Overexpression of Human Immunodeficiency Virus (HIV) coreceptors in Rhodobacter

Structural information revealing the interactions that occur between host and viral proteins in HIV infection is growing. The HIV coreceptors CCR5 and CXCR4 represent promising untapped opportunities for anti-HIV drug development. CCR5 is, in particular, a high-priority target, however structural information is sparse for the host coreceptor proteins involved in viral recognition and host/viral membrane fusion. These coreceptors are integral transmembrane proteins which have proven to be very difficult to express in quantities needed for extensive crystallization trials. In addition, they are known to be extremely heterogeneous both in vivo and when purified from native hosts. Thus, the coreceptors represent extremely difficult protein subjects for the generation of crystals for x-ray diffraction experiments. Methods by which the structures of CCR5 and CXCR4 can be obtained efficiently do not currently exist. We are employing our recently-developed membrane protein expression system, which utilizes the Rhodobacter species of photosynthetic bacteria, to produce these transmembrane proteins. We plan to optimize the expression of CCR5 and CXCR4 in the Rhodobacter system in order to generate adequate amounts of these proteins, significantly improving the opportunities for obtaining high-resolution structural information. We also plan to coexpress proteins that may serve as chaperones to encourage folding and insertion of recombinant and synthetic coreceptor gene products into the developing Rhodobacter intracytoplasmic membranes. The structural information that will be obtained following successful overexpression and crystallization of the CCR5 and CXCR4 coreceptors will provide pharmaceutical companies with the foundation upon which targeted anti-HIV drugs can be designed in a structure-based, rational manner.