Dynamic studies of H-Ras•GTPγS interactions with nucleotide exchange factor Sos reveal a transient ternary complex formation in solution

Sci Rep. 2016 Jul 14:6:29706. doi: 10.1038/srep29706.

Abstract

The cycling between GDP- and GTP- bound forms of the Ras protein is partly regulated by the binding of Sos. The structural/dynamic behavior of the complex formed between activated Sos and Ras at the point of the functional cycle where the nucleotide exchange is completed has not been described to date. Here we show that solution NMR spectra of H-Ras∙GTPγS mixed with a functional fragment of Sos (Sos(Cat)) at a 2:1 ratio are consistent with the formation of a rather dynamic assembly. H-Ras∙GTPγS binding was in fast exchange on the NMR timescale and retained a significant degree of molecular tumbling independent of Sos(Cat), while Sos(Cat) also tumbled largely independently of H-Ras. Estimates of apparent molecular weight from both NMR data and SEC-MALS revealed that, at most, only one H-Ras∙GTPγS molecule appears stably bound to Sos. The weak transient interaction between Sos and the second H-Ras∙GTPγS may provide a necessary mechanism for complex dissociation upon the completion of the native GDP → GTP exchange reaction, but also explains measurable GTP → GTP exchange activity of Sos routinely observed in in vitro assays that use fluorescently-labelled analogs of GTP. Overall, the data presents the first dynamic snapshot of Ras functional cycle as controlled by Sos.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catalytic Domain
  • Guanosine 5'-O-(3-Thiotriphosphate) / chemistry
  • Guanosine 5'-O-(3-Thiotriphosphate) / metabolism*
  • Guanosine Diphosphate / chemistry
  • Guanosine Diphosphate / metabolism
  • Guanosine Triphosphate / chemistry
  • Guanosine Triphosphate / metabolism
  • Humans
  • Kinetics
  • Magnetic Resonance Spectroscopy
  • Models, Molecular
  • Multiprotein Complexes / chemistry
  • Multiprotein Complexes / metabolism*
  • Protein Binding
  • Protein Conformation
  • Proto-Oncogene Proteins p21(ras) / chemistry
  • Proto-Oncogene Proteins p21(ras) / metabolism*
  • SOS1 Protein / chemistry
  • SOS1 Protein / metabolism*
  • Solutions / chemistry

Substances

  • Multiprotein Complexes
  • SOS1 Protein
  • Solutions
  • Guanosine Diphosphate
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • Guanosine Triphosphate
  • HRAS protein, human
  • Proto-Oncogene Proteins p21(ras)