Tat peptide‐calmodulin binding studies and bioinformatics of HIV‐1 protein–calmodulin interactions

Abstract

The human immunodeficiency virus type 1 (HIV-1) genome encodes 18 proteins and 2 peptides. Four of these proteins encode high-affinity calmodulin-binding sites for which direct interactions with calmodulin have already been described. In this study, the HIV-1 proteome is queried using an algorithm that predicts calmodulin-binding sites revealing seven new putative calmodulin-binding sites including residues 34-56 of the transactivator of transcription (Tat). Tat is a 101-residue intrinsically disordered RNA-binding protein that plays a central role in the regulation of HIV-1 replication. Interactions between a Tat peptide (residues 34-56), melittin, a well-characterized calmodulin-binding peptide, and calmodulin were examined by direct binding studies, mass spectrometry, and fluorescence. The Tat peptide binds to both calcium-saturated and apo-calmodulin with a low micromolar affinity. Conformational changes induced in the Tat peptide were determined by circular dichroism, and residues in calmodulin that interact with the peptide were identified by HSQC NMR spectroscopy. Multiple interactions between HIV-1 proteins and calmodulin, a highly promiscuous signal transduction hub protein, may be an important mechanism by which the virus controls cell physiology.

Solution Structure of Calmodulin Bound to the Binding Domain of the HIV-1 Matrix Protein

“…CaM also appears to play a role in simian immunodeficiency virus replication by interacting directly with the MA domain of Gag (27,34,37,(52)(53)(54)(55). In vivo and in vitro studies revealed that CaM interacts with additional HIV-1 proteins like Nef, Tat, and gp160 (27,34,37,52,53,56). Small-angle x-ray scattering studies have provided a global picture of CaM bound to full-length MA (57) or to short peptides derived from the MA protein (58).…”

Insights on the structural characteristics of Vim-TBS (58-81) peptide for future applications as a cell penetrating peptide

“…Helical secondary structures have already been highlighted in the literature as being necessary for cell penetration properties (48,49) because such structures seem essential particularly within the lipid phase of the membrane for its cellular uptake and stabilization. Minimization studies (at a dielectric constant of 4) have revealed that Vim-TBS (58-81) adopts a secondary structure similar to that observed in Tat (48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60), and the same mechanism of cell penetration can be attributed to Vim-TBS (58-81) based on the structure-function and activity relationships (50,51).…”

“…Conformation I, II, and III of Vim-TBS (58-81) correspond to the starting geometry with Φ, Ψ, ω values obtained after MM energy minimization studies. Similarly, final conformations obtained after MM studies for Tat (48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60) and p10 peptides were the starting conformations for MD simulation studies.…”

“…To have an in depth knowledge of its structural characteristics, structural properties of Tat (48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60) and p10 peptides were also studied. Tat (48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60) and p10 were particularly selected as the former is a well studied CPP and the later is a pro-apoptogenic fragment of p21/WAFI protein that is often used as a model cargo to evaluate cell penetrating properties. As structural versatility has been described as an important factor to be considered for deciphering cellular uptake properties by CPPs (26-28) the present work supports the hypothesis that structural plasticity could have a crucial role on its properties and functionality.…”

“…(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60) peptide Vim-TBS (58-81) peptide p10 peptide evaluation of coulomb interactions and Van der Waals interaction a cut off of 0.9 and 1.0 nm respectively was applied. Long range forces were updated every 10fs during generation of the neighbor list.…”