title: Calcium-dependent association of calmodulin with the rubella virus nonstructural protease domain.
abstract: The rubella virus (RUBV) nonstructural (NS) protease domain, a Ca(2+)- and Zn(2+)-binding papain-like cysteine protease domain within the nonstructural replicase polyprotein precursor, is responsible for the self-cleavage of the precursor into two mature products, P150 and P90, that compose the replication complex that mediates viral RNA replication; the NS protease resides at the C terminus of P150. Here we report the Ca(2+)-dependent, stoichiometric association of calmodulin (CaM) with the RUBV NS protease. Co-immunoprecipitation and pulldown assays coupled with site-directed mutagenesis demonstrated that both the P150 protein and a 110-residue minidomain within NS protease interacted directly with Ca(2+)/CaM. The specific interaction was mapped to a putative CaM-binding domain. A 32-mer peptide (residues 1152-1183, denoted as RUBpep) containing the putative CaM-binding domain was used to investigate the association of RUBV NS protease with CaM or its N- and C-terminal subdomains. We found that RUBpep bound to Ca(2+)/CaM with a dissociation constant of 100-300 nm. The C-terminal subdomain of CaM preferentially bound to RUBpep with an affinity 12.5-fold stronger than the N-terminal subdomain. Fluorescence, circular dichroism and NMR spectroscopic studies revealed a "wrapping around" mode of interaction between RUBpep and Ca(2+)/CaM with substantially more helical structure in RUBpep and a global structural change in CaM upon complex formation. Using a site-directed mutagenesis approach, we further demonstrated that association of CaM with the CaM-binding domain in the RUBV NS protease was necessary for NS protease activity and infectivity.
authors.affiliation: Department of Chemistry, Georgia State University, Atlanta, Georgia 30303, USA.
authors.forename: Yubin
authors.initials: Y
authors.lastname: Zhou
authors.forename: Wen-Pin
authors.initials: WP
authors.lastname: Tzeng
authors.forename: Hing-Cheung
authors.initials: HC
authors.lastname: Wong
authors.forename: Yiming
authors.initials: Y
authors.lastname: Ye
authors.forename: Jie
authors.initials: J
authors.lastname: Jiang
authors.forename: Yanyi
authors.initials: Y
authors.lastname: Chen
authors.forename: Yun
authors.initials: Y
authors.lastname: Huang
authors.forename: Suganthi
authors.initials: S
authors.lastname: Suppiah
authors.forename: Teryl K
authors.initials: TK
authors.lastname: Frey
authors.forename: Jenny J
authors.initials: JJ
authors.lastname: Yang
mesh_terms: D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D002147:Calmodulin / Q000737:chemistry*; D002522:Chlorocebus aethiops; D057056:Cysteine Proteases / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012412:Rubella virus / Q000201:enzymology*; D013050:Spectrometry, Fluorescence / Q000379:methods; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000737:chemistry*; D015032:Zinc / Q000737:chemistry
chemical_list: D002147:Calmodulin; D010455:Peptides; D017361:Viral Nonstructural Proteins; D057056:Cysteine Proteases; D015032:Zinc; D002118:Calcium