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 b1 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.