Structure of the His44 --> Ala single point mutant of the distal finger motif of HIV-1 nucleocapsid protein: a combined NMR, molecular dynamics simulation, and fluorescence study.
The nucleocapsid protein (NCp7) of human immunodeficiency virus type 1 (HIV-1) contains two highly conserved CCHC zinc fingers that strongly bind Zn(2+) through coordination of one His and three Cys residues. It has been suggested that NCp7 function is conformation specific since substitution of any of the zinc coordinating residues in the zinc finger motifs leads to subsequent loss of viral infectivity. To further determine the structural requirements necessary for this specific conformation, we investigated by (1)H 2D NMR and molecular dynamics simulations the structure of the distal finger motif of NCp7 in which the zinc coordinating amino acid, His 44, was substituted by a noncoordinating Ala residue. While the fold of the N-terminal part of this mutated peptide was similar to that of the native peptide, an increased lability and significant conformational changes were observed in the vicinity of the His-to-Ala mutation. Moreover, molecular dynamics simulations suggested a mechanism by which the variant peptide can bind zinc ion even though one zinc-coordinating amino acid was lacking. Using the fluorescence of the naturally occurring Trp37 residue, the binding affinity of the variant peptide to the (TG)(3) model oligonucleotide was found to be decreased by about 2 orders of magnitude with respect with the native peptide. Modeling of the DNA:NCp7 complex using structures of the variant peptide suggests that the residues forming a hydrophobic cleft in the native protein are improperly oriented for efficient DNA binding by the variant peptide.
Stote
Roland H
RH
Laboratoire de Chimie Biophysique, ISIS UMR 7006 CNRS, Université Louis Pasteur, 8 allée Gaspard Monge, BP 70028, F-67083 Strasbourg Cedex, France. rstote@isis.u-strasbg.fr
Kellenberger
Esther
E
Muller
Hervé
H
Bombarda
Elisa
E
Roques
Bernard P
BP
Kieffer
Bruno
B
Mély
Yves
Y
D000409:Alanine / Q000737:chemistry* / Q000235:genetics; D020816:Amino Acid Motifs; D036022:Capsid Proteins / Q000737:chemistry* / Q000235:genetics; D015683:Gene Products, gag / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D017354:Point Mutation; D013050:Spectrometry, Fluorescence; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D006639:Histidine; D000409:Alanine