Characterization of the calmodulin binding domain of SIV transmembrane glycoprotein by NMR and CD spectroscopy.
Recent experimental evidence has shown that the C-terminal peptide of the HIV/SIV transmembrane glycoprotein 41 (gp41) can bind very tightly to calmodulin (CaM). These findings imply a potential mechanism for HIV/SIV cytopathogenesis, which involves the uncoupling of some critical cellular signal transduction pathways that are normally mediated by CaM. Here, we present circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy studies of a 28-residue synthetic peptide, SIV-L, corresponding to the C-terminal portion of the SIV transmembrane glycoprotein gp41. CD studies recorded in aqueous solution show a dramatic increase in the amount of alpha-helical structure of the SIV-L peptide upon binding to calcium-CaM. Two-dimensional NMR experiments were performed to determine the secondary structure of the peptide in 25% aqueous trifluoroethanol solution. In this alpha-helix inducing solvent, the observed nuclear Overhauser effects, as well as the alpha 1H and alpha 13C chemical shift changes, demonstrate that a continuous alpha-helix is formed from W3 to L28, although there is some distortion around P17. This result is in accordance with those obtained for many other CaM-binding peptides. Subsequent one-dimensional NMR titration experiments of calcium-CaM and the SIV-L peptide suggest that the peptide can bind to CaM with a 1:1 stoichiometry and that the peptide binding involves both the N- and C-lobe of CaM. However, gel mobility shift assays suggest that the peptide CaM interaction may be more complicated, as oligomeric forms of CaM and the SIV-L peptide were found. These studies provide a potential molecular basis for HIV/SIV cytopathogenesis.
Yuan
T
T
Department of Biological Sciences, University of Calgary, Alberta, Canada.
Mietzner
T A
TA
Montelaro
R C
RC
Vogel
H J
HJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D009682:Magnetic Resonance Spectroscopy; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D012191:Retroviridae Proteins / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D015302:Simian Immunodeficiency Virus; D013050:Spectrometry, Fluorescence; D014270:Trifluoroethanol / Q000494:pharmacology
D002147:Calmodulin; D008562:Membrane Glycoproteins; D012191:Retroviridae Proteins; C082614:SIV envelope protein gp41; D014270:Trifluoroethanol