Structural and dynamic properties of the HIV-1 tat transduction domain in the free and heparin-bound states.
An 11-residue basic domain of the HIV-1 tat protein, termed the tat transduction domain (TTD), has been shown to mediate transfer of biomolecules across biological membranes. The mechanism of TTD-mediated membrane translocation is currently unknown but thought to involve binding to heparan sulfate, which is found in proteoglycans that are ubiquitously present on cell surfaces. To study the mechanism of TTD-mediated membrane translocation, the TTD was fused to the C-terminus of a model cargo protein, the IgG binding domain of streptococcal protein G (PG) to form PG-TTD. NMR studies of PG-TTD in the free state indicated that the structure of the PG moiety of PG-TTD was not perturbed by the presence of the TTD and that the TTD moiety is in an extended conformation. Heteronuclear relaxation measurements of PG-TTD in the free state show that the TTD moiety of PG-TTD is relatively mobile (e.g., the average S(2) value of the TTD and PG core are approximately 0.54 and approximately 0.84, respectively). PG-TTD has been shown to bind to heparin by isothermal titration calorimetry (K(D) = 0.37 microM, Delta H = -12 kcal/mol, Delta S = -11 cal/mol/T). NMR spectroscopy demonstrated that heparin binds to the TTD moiety of PG-TTD. The heteronuclear relaxation measurements of PG-TTD in complex with heparin show that the TTD becomes less dynamic when bound to heparin (average S(2) value of the TTD is 0.69 in the presence of heparin). A model for the first step of TTD-mediated entry into cells is presented.
Hakansson
Susanna
S
Department of Biochemistry and Molecular Biology, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
Caffrey
Michael
M
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D015696:Gene Products, tat / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006493:Heparin / Q000378:metabolism*; D006497:Heparan Sulfate / Q000378:metabolism; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014161:Transduction, Genetic; D054322:tat Gene Products, Human Immunodeficiency Virus
D001426:Bacterial Proteins; D015696:Gene Products, tat; D006493:Heparin; D006497:Heparan Sulfate; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; C034284:IgG Fc-binding protein, Streptococcus