[Show abstract][Hide abstract] ABSTRACT: Identification of the signal peptide-binding domain within SecA ATPase is an important goal for understanding the molecular basis of SecA preprotein recognition as well as elucidating the chemo-mechanical cycle of this nanomotor during protein translocation. In this study, Forster resonance energy transfer methodology was employed to map the location of the SecA signal peptide-binding domain using a collection of functional monocysteine SecA mutants and alkaline phosphatase signal peptides labeled with appropriate donor-acceptor fluorophores. Fluorescence anisotropy measurements yielded an equilibrium binding constant of 1.4 or 10.7 muM for the alkaline phosphatase signal peptide labeled at residue 22 or 2, respectively, with SecA, and a binding stoichiometry of one signal peptide bound per SecA monomer. Binding affinity measurements performed with a monomer-biased mutant indicate that the signal peptide binds equally well to SecA monomer or dimer. Distance measurements determined for 13 SecA mutants show that the SecA signal peptide-binding domain encompasses a portion of the preprotein cross-linking domain but also includes regions of nucleotide-binding domain 1 and particularly the helical scaffold domain. The identified region lies at a multidomain interface within the heart of SecA, surrounded by and potentially responsive to domains important for binding nucleotide, mature portions of the preprotein, and the SecYEG channel. Our FRET-mapped binding domain, in contrast to the domain identified by NMR spectroscopy, includes the two-helix finger that has been shown to interact with the preprotein during translocation and lies at the entrance to the protein-conducting channel in the recently determined SecA-SecYEG structure.
[Show abstract][Hide abstract] ABSTRACT: The pH (low) insertion peptide (pHLIP) has exceptional characteristics: at neutral pH it is an unstructured monomer in solution or when bound to lipid bilayer surfaces, and it inserts across a lipid bilayer as a monomeric alpha-helix at acidic pH. The peptide targets acidic tissues in vivo and may be useful in cancer biology for delivery of imaging or therapeutic molecules to acidic tumors. To find ways to vary its useful properties, we have designed and analyzed pHLIP sequence variants. We find that each of the Asp residues in the transmembrane segment is critical for solubility and pH-dependent membrane insertion of the peptide. Changing both of the Asp residues in the transmembrane segment to Glu, inserting an additional Asp into the transmembrane segment, or replacing either of the Asp residues with Ala leads to aggregation and/or loss of pH-dependent membrane insertion of the peptide. However, variants with either of the Asp residues changed to Glu remained soluble in an aqueous environment and inserted into the membrane at acidic pH with a higher pK(app) of membrane insertion.
[Show abstract][Hide abstract] ABSTRACT: Perturbations of the chemical shifts of a small subset of residues in the catalytically active domain of Escherichia coli signal peptidase I (SPase I) upon binding signal peptide suggest the contact surface on the enzyme for the substrate. SPase I, an integral membrane protein, is vital to preprotein transport in prokaryotic and eukaryotic secretory systems; it binds and proteolyses the N-terminal signal peptide of the preprotein, permitting folding and localization of the mature protein. Employing isotopically labeled C-terminal E. coli SPase I Delta2-75 and an unlabeled soluble synthetic alkaline phosphatase signal peptide, SPase I Delta2-75 was titrated with the signal peptide and 2D (1)H-(15)N heteronuclear single-quantum correlation nuclear magnetic resonance spectra revealed chemical shifts of specific enzyme residues sensitive to substrate binding. These residues were identified by 3D HNCACB, 3D CBCA(CO)NH, and 3D HN(CO) experiments. Residues Ile80, Glu82, Gln85, Ile86, Ser88, Gly89, Ser90, Met91, Leu95, Ile101, Gly109, Val132, Lys134, Asp142, Ile144, Lys145, and Thr234, alter conformation and are likely all in, or adjacent to, the substrate binding site. The remainder of the enzyme structure is unperturbed. Ramifications for conformational changes for substrate docking and catalysis are discussed.
