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A triad of lys12, lys41, arg78 spatial domain, a novel identified heparin binding site on tat protein, facilitates tat-driven cell adhesion.

Ai J, Xin X, Zheng M, Wang S, Peng S, Li J, Wang L, Jiang H, Geng M - PLoS ONE (2008)

Bottom Line: However, findings that deletion or substitution of the basic domain inhibits but does not completely eliminate Tat-heparin interactions suggest that the basic domain is not the sole Tat heparin binding site.This domain was also found to facilitate Tat-driven β1 integrin activation, producing subsequent SLK cell adhesion in an HSPG-dependent manner, but was not involved in Tat internalization.The identification of this new heparin binding site may foster further insight into the nature of Tat-heparin interactions and subsequent biological functions, facilitating the rational design of new therapeutics against Tat-mediated pathological events.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Glycobiology, Marine Drug and Food Institute, Ocean University of China, Qingdao, People's Republic of China.

ABSTRACT
Tat protein, released by HIV-infected cells, has a battery of important biological effects leading to distinct AIDS-associated pathologies. Cell surface heparan sulfate protoglycans (HSPGs) have been accepted as endogenous Tat receptors, and the Tat basic domain has been identified as the heparin binding site. However, findings that deletion or substitution of the basic domain inhibits but does not completely eliminate Tat-heparin interactions suggest that the basic domain is not the sole Tat heparin binding site. In the current study, an approach integrating computational modeling, mutagenesis, biophysical and cell-based assays was used to elucidate a novel, high affinity heparin-binding site: a Lys12, Lys41, Arg78 (KKR) spatial domain. This domain was also found to facilitate Tat-driven β1 integrin activation, producing subsequent SLK cell adhesion in an HSPG-dependent manner, but was not involved in Tat internalization. The identification of this new heparin binding site may foster further insight into the nature of Tat-heparin interactions and subsequent biological functions, facilitating the rational design of new therapeutics against Tat-mediated pathological events.

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Computational docking model of HIV-Tat with heparin-derived fragments.Predicted interaction modes between HIV-Tat and heparin-derived fragments, including di- (A, B and C), tetra- (D), hexa- (E), and octa-saccharide (F) were indicated. Tat was represented in surface and oligosaccharides in stick. The Tat surface was colored by electrostatic potential in A. In B–F, it was colored in white, with the KKR region and basic domain highlighted in green and yellow, respectively. A close-up view of the binding interface between the di-saccharide and the KKR (Lys12, Lys41 and Arg78) region was shown in C, where the surface was set to transparent, the protein main chain was shown in ‘worm’ representation, and hydrogen-bonds are represented by yellow dotted lines.
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pone-0002662-g001: Computational docking model of HIV-Tat with heparin-derived fragments.Predicted interaction modes between HIV-Tat and heparin-derived fragments, including di- (A, B and C), tetra- (D), hexa- (E), and octa-saccharide (F) were indicated. Tat was represented in surface and oligosaccharides in stick. The Tat surface was colored by electrostatic potential in A. In B–F, it was colored in white, with the KKR region and basic domain highlighted in green and yellow, respectively. A close-up view of the binding interface between the di-saccharide and the KKR (Lys12, Lys41 and Arg78) region was shown in C, where the surface was set to transparent, the protein main chain was shown in ‘worm’ representation, and hydrogen-bonds are represented by yellow dotted lines.

Mentions: Computational modeling was first employed with the aim of identifying the potential heparin binding sites. Here, the homology model of Tat protein (Tat-III) constructed on the basis of NMR data was utilized as the target structure, and several heparin oligosaccharides including di-, tetra-, hexa-, and octa-saccharides were selected as docking probes. In the disaccharide heparin model, heparin was noted to preferentially interact with a triad of basic residues (Lys12, Lys41 and Arg78 (KKR); Figure 1, highlighted in green). For better resolution, a closeup view of the binding interface was developed. In the zoomed view, It was evident that the 2-O-sulfate of the glucuronic acid ring was in close proximity to Lys41 in Nζ, which itself is engaged in two tight salt bridges (2.84 and 2.88 Å, respectively). Nζ atom of Lys12, in a like manner, was involved in two hydrogen bonds, established with the 3-O (3.30 Å) and 2-O (2.74 Å) of the glucuronic acid ring. Another strong salt bridge (2.77 Å) was observed between the 5-carboxylate group and Nη of Arg78. The binding free energy of the disaccharide was further calculated as −8.84 kcal/mol, indicative of the nanomolar or even lower level of the binding affinity of Tat-III for heparin.


