This project set out to determine the three dimensional structure of Nef-SH3 complex using X-ray crystallography techniques. Since the full length Nef failed to crystallize despite significant efforts, we decided to identify the well folded domain of Nef and to discard the remaining unstructured region in order to facilitate crystallization. Applying the techniques of limited proteolysis and mass spectrometry, we have successfully defined the boundaries of the folded Nef structure which is required for interacting with SH3 domains. Furthermore, we found that, in addition to the PxxP motif, the carboxyl-terminal region of Nef is also involved in the interaction with SH3 domains. It should be noted that limited proteolysis of the two different proteins usually resulted in very complicated pattern on SDS-PAGE. Mass spectroscopy was used to assign the protein fragments to the corresponding amino acid sequence and was essential for interpreting the proteolysis results. The Nef construct tailored on the basis of these findings was found to be well folded and was subsequently crystallized. The crystals of Nef in complex with an SH3 domain diffracted to better than 3 A and the structure determination is now underway. The structural information may be useful for structure based drug design of anti HIV drugs aimed at disruption of the critical interactions between Nef and its cellular ligands.
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