Identification Of A Novel Heparin Binding Site On Tat Protein And Its Function Study

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, and immune suppression and increased tumorigenesis in AIDS patients.Tat is a polypeptide of 86–102 amino acids depending on the viral strain. Amino acids 1–72 are endowed with full transactivating activity, 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-Lys-Asp (RGD) motif responsible for binding to integrin receptors and subsequent cell adhesion.Tat-driven activities depend precisely on its interaction with cell surface heparan sulfate (HS) and heparin through their negatively charged sulfate groups. The basic domain has long been recognized as the sole contributor mediating this interaction via its heparin binding properties. However, 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. This led us to hypothesize that other heparin binding sites might exist.Toward this end, we sought to Toward this end, we sought to identify novel Tat heparin binding sites using the molecular simulation for appreciable prediction, combined with the surface plasmon resonance (SPR)-based competitive inhibition assay and cell-based functional evaluation with the introduction of several relevant targeted mutant Tat proteins. Encouragingly, a triad of basic residues—Lys12, Lys41 and Arg78 (KKR)—that are spatially enclosed rather than sequentially oriented, was thus identified as a hereto unrecognized high-affinity heparin binding site which help facilitate Tat-drivenβ1 integrin activation and subsequent adhesion in an heparan sulfate protoglycan (HSPG)-dependent manner. Clearly, our findings will facilitate a crucial touch on a precise understanding of the comprehensive roles of Tat as well as aid in design of new therapeutic agents.Molecular simulation predicts that Lys12, Lys41, and Arg78 comprise a novel Tat heparin binding siteIn the present study, a homology model of Tat-III constructed using its NMR structure docked with heparin oligos probes provided a promising candidate motif for heparin binding in addition to the well-recognized basic domain. Further analysis of the free energy of heparin-Tat-III interactions and detailed assessment of their atomic associations, coupled with flexible molecular docking schemes substantially predicted the confluence of a triad of basic residues Lys12, Lys41, Arg78 (designated as KKR triad) into a previously unrecognized binding site for heparin.Preparation of Wild Type HIV-1 Tat and Its MutantsTo characterize the Tat KKR motif engaged with heparin, a series of Tat mutants were expressed in E. coli as GST-Tat fusion proteins, purified from the bacterial lysates by glutathione-agarose affinity chromatography and checked by SDS-PAGE. The identification of mutant products was further carried out by MS-MS spectral analysis. In addition, the similarity of secondary structure of the mutated products to the native Tat protein was predicted using a position-specific scoring method.Surface plasmon resonance confirms that the KKR domain is a spatially enclosed Tat heparin binding siteThe SPR biosensor assay is an accurate and powerful method of dig the molecule-molecule interactions, while mutation studies provide a wealth of information about biological functions. By combining these two approaches, we successfully defined the intrinsic properties of the KKR domain and characterized its functional importance. Using the SPR assay, a multiphasic binding mode between Tat and heparin was observed. With mutation of the basic domain, a monophasic binding series became evident. A complete loss of binding capacity for heparin was then noted with double mutation of both KKR and the basic domain. These findings collectively support the idea that the Tat protein offers two binding sites for heparin: the well-recognized basic domain, and the newly identified KKR region. This model is congruent with our simulation-based predictions. Notably, the observation that mutants with either a single or a double substitution of KKR residues does not dramatically affect heparin binding affinity, while a triple mutation significantly did, highlights the importance of all residues in this triad for functionality.The KKR spatial triad binds specifically to heparin in a high affinity manner Delineation of the intrinsic properties of the newly identified binding site is important since it assists in unraveling the nature of the domain's interaction with heparin. By kinetics analysis, the dissociation constant (KD value) was calculated as 5.31pM, which represents an extremely high-affinity binding interaction (~10,000 times that of the basic domain). The disparity in binding affinity between the KKR and the basic domain, in principle, should allow heparin to preferentially occupy KKR. We next conducted a series of experiments evaluating heparin-mediated competitive inhibition of Tat-heparin binding for further confirmation. Notably, mutation of the basic domain (GST-Tat(G48-R57)A) produced an IC50 value (0.659μg/ml) comparable to that of GST-Tat (0.717μg/ml), while mutation of the KKR region (GST-TatK (12,41)A/R78A) yielded an IC50 value of 1.574μg/ml, almost twice that of GST-Tat. These data further support the notion that the KKR spatial domain contributes dramatically to heparin binding, and represents an extremely high-affinity heparin binding site. In silico simulations clearly demonstrate that heparin can specifically recognize the KKR region when the disaccharide probe was used. As the length of the oligosaccharide increases, the oligomer stretches toward and along the basic domain: it begins to form interactions with Arg49 when the tetramer probe was used, approaches Arg52 as the probe size increases to 6 sugar unit, and reaches Gln54 as the sugar length is 8. Ideally, heparin at its typical length would simultaneously bind to both KKR and the whole basic domain, with residue Arg49 serving as the'knot/link'between the two regions.Another important feature shaped by the KKR domain is its specificity for heparin, as is evident from the observation that only heparin/HS but not other GAGs including CSA, CSC and HA were able to produce a dose-response-based competitive inhibitory effect on Tat-heparin interactions. Because sugar-protein interactions are predominately dependent on saccharide composition and the extent and distribution of sulfation of the sugar backbone, the high affinity of Tat for heparin may be a product of both its defined sulfate groups and specific saccharide composition. However, though heparin and HS are comprised of same saccharide composition, HS still displays differences from heparin in binding affinity for KKR triad, which clearly points to the importance of the extent of sulfation of sugar chains in specificity of sugar-protein recognition.The KKR spatial region is not involved in Tat-driven internalizationIt is well-known that Tat-regulated function mostly precisely depends on the association of Tat with cell surface heparin sulfate/heparin via the negatively charged sulfate group. Of note, Tat-driven internalization indispensably counts on Tat's heparin-binding properties. The substitution of KKR region with alanine (GST-TatK(12,41)A/R78A), however, failed to affect internalization at all. This helps exclude the involvement of the KKR spatial domain in Tat protein internalization.The KKR spatial region contributes to Tat-mediated SLK cell adhesion viaβ1 integrin activation in a cell surface HSPG-dependent mannerRegarding integrin activation and subsequent adhesion, one important issue merits further addressed: the KKR triad matches quite well in a similar pattern with RGD-driven events that depend on its engagement with cell surface HS [4]. However, a complete loss of integrin activation and subsequent cell adhesion via the replacement of the RGD region, but only a partial loss observed with KKR substitution, convincingly support the concept that the KKR is an essential but not dominant component in mediating cell adhesion events as RGD does. This triad, alternatively resembling the basic domain, cooperates with the RGD motif in integrin activation and subsequent adhesion.In sum, we herein show for the first time that a triad of Lys12, Lys41, Arg78 domain is a novel, high-affinity, spatially enclosed heparin binding site on Tat, which we have further defined as important in facilitating Tat-driven integrin activation and subsequent cell adhesion in an HSPG-dependent manner. Of note, this newly identified heparin binding site functions similarly to the basic domain as a participator in Tat-triggered cell adhesion on one hand, but behaves quite differently from the basic domain in the internalization process. Particularly, the findings that in addition to the commonly accepted basic domain, the KKR region is an extremely high affinity heparin binding site appeal a careful consideration when interpreting Tat-driven adhesion events.Further characterization of functional synergy of KKR-mediated Tat-heparin interactions will add a crucial component of understanding AIDS-associated pathological in particular and Tat-driven battery of biological events in general. Also, challenging efforts towards the interest in the KKR-targeted rational designing for novel therapeutics are further needed.