A triad of lys12, lys41, arg78 spatial domain, a novel identified heparin binding site on tat protein, facilitates tat-driven cell adhesion.

Ai J, Xin X, Zheng M, Wang S, Peng S, Li J, Wang L, Jiang H, Geng M - PLoS ONE (2008)

Computational docking model of HIV-Tat with heparin-derived fragments.Predicted interaction modes between HIV-Tat and heparin-derived fragments, including di- (A, B and C), tetra- (D), hexa- (E), and octa-saccharide (F) were indicated. Tat was represented in surface and oligosaccharides in stick. The Tat surface was colored by electrostatic potential in A. In B–F, it was colored in white, with the KKR region and basic domain highlighted in green and yellow, respectively. A close-up view of the binding interface between the di-saccharide and the KKR (Lys12, Lys41 and Arg78) region was shown in C, where the surface was set to transparent, the protein main chain was shown in ‘worm’ representation, and hydrogen-bonds are represented by yellow dotted lines.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3278312&req=5

pone-0002662-g001: Computational docking model of HIV-Tat with heparin-derived fragments.Predicted interaction modes between HIV-Tat and heparin-derived fragments, including di- (A, B and C), tetra- (D), hexa- (E), and octa-saccharide (F) were indicated. Tat was represented in surface and oligosaccharides in stick. The Tat surface was colored by electrostatic potential in A. In B–F, it was colored in white, with the KKR region and basic domain highlighted in green and yellow, respectively. A close-up view of the binding interface between the di-saccharide and the KKR (Lys12, Lys41 and Arg78) region was shown in C, where the surface was set to transparent, the protein main chain was shown in ‘worm’ representation, and hydrogen-bonds are represented by yellow dotted lines.
Mentions: Computational modeling was first employed with the aim of identifying the potential heparin binding sites. Here, the homology model of Tat protein (Tat-III) constructed on the basis of NMR data was utilized as the target structure, and several heparin oligosaccharides including di-, tetra-, hexa-, and octa-saccharides were selected as docking probes. In the disaccharide heparin model, heparin was noted to preferentially interact with a triad of basic residues (Lys12, Lys41 and Arg78 (KKR); Figure 1, highlighted in green). For better resolution, a closeup view of the binding interface was developed. In the zoomed view, It was evident that the 2-O-sulfate of the glucuronic acid ring was in close proximity to Lys41 in Nζ, which itself is engaged in two tight salt bridges (2.84 and 2.88 Å, respectively). Nζ atom of Lys12, in a like manner, was involved in two hydrogen bonds, established with the 3-O (3.30 Å) and 2-O (2.74 Å) of the glucuronic acid ring. Another strong salt bridge (2.77 Å) was observed between the 5-carboxylate group and Nη of Arg78. The binding free energy of the disaccharide was further calculated as −8.84 kcal/mol, indicative of the nanomolar or even lower level of the binding affinity of Tat-III for heparin.

Bottom Line: However, findings that deletion or substitution of the basic domain inhibits but does not completely eliminate Tat-heparin interactions suggest that the basic domain is not the sole Tat heparin binding site.This domain was also found to facilitate Tat-driven β1 integrin activation, producing subsequent SLK cell adhesion in an HSPG-dependent manner, but was not involved in Tat internalization.The identification of this new heparin binding site may foster further insight into the nature of Tat-heparin interactions and subsequent biological functions, facilitating the rational design of new therapeutics against Tat-mediated pathological events.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Glycobiology, Marine Drug and Food Institute, Ocean University of China, Qingdao, People's Republic of China.

ABSTRACT
Tat protein, released by HIV-infected cells, has a battery of important biological effects leading to distinct AIDS-associated pathologies. Cell surface heparan sulfate protoglycans (HSPGs) have been accepted as endogenous Tat receptors, and the Tat basic domain has been identified as the heparin binding site. However, findings that deletion or substitution of the basic domain inhibits but does not completely eliminate Tat-heparin interactions suggest that the basic domain is not the sole Tat heparin binding site. In the current study, an approach integrating computational modeling, mutagenesis, biophysical and cell-based assays was used to elucidate a novel, high affinity heparin-binding site: a Lys12, Lys41, Arg78 (KKR) spatial domain. This domain was also found to facilitate Tat-driven β1 integrin activation, producing subsequent SLK cell adhesion in an HSPG-dependent manner, but was not involved in Tat internalization. The identification of this new heparin binding site may foster further insight into the nature of Tat-heparin interactions and subsequent biological functions, facilitating the rational design of new therapeutics against Tat-mediated pathological events.

Show MeSH
Related in: MedlinePlus