role of these clusters were not understood.

Purpose of the Study:

• To elucidate the structure of the RicAFT complex and its constituent proteins.
• To determine the arrangement and ligation of the two [4Fe-4S]+2 clusters within the complex.
• To understand the functional significance of the iron-sulfur clusters for Ric-associated phenotypes and RNA maturation.

Main Methods:

• X-ray crystallography was used to determine the structures of a RicAF heterotetramer and a RicA dimer.
• Biochemical analysis was performed to characterize the iron-sulfur cluster ligation and retention by individual RicT monomers.
• Mutational analysis was employed to assess the importance of cluster-ligating cysteine residues for various cellular processes.

Main Results:

• The RicAFT complex is a 1:1:1 heterotrimer.
• X-ray crystal structures revealed the quaternary structure of RicAF and RicA.
• Cluster 1 is ligated solely by RicT, while Cluster 2 involves cysteine residues from RicT, RicA, and RicF, indicating proximity and an interfacial location.
• The cluster-ligating cysteine residues are essential for cggR-gapA mRNA maturation, ricF transcript stability, competence, biofilm formation, and sporulation.

Conclusions:

• The study reveals the detailed structure of the RicAFT complex and the novel arrangement of its two iron-sulfur clusters.
• The iron-sulfur clusters and their specific ligation by RicA, RicF, and RicT are critical for multiple developmental pathways and RNA maturation in Bacillus subtilis.
• Distinct regulatory roles for RicAFT, RicAF, RicA, and RicT monomers in vivo are suggested.