The Conformation of a Signal Peptide Bound by Escherichia coli Preprotein Translocase SecA (original) (raw)
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Biochemistry, 2010
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 chemomechanical cycle of this nanomotor during protein translocation. In this study, Förster 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 μM 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 monomerbiased 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 nucleotidebinding 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. Proteins are secreted across or integrated into biological membranes by means of a variety of protein translocation systems that have been characterized over the past several decades. In Escherichia coli, the major pathway for protein secretion is the general secretion (Sec) pathway that is composed of two fundamental components: the SecYEG heterotrimeric complex that comprises the protein-conducting channel and the SecA ATPase nanomotor that drives † This work was supported by Grants GM42033 and GM37639 from the National Institutes of Health to D.B.O. and D.A.K., respectively, and from the Patrick and Catherine Weldon Donaghue Medical Research Foundation (DF#00-118) and the National Science Foundation (MCB-031665) to I.M.
Biophysical Journal, 1994
Although the central role of the signal sequence in protein export is well established, the molecular details underlying signal sequence in vivo function remain unclear. As part of our continuing effort to relate signal sequence phenotypes to specific biophysical properties, we have carried out an extensive characterization of the secondary structure and lipid interactions for a family of peptides corresponding to the wild-type E. coli LamB signal sequence, and mutants that harbor charged residue point mutations in the hydrophobic core region. We used membrane-resident fluorescence quenching according to the parallax method to determine the relative depth of insertion of tryptophan-labeled analogs of these peptides into the acyl chain region of bilayer vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-snglycero-3-phosphoglycerol. Also, restriction of acyl chain motion upon peptide binding was evaluated using steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. Each of these peptides showed evidence of insertion into the acyl chain region, although most likely not in a transmembrane orientation. The mutant peptides were shown to have a reduced insertion potential relative to the wild-type peptide. Furthermore, tryptophan spectral properties indicated that insertion of the wild-type and mutant peptides enhances bilayer hydration. This effect was particularly pronounced with peptides harboring negatively charged aspartate point substitutions. The results are discussed in relation to the potential roles of signal sequences in mediating protein translocation.
Biochemistry, 2001
SecA ATPase is an essential component of the Sec-dependent protein translocation machinery. Upon interaction with the plasma membrane containing SecYE, preprotein, and ATP, SecA undergoes cycles of membrane insertion and retraction resulting in the translocation of segments of the preprotein to the trans side of the membrane. To study the structural basis of SecA function, we employed fluorescence spectroscopy along with collisional quenchers with a set of SecA proteins containing single tryptophan substitutions. Our data show that among the seven naturally occurring tryptophan residues of Escherichia coli SecA, only the three tryptophan residues contained within the C-domain contributed significantly to the fluorescence signal, and they occupied distinct local environments in solution: Trp723 and Trp775 were found to be relatively solvent accessible and inaccessible, respectively, while Trp701 displayed an intermediate level of solvent exposure. Exposure to increased temperature or interaction with model membranes or signal peptide elicited a similar conformational response from SecA based upon the fluorescence signals of the SecA-W775F and SecA-W723F mutant proteins. Specifically, Trp775 became more solvent exposed, while Trp723 became less solvent accessible under these conditions, indicating similarities in the overall conformational change of the C-domain promoted by temperature or translocation ligands. Only Trp701 did not respond in parallel to the different conditions, since its solvent accessibility changed only in the presence of signal peptide. These results provide the first detailed structural information about the C-domain of SecA and its response to translocation ligands, and they provide insight into the conformational changes within SecA that drive protein translocation.
