Homonuclear ¹H‐NMR assignment and structural characterization of human immunodeficiency virus type 1 Tat Mal protein

Abstract

The transacting transcriptional activator (Tat) is a viral protein essential for activation of the human immunodeficiency virus (HIV) genes, and it plays an important role in HIV induced immunodeficiency. We report the NMR structural characterization of the active Tat Mal variant that belongs to a highly virulent D-subtype HIV type-1 (HIV-1) strain (Mal) found mainly in Africa. A full Tat Mal protein (87 residues) is synthesized. This synthetic protein is active in a transactivation assay with HeLa cells infected with the HIV long terminal repeated noncoding sequences of the HIV-1 provirus (LTR) lac Z gene. Homonuclear (1)H-NMR spectra allows the sequential assignment of the Tat Mal spin systems. Simulating annealing generates 20 conformers with similar folding. The geometry of the mean structure is optimized with energy minimization to obtain a final structure. As the European variant (Tat Bru) the N-terminal region of Tat Mal constitutes the core, and there is a hydrophobic pocket composed of the conserved Trp 11 interacting with several aromatic residues. The two functional regions of Tat (basic and the cysteine-rich regions) are well exposed to the solvent. A short alpha-helix is observed in region V adjacent to the basic region. This alpha helix induces local structural variations compared to the NMR structure of Tat Bru, and it brings the cysteine-rich and basic regions closer. This study suggests that similar folding exists among Tat variants.

The Glutamine-rich Region of the HIV-1 Tat Protein Is Involved in T-cell Apoptosis

“…The low intensity of the negative bands at 200 nm suggests that the three proteins are not random-coil but rather structured with ␤-turns as the main secondary structures. This is in agreement with the Tat Mal structure determined by two-dimensional NMR (19). Tat Ug05RP and Tat Mal have similar CD spectra, whereas differences are observed in the CD spectrum of Tat Ug11LTS (Fig.…”

“…Interestingly, we found that there is just 5% ␣-helix structure (three to four residues) in both Tat Mal and Tal Ug05RP, but not in Tat Ug11LTS. Molecular modeling of Ug05RP using the NMR structure of Tat Mal (19) showed that it is possible to obtain a structure similar to that of Tat Mal despite the mutations in these two proteins, in agreement with the CD spectra (Fig. 5).…”

“…Structural Studies-Ug05RP and Ug11LTS share 87% sequence homology with Tat Mal, the two-dimensional NMR structure of which has already been defined (19), and 91% sequence homology between themselves. We compared the CD spectra of Tat Mal with those of the Ugandan Tat variants (Fig.…”

“…The Tat Mal structure was obtained from NMR studies (Protein Data Bank code 1K5K) (19). Models were optimized with CVFF (consistent valence force field) in terms of the internal energies using the van der Waals energy to monitor each step of the model.…”

“…Region IV (residues 49 -59) is rich in basic residues and has a highly conserved sequence that is important for trans-activation and binding to the nucleotide trans-activator-responsive region (TAR) sequence (11) and in the uptake of Tat by cells (18). Region V (residues 60 -72) is a glutamine-rich region that forms an ␣-helix in the subtype D Tat NMR structure (19). Tat-TAR binding induces the formation of an ␣-helix in region V (11).…”