HIV Integrase Binding Interfaces by H/D Exchange
University of California, San Diego
Basic Biomedical Sciences
Treating the rapidly mutating HIV retrovirus in AIDS patients requires a cocktail of several drugs that interfere with the actions of the 3 HIV proteins that create and process the viral DNA. If a single-site mutation in one of these proteins renders one drug ineffective, drugs acting on other regions in the 3 proteins can still control the retrovirus. Unfortunately, multiple-drug-resistant HIV is emerging, so there is a continuing need to identify new regions in the HIV proteins that can lead to the design of new inhibitory drugs. One of the 3 proteins, HIV integrase, processes the ends of the viral DNA and then inserts them into the chromosome of the host (human) cell. The infected cell then produces hordes of new viruses, which include mutants that are hard for the immune system to recognize. Unlike the other 2 HIV proteins, HIV integrase has no direct human counterpart, so integrase inhibitors may result in fewer side effects, potentially allowing sufficiently high doses to block insertion of the viral DNA into the host cell DNA, and hence providing a cure for AIDS. At this point, however, only one inhibitor of HIV integrase has been developed for clinical use.
The lack of 3-dimensional structural information on the biologically active integrase assembly has hindered the development of integrase inhibitors. HIV integrase must interact with viral DNA in order to form the biologically active complex that performs the insertion reaction. Therefore, determining integrase-DNA interactions is key for understanding biological function. Despite over 15 years of effort, no structures have been obtained for DNA bound to HIV integrase or portions of HIV integrase.
In this pilot proposal, we will use a technique termed hydrogen/deuterium exchange mass spectrometry (DXMS) to locate regions of HIV integrase that interact with DNA. DXMS examines interaction of a protein with the surrounding water molecules, distinguishing the outside surface of a protein from its interior. When a protein interacts with another molecule, such as DNA, the protein surface that contacts the DNA becomes protected from water molecules. With DXMS, we can compare the interactions of a protein with water in the absence and presence of DNA, revealing regions of the protein that contact DNA.
We believe that DXMS will be effective in determining DNA-interaction surfaces of HIV integrase where other methods have failed because DXMS can be used to investigate proteins that are too large, poorly soluble, impure, or scarce to be studied by other methods. Two of these methods, NMR spectroscopy and x-ray crystallography, have been greatly hindered by problems with integrase solubility, partly because they require high protein concentrations. DXMS uses much lower protein concentrations and allows the experimental conditions to be tuned to the protein under study rather than be constricted by the needs of the experimental technique.
We will apply DXMS to the study of each of the 3 protein domains of HIV integrase and their interactions with DNA. At least some of these identified DNA-binding surfaces may also occur in the full integrase-DNA assembly. These surfaces define new regions as targets for the development of new integrase inhibitors, a crucial need in the search for new drugs to combat AIDS.