Transient structure and SH3 interaction sites in an intrinsically disordered fragment of the hepatitis C virus protein NS5A

J Mol Biol. 2012 Jul 20;420(4-5):310-23. doi: 10.1016/j.jmb.2012.04.023. Epub 2012 Apr 26.

Abstract

Understanding the molecular mechanisms involved in virus replication and particle assembly is of primary fundamental and biomedical importance. Intrinsic conformational disorder plays a prominent role in viral proteins and their interaction with other viral and host cell proteins via transiently populated structural elements. Here, we report on the results of an investigation of an intrinsically disordered 188-residue fragment of the hepatitis C virus non-structural protein 5A (NS5A), which contains a classical poly-proline Src homology 3 (SH3) binding motif, using sensitivity- and resolution-optimized multidimensional NMR methods, complemented by small-angle X-ray scattering data. Our study provides detailed atomic-resolution information on transient local and long-range structure, as well as fast time scale dynamics in this NS5A fragment. In addition, we could characterize two distinct interaction modes with the SH3 domain of Bin1 (bridging integrator protein 1), a pro-apoptotic tumor suppressor. Despite being largely disordered, the protein contains three regions that transiently adopt α-helical structures, partly stabilized by long-range tertiary interactions. Two of these transient α-helices form a noncanonical SH3-binding motif, which allows low-affinity SH3 binding. Our results contribute to a better understanding of the role of the NS5A protein during hepatitis C virus infection. The present work also highlights the power of NMR spectroscopy to characterize multiple binding events including short-lived transient interactions between globular and highly disordered proteins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / metabolism*
  • Binding Sites
  • Electron Spin Resonance Spectroscopy
  • Humans
  • Magnetic Resonance Spectroscopy
  • Nuclear Proteins / metabolism*
  • Proline / chemistry*
  • Protein Binding
  • Protein Structure, Tertiary
  • Scattering, Small Angle
  • Tumor Suppressor Proteins / metabolism*
  • Viral Nonstructural Proteins / chemistry*
  • Viral Nonstructural Proteins / genetics
  • Viral Nonstructural Proteins / metabolism*
  • X-Rays
  • src Homology Domains

Substances

  • Adaptor Proteins, Signal Transducing
  • BIN1 protein, human
  • NS-5 protein, hepatitis C virus
  • Nuclear Proteins
  • Tumor Suppressor Proteins
  • Viral Nonstructural Proteins
  • Proline