A multi-residue electrostatic clamp in HIV-1 CRF01_AE Tat underlies high-affinity engagement with NMDA and hDAT receptors

Introduction HIV-1 CRF01_AE is an epidemic in Southeast Asia associated with rapid HIV disease progression. Despite effective antiretroviral therapy, serious non-AIDS-defining complications, including HIV-associated neurocognitive disorder, remain a clinical burden. HIV transactivator of transcription (Tat) protein is a mediator of neurotoxicity known to vary by subtype. Subtype B Tat-mediated neurovirulence has been linked to an arginine residue at position 57 (R57) as a critical structural anchor. However, the CRF01_AE variant lacks the motif, presenting a structural paradox regarding its neuropathogenicity. Methods To elucidate the functional mechanisms of the CRF01_AE Tat exon 1 domain, comprehensive in silico biophysical modeling and protein-protein docking were conducted. Thermodynamic profiling, residue-level mutational scanning, and normal mode analysis (NMA) were utilized to evaluate the epistatic architecture and kinetic stability of the protein when bound to human N-methyl-d-aspartate (NMDA) and dopamine transporter (hDAT) receptors against subtypes B and C. Results Our computational profiling revealed that the Philippine CRF01_AE Tat exon 1, lacking the R57 motif, evolved a stabilizing, multi-residue lateral electrostatic clamp (K53 and H54), alongside other electropositive substitution mutations. CRF01_AE Tat exhibited high nanomolar affinity for the NMDA ( ΔG = -9.6 kcal/mol; K d = 1.7 × 10 –7 M) and hDAT ( ΔG = -10.6 kcal/mol; K d = 3.2 x 10 –8 M) neuroreceptors. NMA predicted that the epistatic adaptations specific to CRF01_AE would mechanically lock the central basic domain (residues 40–55) into the neuroreceptor cleft. This was in contrast with subtype B, which relied on the R57 insertion anchor, allowing greater localized conformational freedom at the docking interface. Conclusion The predicted CRF01_AE Tat structure suggests a strong binding affinity and enhanced kinetic stability with both NMDA and hDAT neuroreceptors. These computational findings indicate that structural adaptations in CRF01_AE Tat may alter neuroreceptor engagement and could contribute to its neurovirulence in HAND.