We have previously determined structures of the amino-terminal core domain of the HIV- 1 capsid protein (Gag residues 133-283; CA133-283), both free in solution 1, and in complex with human cyclophilin A (CypA)2. The CA133-283/CypA structure revealed how capsid makes its essential packaging interaction with cyclophilin. A, and also suggested how the amino-terminal domain of capsid participates in forming the outer shell of the viral core structure. We now propose to determine the X-ray crystal structure of the carboxyl-terminal assembly domain of HIV- I capsid (Gag residues 284-363; CA284-363). This domain helps mediate Gag assembly and is of particular interest because it contains a 20 amino acid stretch, termed the major homology region (MHR), which is highly conserved across retroviridae 3-5. Genetic analyses have demonstrated that both the MHR and other regions of the capsid carboxyl-terminal domain are essential for HIV- 1 replication 6-8. We have developed efficient expression and purification systems for CA284.363, and grown crystals of both the native and selenomethionine- substituted proteins. Very recently, we have collected complete data sets at 2.1 A resolution, for structure determination by multiwavelength anomalous dispersion. Since we have shown that this domain is essential for capsid dimerization in vitro 9, we anticipate that the CA284-363 structure will reveal both the domain's fold and the interactions that mediate dimerization of the full-length capsid protein. The crystal structure of the CA133-283/CypA complex suggests that capsid molecules may form the shell of the viral capsid core by assembling into planar strips that are stabilized by two crystallographically defined CA-CA interactions 2. We hypothesize that these strips then align via a third interface to form the surface of the capsid shell. To test this hypothesis, we have designed an ensemble of 15 mutations that will be used to """"""""alanine scan"""""""" the capsid protein surface. The mutant viruses will be assayed for their ability to assemble, package viral proteins, mature, and replicate in culture, thereby defining the function(s) of the different ca sid surfaces in HIV- I replication.
| Worthylake, D K; Wang, H; Yoo, S et al. (1999) Structures of the HIV-1 capsid protein dimerization domain at 2.6 A resolution. Acta Crystallogr D Biol Crystallogr 55:85-92 |
| Gamble, T R; Yoo, S; Vajdos, F F et al. (1997) Structure of the carboxyl-terminal dimerization domain of the HIV-1 capsid protein. Science 278:849-53 |
