Calcium-dependent association of calmodulin with the rubella virus nonstructural protease domain

J Biol Chem. 2010 Mar 19;285(12):8855-68. doi: 10.1074/jbc.M109.097063. Epub 2010 Jan 19.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites
  • Calcium / chemistry*
  • Calmodulin / chemistry*
  • Chlorocebus aethiops
  • Cysteine Proteases / chemistry
  • Magnetic Resonance Spectroscopy / methods
  • Mutagenesis, Site-Directed
  • Peptides / chemistry
  • Protein Structure, Tertiary
  • Rubella virus / enzymology*
  • Spectrometry, Fluorescence / methods
  • Vero Cells
  • Viral Nonstructural Proteins / chemistry*
  • Zinc / chemistry

Substances

  • Calmodulin
  • Peptides
  • Viral Nonstructural Proteins
  • Cysteine Proteases
  • Zinc
  • Calcium