The
HIV-1 protein Rev facilitates the nuclear export of intron-containing
viral mRNAs by recognizing a structured RNA site, the Rev-response-element
(RRE), contained in an intron. Rev assembles as a homo-oligomer on
the RRE using its α-helical arginine-rich-motif (ARM) for RNA
recognition. One unique feature of this assembly is the repeated use
of the ARM from individual Rev subunits to contact distinct parts
of the RRE in different binding modes. How the individual interactions
differ and how they contribute toward forming a functional complex
is poorly understood. Here we examine the thermodynamics of Rev–ARM
peptide binding to two sites, RRE stem IIB, the high-affinity site
that nucleates Rev assembly, and stem IA, a potential intermediate
site during assembly, using NMR spectroscopy and isothermal titration
calorimetry (ITC). NMR data indicate that the Rev–IIB complex
forms a stable interface, whereas the Rev–IA interface is highly
dynamic. ITC studies show that both interactions are enthalpy-driven,
with binding to IIB being 20–30 fold tighter than to IA. Salt-dependent
decreases in affinity were similar at both sites and predominantly
enthalpic in nature, reflecting the roles of electrostatic interactions
with arginines. However, the two interactions display strikingly different
partitioning between enthalpy and entropy components, correlating
well with the NMR observations. Our results illustrate how the variation
in binding modes to different RRE target sites may influence the stability
or order of Rev–RRE assembly and disassembly, and consequently
its function
