STRUCTURAL FEATURES OF HALOPHILICITY DERIVED FROM THE CRYSTAL-STRUCTURE OF DIHYDROFOLATE-REDUCTASE FROM THE DEAD-SEA HALOPHILIC ARCHAEON, HALOFERAX-VOLCANII

Background: The proteins of halophilic archaea require high salt conce ntrations both for stability and for activity, whereas they denature a t low ionic strength. The structural basis for this phenomenon is not yet well understood. The crystal structure of dihydrofolate reductase (DHFR) from Haloferax volcanii (hv-DHFR) reported here provides the th ird example of a structure of a protein from a halophilic organism. Th e enzyme is considered moderately halophilic, as it retains activity a nd secondary structure at monovalent salt concentrations as low as 0.5 M. Results: The crystal structure of hv-DHFR has been determined at 2 .6 Angstrom resolution and reveals the same overall fold as that of ot her DHFRs. The structure is in the apo state, with an open conformatio n of the active-site gully different from the open conformation seen i n other DHFR structures. The unique feature of hv-DHFR is a shift of t he or helix encompassing residues 46-51 and an accompanied altered con formation of the ensuing loop relative to other DHFRs. Analysis of the charge distribution, amino acid composition, packing and hydrogen-bon ding pattern in hv-DHFR and its non-halophilic homologs has been perfo rmed. Conclusions: The moderately halophilic behavior of hv-DHFR is co nsistent with the lack of striking structural features expected to occ ur in extremely halophilic proteins. The most notable feature of halop hilicity is the presence of clusters of non-interacting negatively cha rged residues. Such clusters are associated with unfavorable electrost atic energy at low salt concentrations, and may account for the instab ility of hv-DHFR at salt concentrations lower than 0.5 M. With respect to catalysis, the open conformation seen here is indicative of a conf ormational transition not reported previously. The impact of this conf ormation on function and/or halophilicity is unknown.