A Triad of Lys12, Lys41, Arg78 Spatial Domain, a Novel Identified Heparin Binding Site on Tat Protein, Facilitates Tat-Driven Cell Adhesion

Jing Ai,Meiyu Geng,Jing Li,Shuai Wang,Xianliang Xin,Limei Wang,Shuying Peng,Mingyue Zheng,Hualiang Jiang,Maxim Antopolsky

doi:10.1371/journal.pone.0002662

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.

Highlights

The transactivating factor of the HIV-1 virus (Tat), released from human immunodeficiency virus type 1 (HIV-1) infected cells, has been pointed towards a variety of important biological functions related to the distinct AIDS-associated pathologies in AIDS patients, including neuropathies[1], and immune suppression[2,3] and increased tumorigenesis in AIDS patients[3].Tat is a polypeptide of 86–102 amino acids depending on the viral strain[4]
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
Rusnati et al.[9] previously recognized that the neutralization of six arginine residues within the basic domain was not sufficient to abolish the heparin binding capacity of Tat. They suggested that the remaining lysine residues in the basic region and/or other basic amino acids scattered on the surface of the Tat protein contributed to the residual heparin binding capacity they observed

Summary

Introduction

The transactivating factor of the HIV-1 virus (Tat), released from human immunodeficiency virus type 1 (HIV-1) infected cells, has been pointed towards a variety of important biological functions related to the distinct AIDS-associated pathologies in AIDS patients, including neuropathies[1], and immune suppression[2,3] and increased tumorigenesis in AIDS patients[3].Tat is a polypeptide of 86–102 amino acids depending on the viral strain[4]. Amino acids 1–72 are endowed with full transactivating activity[5], present within a basic domain (amino acids 48–57) constituted by a stretch of repeated Arg and Lys residues critical for a number of biological functions, while amino acids 72–86 of the carboxy terminal region contain an Arg-GlyAsp (RGD) motif responsible for binding to integrin receptors and subsequent cell adhesion[4,6]. Tat-driven activities depend precisely on its interaction with cell surface heparan sulfate (HS) and heparin through their negatively charged sulfate groups[7]. Recent limited studies have challenged this notion with the demonstration that deletion or substitution of the basic domain really affects, but does not completely eliminate Tat-heparin interactions[9]. This led us to hypothesize that other heparin binding sites might exist

Methods

Results

Conclusion