SUNY at Albany
June 19-23, 2001
Calorimetric Studies of Domain Interactions in HIV-1 Capsid Protein
HIV-1 is initially assembled by the association of approximately 1200 molecules of the Gag polyprotein into a spherical immature virion at the cell membrane. Following budding through the cell membrane, the viral encoded protease is activated, and cleaves the Gag polyprotein into three structural domains, MA, CA, and NC. The MA remains membrane bound while the CA and NC domains collapse to form the conical core of the virion. CA is composed of N- and C- terminal structural domains connected by a short "linker" region. Within the core, there appears to be little interaction between the N- and C-terminal domains, however in vitro assembly studies have provided evidence for interdomain interactions. To determine whether or not the N- and C- terminal domains interact, we have used differential scanning calorimetry (DSC) to study the thermally-induced unfolding of the intact HIV-1 CA, its isolated N-terminal and C-terminal domains as well as binary combination of CA and its domains.
The isolated N- and C-terminal domains unfold according to a two-state mechanism at 53.4» and 60.4»C, respectively. The intact CA protein shows two transitions, one at 52.3»C whose unfolding parameters are close to those for the isolated N-domain, and a new transition at 47.8»C which represents a lowering of the melting temperature of the C-terminal domain. Thus, the C-terminal domain undergoes a strong destabilization due to interactions with the N-domain. This may be related to a difference between the conformational states of the "linker" region in the isolated C-domain compared to the intact CA protein.
In contrast, a comparison of the sum of the unfolding entropy of isolated domains, the entropy of the binary composition and the unfolding entropy of the intact CA reveals that the unfolding entropy of the intact CA is significantly higher than for all others. Based on these observations we suggest that the decreased conformational flexibility of the C-domain within the intact CA protein is a result of interdomain interactions. A point mutation (M185A) in the principal dimer interface of the intact capsid protein slightly increases the melting temperature of both domains and does not change the difference between domain stability in comparison with wild CA protein. This indicates that the C-domain dimerization motif and interdomain interface are distinct entities in intact CA protein.
Irina I. Protassevitch* (1,2), Jason Lanman (1) and Peter E. Prevelige Jr (1)
Department of Microbiology (1) and Center for Biophysical Sciences and Engineering (2),