Abstract
MecA is an adaptor protein that regulates the assembly and activity of the ATP-dependent ClpCP protease in Bacillus subtilis. MecA contains two domains. Although the amino-terminal domain of MecA recruits substrate proteins such as ComK and ComS, the carboxyl-terminal domain (residues 121-218) has dual roles in the regulation and function of ClpCP protease. MecA-(121-218) facilitates the assembly of ClpCP oligomer, which is required for the protease activity of ClpCP. This domain was identified to be a non-recycling degradation tag that targets heterologous fusion proteins to the ClpCP protease for degradation. To elucidate the mechanism of MecA, we determined the crystal structure of MecA-(121-218) at 2.2 A resolution, which reveals a previously uncharacterized alpha/beta fold. Structure-guided mutagenesis allows identification of surface residues that are essential for the function of MecA. We also solved the structure of a carboxyl-terminal domain of YpbH, a paralogue of MecA in B. subtilis, at 2.4 A resolution. Despite low sequence identity, the two structures share essentially the same fold. The presence of MecA homologues in other bacterial species suggests conservation of a large family of unique degradation tags.
Highlights
Translocate the substrate proteins to the proteolytic chamber within the double ring of ClpP heptamers for degradation (3). Among these ATPases, ClpC is intriguing because it requires an adaptor protein to stimulate its ATPase activity and to facilitate its oligomerization and activation
We demonstrated that MecA-(121–218) is a nonrecycling degradation tag that targets heterologous fusion proteins for degradation by the ClpCP protease
We showed that the C-terminal domain of YpbH, which share a sequence identity of 31% and sequence similarity of 63% with MecA-(121–218), functions as MecA-(121–218), both for assembly of the ClpCP protease and as a non-recycling degradation tag
Summary
Protein Preparation—All clones were generated using a standard PCR-based cloning strategy, and the identities of individual clones were verified through double-strand plasmid sequencing. The soluble fraction of the E. coli lysate was purified through a glutathione-Sepharose column and cleaved by the PreScissionTM protease (GE Healthcare) wherever necessary. The soluble fraction of maltose-binding protein fusion ComK in the E. coli extracts was purified over amylose resin (New England Biolabs) and cleaved by the PreScission protease (GE Healthcare). All MecA variants and ComK were added at 6 M final concentration. Crystals of MecA-(121–218) were grown at 18 °C by mixing an equal volume of the protein (10 mg/ml) with reservoir solution containing 14% (w/v) polyethylene glycol 3350, 300 mM CaCl2, 4% ethylene glycol, and 0.1 M Bis-Tris, pH 6.2. Crystals of YpbH-(101–194) were grown at 18 °C by mixing an equal volume of the protein (10 mg/ml) with reservoir solution containing 1.85 M (NH4)2SO4, 2% polyethylene glycol 400, and 100 mM HEPES at pH 7.5
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