Characterization of the allosteric mechanism of HIV-1 proteinsgp120 and NEF

As of December 2004 the World Health Organization estimated that 39.4 million people are living with HIV. Although the current pharmaceuticals are effective, resistance to them is becoming widespread. One way to overcome the problem of resistance is to target different areas of the viral life cycle. Inhibiting the interaction of the viral envelope protein with its cellular receptors can interrupt cellular entry; specifically the viral protein gp 120 interacts with the CD4 receptor on the cell surface mediating binding of the co-receptor CCR5 to gp120. In addition another viral protein, Nef, interacts with multiple cellular proteins to alter the cellular environment to make it more conducive for viral production. These interactions are highly controlled and take place in a concerted fashion. How do these two proteins carry out their functions? What conformational changes take place, and what is the nature of these conformational changes? This study has characterized the structural stability of these proteins by differential scanning calorimetry (DSC) and their binding properties by isothermal titration calorimetry (ITC). DSC data for gp120 show that it has a stable core and regions of instability with an unfolding transition centered at 59.2°C. ITC data show an increase in binding affinity and a decrease in enthalpy and entropy when gp120 is pre-bound to either sCD4 or 17b (CCR5 mimic). This change is partly due to the structuring of a previously unstructured region. DSC data for the full length and protease cleaved Nef show that the N-terminus adds 3 kcal/mol of energy to the stability of the protein and is an integral part of the protein with transitions centered at 44.7°C and 53.4°C respectively. ITC experiments show that hydrophobic surface area is exposed upon binding of Lck SH3 perhaps activating other sites. These results suggest that gp120 and Nef use an allosteric mechanism to carry out their function and that it may be possible to exploit these mechanisms in drug design.