Details

Autor(en) / Beteiligte

• Zhou, Yubin
• Tzeng, Wen-Pin
• Wong, Hing-Cheung
• Ye, Yiming
• Jiang, Jie
• Chen, Yanyi
• Huang, Yun
• Suppiah, Suganthi
• Frey, Teryl K.
• Yang, Jenny J.

Titel

Calcium-dependent Association of Calmodulin with the Rubella Virus Nonstructural Protease Domain

Ist Teil von

• The Journal of biological chemistry, 2010-03, Vol.285 (12), p.8855-8868

Ort / Verlag

United States: Elsevier Inc

Erscheinungsjahr

2010

Quelle

MEDLINE

Beschreibungen/Notizen

• The rubella virus (RUBV) nonstructural (NS) protease domain, a Ca2+- and Zn2+-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 Ca2+-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 Ca2+/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 Ca2+/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 Ca2+/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.