ABSTRACT
The COP9 (Constitutive photomorphogenesis 9) signalosome (CSN), a large multiprotein complex that resembles the 19S lid of the 26S proteasome, plays a central role in the regulation of the E3-cullin RING ubiquitin ligases (CRLs). The catalytic activity of the CSN complex, carried by subunit 5 (CSN5/Jab1), resides in the deneddylation of the CRLs that is the hydrolysis of the cullin-neural precursor cell expressed developmentally downregulated gene 8 (Nedd8)isopeptide bond. Whereas CSN-dependent CSN5 displays isopeptidase activity, it is intrinsically inactive in other physiologically relevant forms. Here we analyze the crystal structure of CSN5 in its catalytically inactive form to illuminate the molecular basis for its activation state. We show that CSN5 presents a catalytic domain that brings essential elements to understand its activity control. Although the CSN5 active site is catalytically competent and compatible with di-isopeptide binding, the Ins-1 segment obstructs access to its substrate-binding site, and structural rearrangements are necessary for the Nedd8-binding pocket formation. Detailed study of CSN5 by molecular dynamics unveils signs of flexibility and plasticity of the Ins-1 segment. These analyses led to the identification of a molecular trigger implicated in the active/inactive switch that is sufficient to impose on CSN5 an active isopeptidase state. We show that a single mutation in the Ins-1 segment restores biologically relevant deneddylase activity. This study presents detailed insights into CSN5 regulation. Additionally, a dynamic monomer-dimer equilibrium exists both in vitro and in vivo and may be functionally relevant.
Subject(s)
Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/metabolism , Peptide Hydrolases/chemistry , Peptide Hydrolases/metabolism , Amino Acid Sequence , Arginine/chemistry , COP9 Signalosome Complex , Catalytic Domain , Crystallography, X-Ray , Enzyme Activation , Humans , Intracellular Signaling Peptides and Proteins/genetics , Models, Molecular , Molecular Dynamics Simulation , Molecular Sequence Data , Mutagenesis, Site-Directed , NEDD8 Protein , Peptide Hydrolases/genetics , Protein Multimerization , Protein Structure, Quaternary , Protein Structure, Tertiary , Protein Subunits , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Ubiquitins/metabolism , Zinc/metabolismABSTRACT
The protein Rv0020c from Mycobacterium tuberculosis, also called FhaA, is one of the major substrates of the essential Ser/Thr protein kinase (STPK) PknB. The protein is composed of three domains and is phosphorylated on a unique site in its N terminus. We solved the solution structure of both N- and C-terminal domains and demonstrated that the approximately 300 amino acids of the intermediate domain are not folded. We present evidence that the FHA, a phosphospecific binding domain, of Rv0020c does not interact with the phosphorylated catalytic domains of PknB, but with the phosphorylated juxtamembrane domain that links the catalytic domain to the mycobacterial membrane. We also demonstrated that the degree and the pattern of phosphorylation of this juxtamembrane domain modulates the affinity of the substrate (Rv0020c) toward its kinase (PknB).