Chemical Biology & Drug Design 08/2008; 72(2):140-6. DOI:10.1111/j.1747-0285.2008.00685.x · 2.49 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Useful solution nuclear magnetic resonance (NMR) data can be obtained from full-length, enzymatically active type I signal peptidase (SPase I), an integral membrane protein, in detergent micelles. Signal peptidase has two transmembrane segments, a short cytoplasmic loop, and a 27-kD C-terminal catalytic domain. It is a critical component of protein transport systems, recognizing and cleaving amino-terminal signal peptides from preproteins during the final stage of their export. Its structure and interactions with the substrate are of considerable interest, but no three-dimensional structure of the whole protein has been reported. The structural analysis of intact membrane proteins has been challenging and only recently has significant progress been achieved using NMR to determine membrane protein structure. Here we employ NMR spectroscopy to study the structure of the full-length SPase I in dodecylphosphocholine detergent micelles. HSQC-TROSY spectra showed resonances corresponding to approximately 3/4 of the 324 residues in the protein. Some sequential assignments were obtained from the 3D HNCACB, 3D HNCA, and 3D HN(CO) TROSY spectra of uniformly 2H, 13C, 15N-labeled full-length SPase I. The assigned residues suggest that the observed spectrum is dominated by resonances arising from extramembraneous portions of the protein and that the transmembrane domain is largely absent from the spectra. Our work elucidates some of the challenges of solution NMR of large membrane proteins in detergent micelles as well as the future promise of these kinds of studies.
[Show abstract][Hide abstract] ABSTRACT: SecA, an ATPase crucial to the Sec-dependent translocation machinery in Escherichia coli, recognizes and directly binds the N-terminal signal peptide of an exported preprotein. This interaction plays a central role in the targeting and transport of preproteins via the SecYEG channel. Here we identify the signal peptide binding groove (SPBG) on SecA addressing a key issue regarding the SecA-preprotein interaction. We employ a synthetic signal peptide containing the photoreactive benzoylphenylalanine to efficiently and specifically label SecA containing a unique Factor Xa site. Comparison of the photolabeled fragment from the subsequent proteolysis of several SecAs, which vary only in the location of the Factor Xa site, reveals one 53 residue segment in common with the entire series. The covalently modified SecA segment produced is the same in aqueous solution and in lipid vesicles. This spans amino acid residues 269 to 322 of the E. coli protein, which is distinct from a previously proposed signal peptide binding site, and contributes to a hydrophobic peptide binding groove evident in molecular models of SecA.
[Show abstract][Hide abstract] 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 Î"2-75) 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 2 H, 13 C, 15 N-labeled full length SPase I on the 2D 1 H- 15 N 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 .
[Show abstract][Hide abstract] ABSTRACT: SecA, the peripheral subunit of the Escherichia coli preprotein translocase, interacts with a number of ligands during export, including signal peptides, membrane phospholipids, and nucleotides. Using fluorescence resonance energy transfer (FRET), we studied the interactions of wild-type (WT) and mutant SecAs with IAEDANS-labeled signal peptide, and how these interactions are modified in the presence of other transport ligands. We find that residues on the third alpha-helix in the preprotein cross-linking domain (PPXD) are important for the interaction of SecA and signal peptide. For SecA in aqueous solution, saturation binding data using FRET analysis fit a single-site binding model and yielded a Kd of 2.4 microM. FRET is inhibited for SecA in lipid vesicles relative to that in aqueous solution at a low signal peptide concentration. The sigmoidal nature of the binding curve suggests that SecA in lipids has two conformational states; our results do not support different oligomeric states of SecA. Using native gel electrophoresis, we establish signal peptide-induced SecA monomerization in both aqueous solution and lipid vesicles. Whereas the affinity of SecA for signal peptide in an aqueous environment is unaffected by temperature or the presence of nucleotides, in lipids the affinity decreases in the presence of ADP or AMP-PCP but increases at higher temperature. The latter finding is consistent with SecA existing in an elongated form while inserting the signal peptide into membranes.