Capacity of simian immunodeficiency virus strain mac Nef for high-affinity Src homology 3 (SH3) binding revealed by ligand-tailored SH3 domains.
The simian immunodeficiency virus (SIV) Nef protein contains a consensus Src-homology 3 (SH3) binding motif. However, no SH3-domain proteins showing strong binding to SIV Nef have yet been found, and its potential capacity for high-affinity SH3 binding has therefore remained unproven. Here we have used phage-display-assisted protein engineering to develop artificial SH3 domains that bind tightly to SIV strain mac (SIVmac) Nef. Substitution of six amino acids in the RT loop region of Hck-SH3 with the sequence E/DGWWG resulted in SH3 domains that bound in vitro to SIVmac Nef much better than the natural Hck- or Fyn-SH3 domains. These novel SH3 domains also efficiently associated with SIVmac Nef when co-expressed in 293T cells and displayed a strikingly differential specificity when compared with SH3 domains similarly targeted for binding to human immunodeficiency virus type 1 (HIV-1) Nef. Thus, SIVmac Nef is competent for high-affinity SH3 binding, but its natural SH3 protein partners are likely to be different from those of HIV-1 Nef.
Hiipakka
Marita
M
Institute of Medical Technology and Tampere University Hospital, FIN-33014 University of Tampere, Finland1.
Saksela
Kalle
K
Institute of Medical Technology and Tampere University Hospital, FIN-33014 University of Tampere, Finland1.
D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D015693:Gene Products, nef / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D008024:Ligands; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D015302:Simian Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*; D018909:src Homology Domains / Q000235:genetics / Q000502:physiology*
D015693:Gene Products, nef; D008024:Ligands