Functional and structural analysis of signal peptide interactions with SecA and SPase I of the Sec-dependent transport system in Escherichia coli

Date of Completion

January 2007

Keywords

Chemistry, Biochemistry

Degree

Ph.D.

Abstract

Many proteins which are synthesized in the cytoplasm of cells are ultimately found in noncytoplasmic locations. This process requires several proteins which comprise the cellular transport pathway, and a signal peptide at the amino-terminus of the secreted protein to direct entry into this pathway. SecA, an ATPase crucial to the Sec-dependent transport process in Escherichia coli, directly binds the signal peptide and other ligands, and plays a central role in transport of the preproteins through the SecYEG channel. Type I signal peptidase (SPase 1), a membrane protein, is also a key component of the translocation pathway, and cleaves the signal peptide from the preprotein at the final step of transport. Even though these proteins are implicated in vital stages of the translocation pathway, the characteristics of SecA- and SPase-signal peptide interactions have not been defined. ^ In this study, using Fluorescence Resonance Energy Transfer (FRET) we established binding between SecA and an IAEDANS-labeled signal peptide. We find that the SecA affinity for signal peptide is controlled through interactions with lipid vesicles and nucleotides, and temperature. Native gel electrophoresis shows that SecA exists as a dimer and that signal peptide induces monomerization in aqueous and lipid environments. In a separate set of experiments, employing a synthetic signal peptide and a series of SecAs with unique Factor Xa cleavage sites, we localized the signal peptide binding groove (SPBG) on SecA. ^ NMR analysis of the isotopically labeled soluble domain of the SPase I (SPase I Δ2-75) identified 18 residues that are altered with signal peptide binding, all located in one groove on the surface of the protein. We show, for the first time, that the cleavage region and the C-terminus of the core region, only, interact with SPase I. Also, using NMR we were able to identify some residues of uniformly 2H, 13C, 15N-labeled full length SPase I on the 2D 1H- 15N HSQC spectrum via CBCA(CO)NH and HNCACB sequential assignments. All identified residues were located in the soluble domain of the protein. ^ These findings elucidate how the signal peptide interact with key proteins of the protein transport pathway in E. coli. ^

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