Complex interactions of HIV-1 nucleocapsid protein with oligonucleotides.
The HIV-1 nucleocapsid (NC) protein is a small, basic protein containing two retroviral zinc fingers. It is a highly active nucleic acid chaperone; because of this activity, it plays a crucial role in virus replication as a cofactor during reverse transcription, and is probably important in other steps of the replication cycle as well. We previously reported that NC binds with high-affinity to the repeating sequence d(TG)n. We have now analyzed the interaction between NC and d(TG)4 in considerable detail, using surface plasmon resonance (SPR), tryptophan fluorescence quenching (TFQ), fluorescence anisotropy (FA), isothermal titration calorimetry (ITC) and electrospray ionization Fourier transform mass spectrometry (ESI-FTMS). Our results show that the interactions between these two molecules are surprisngly complex: while the K(d) for binding of a single d(TG)4 molecule to NC is only approximately 5 nM in 150 mM NaCl, a single NC molecule is capable of interacting with more than one d(TG)4 molecule, and conversely, more than one NC molecule can bind to a single d(TG)4 molecule. The strengths of these additional binding reactions are quantitated. The implications of this multivalency for the functions of NC in virus replication are discussed.
Fisher
Robert J
RJ
Protein Chemistry Laboratory, SAIC-Frederick, Inc. NCI Frederick, Frederick, MD 21702, USA. fisher@ncifcrf.gov
Fivash
Matthew J
MJ
Stephen
Andrew G
AG
Hagan
Nathan A
NA
Shenoy
Shilpa R
SR
Medaglia
Maxine V
MV
Smith
Lindsey R
LR
Worthy
Karen M
KM
Simpson
John T
JT
Shoemaker
Robert
R
McNitt
Karen L
KL
Johnson
Donald G
DG
Hixson
Catherine V
CV
Gorelick
Robert J
RJ
Fabris
Daniele
D
Henderson
Louis E
LE
Rein
Alan
A
D001665:Binding Sites; D001667:Binding, Competitive; D002151:Calorimetry; D036022:Capsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005453:Fluorescence; D005454:Fluorescence Polarization; D015683:Gene Products, gag / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D020349:Surface Plasmon Resonance; D014364:Tryptophan / Q000737:chemistry; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D009838:Oligodeoxyribonucleotides; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D014364:Tryptophan