CRYSTAL STRUCTURES OF B SUBTILIS SECA MUTANTS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. SecA is a bacterial ATPase involved in protein translocation through the cytoplasmic membrane. SecA uses the energy derived from cycles of ATP binding and hydrolysis to drive translocation of preproteins through the translocase SecYEG. However, the details of the mechanism of this process are unclear. A crystal structure of an ATP-bound state of SecA would provide the necessary structural information to decipher a mechanism for this mechanoenzyme. Because non-hydrolyzable analogs such as AMP-PNP have only remedial binding affinity in this system, we have employed active-site mutagenesis to obtain an ATP-bound form of SecA. SecA shares structural homology to the ABC transporter family as well as F1-ATPase, and thus may exhibit a similar mechanism in coupling ATP binding and hydrolysis to mechanical movements. Previously in ABC transporters it was observed that mutagenesis of the catalytic glutamate in the active site to glutamine rendered the protein inactive while maintaining its ability to bind ATP, thereby locking it into an ATP-bound state. Wild-type SecA from B. subtilis has an analogous glutamate in the SecA active site hypothesized to be the catalytic glutamate in the ATP hydrolysis reaction. The same mutant was made in BsSecA (E208Q) and tested for ATPase activity and binding efficiency. While reducing ATPase activity to that of background levels, the E208Q mutation also inhibits any nucleotide binding. This result led us to investigate why this mutation would create such a drastic change in nucleotide affinity. The crystal structure of BsSecA E208Q was solved at Brookhaven NSLS (X12B) to about 3.4¿¿¿ . However, the low resolution was not sufficient to decipher how and why the mutation caused a loss of nucleotide affinity. Improving the resolution of this structure would allow us to investigate changes in cooperative hydrogen bonding patterns and any stereochemical shifts involved in this phenomenon. A double mutant of BsSecA, E208Q R489K, is shown to restore nucleotide binding. Preliminary data collection of apo crystals of this double mutant diffracted to about 3.3¿¿¿ at beamlines such as X12B and X4A. These crystals were then tested at the Advanced Photon Source 24a-ID and the data were significantly improved to 3.0¿¿¿ . This resolution was sufficient to see well-defined electron density, and a similar resolution for the single mutant is needed to confidently assess the structure. Data collection of BsSecA E208Q at a beamline at the NSLS with a high flux beam such as X29 or X25 would greatly improve the crystal structure of this mutant and lead to an understanding of the SecA active site interactions. In addition, crystals of the double mutant containing MgATP were grown and are ready to be collected for diffraction. A structure of BsSecA bound to MgATP would shed light on the mechanistic properties of this molecular machine, providing a functional description of how SecA and related ATPases utilize ATP to convert nucleotide binding and hydrolysis to mechanical energy. High-resolution structures of these mutants are necessary to finalize data for publication. Six to eight hours of beamtime on X25 or X29 would provide sufficient time for minimal screening of crystals to find the best quality diffraction and also data collection of the best crystals.