Towards a Determination of the SecA Signal Peptide Binding Site
SecA is an essential motor protein found in bacteria that uses ATP hydrolysis to transport preproteins across or into the cytoplasmic membrane. Before this translocation occurs, SecA must recognize and bind an N-terminal signal sequence of the newly synthesized preprotein. The location of this binding site on SecA and the orientation of the bound signal peptide is currently a matter of debate. Attempts at obtaining a high resolution X-ray crystal structure of signal peptide bound to SecA in order to elucidate the binding site have been inhibited by signal sequence hydrophobicity and aggregation. In this study, we have created a mutant E.coli SecA protein with the signal sequence KRR-LamB attached to the C-terminus using DNA recombination technology with the aim to obtain an X-ray crystal structure of SecA self-binding the attached signal peptide. Fluorescence anisotropy assays with a dyelabeled signal peptide and SecA or SecA-KRR-LamB proteins were used to analyze the extent of self-binding in two SecA-KRR-LamB constructs. Both the full length SecA-KRR-LamB and the truncated version lacking the C-terminus exhibit significant self-binding of the attached signal peptide. The functionality of the SecAattached KRR-LamB signal peptide was tested by engineering a SecA-KRR-LamB-PhoA chimera in which the alkaline phosphatase gene lacking its endogenous signal sequence was fused downstream of the SecA-attached KRR-LamB signal sequence, and the ability of the latter signal peptide to transport alkaline phosphatase across the membrane where it is enzymatically active in vivo was assayed. Both the full-length and truncated construct show self-binding and are able to facilitate translocation of the fused preprotein substrate to different extents. First and foremost, I would like to thank my advisor, Professor Donald Oliver, for giving me the opportunity to work in his lab and produce the work that turned into this thesis. I greatly appreciate his academic insight, life advice, and overall support. A special thank you goes to the readers of my thesis-Professors Manju Hingorani and Ishita Mukerjifor taking the time to look over my work and provide feedback. I highly value their input. Thank you to the MB&B department as a whole-faculty, staff, students-it has been an enriching few years. To the members of the Oliver Lab, past and present-thanks for everything. My lab experience would not have been nearly as positive without you in it. Qi, it has been an exceptional pleasure collaborating with you on this project, I hope you didn't mind my constant questions too much. Zeliang, Christine, Tithi, Sudipta, Jenn, Lorry and Stephanie, thank you. This process would not have been tolerable without the Cave-Zack, Ryu, Jaewon, and Corey-and of course all those who associate with the Cave. It has been a blast living with you guys. Thanks to my family for backing me in whatever I do, especially my Aunt Ellen and Uncle Lou. I appreciate beyond words your constant support, enthusiasm, and belief. Finally, to Kat, the person who has kept me reasonably sane throughout my two years of often frustrating lab work and my semester of writing. Thank you. I can't imagine these past years without you.
Proteins: Structure, Function, and Bioinformatics, 2012
A protein destined for export from the cell cytoplasm is synthesized as a preprotein with an aminoterminal signal peptide. In Escherichia coli, signal peptides that guide preproteins into the SecYEG protein conduction channel are typically subsequently removed by signal peptidase I. To understand the mechanism of this critical step, we have assessed the conformation of the signal peptide when bound to signal peptidase by solution NMR. We employed a soluble form of signal peptidase without its two transmembrane domains (SPase I Δ2-75) and the E. coli alkaline phosphatase signal peptide. Using a transferred NOE approach, we found clear evidence of weak peptide-enzyme complex formation. The peptide adopts a "U-turn" shape originating from the proline residues within the primary sequence that is stabilized by its interaction with the peptidase and leaves key residues of the cleavage region exposed for proteolysis. In dodecylphosphocholine (DPC) micelles the signal peptide also adopts a U-turn shape comparable to that observed in association with the enzyme. In both environments this conformation is stabilized by the signal peptide phenylalanine side chain-interaction with enzyme or lipid mimetic. Moreover, in the presence of DPC, the N-terminal core region residues of the peptide adopt a helical motif and, based on PRE (paramagnetic relaxation enhancement) experiments, are shown to be buried within the membrane. Taken together, this is consistent with proteolysis of the preprotein occurring while the signal peptide remains in the bilayer and the enzyme active site functioning at the membrane surface.
Biochemistry, 2016
Signal peptides are critical for the initiation of protein transport in bacteria by virtue of their recognition by the SecA ATPase motor protein followed by their transfer to the lateral gate region of the SecYEG protein-conducting channel complex. In this study we have constructed and validated the use of signal peptide-attached SecA chimeras for conducting structural and functional studies on the initial step of SecA signal peptide interaction. We utilized this system to map the location and orientation of the bound alkaline phosphatase and KRRLamB signal peptides to a peptide-binding groove adjacent to the two-helix finger sub-domain of SecA. These results support the existence of a single conserved SecA signal peptide-binding site that positions the signal peptide parallel to the two-helix finger sub-domain of SecA, and they are also consistent with the proposed role of this sub-domain in the transfer of the bound signal peptide from SecA into the protein-conducting channel of S...
1997
SecA is the peripheral subunit of the preprotein translocaseof Escherichia coli . SecA consists of two independently folding domains, i.e., the N-domain bearing the high-affinity nucleotide binding site (NBS-I) and the C-domain that harbors the low-affinity NBS-II. ATP induces SecA insertion into the membrane during preprotein translocation. Domain-specific monoclonal antibodies (mAbs) were developed to analyze the functions of the SecA domains in preprotein translocation. The antigen binding sites of the obtained mAbs were confined to five epitopes. One of the mAbs, i.e., mAb 300-1K5, recognizes an epitope in the C-domain in a region that has been implicated in membrane insertion. This mAb, either as IgG or as Fab, completely inhibits in Vitro proOmpA translocation and SecA translocation ATPase activity. It prevents SecA membrane insertion and, more strikingly, reverses membrane insertion and promotes the release of SecA from the membrane. Surface plasmon resonance measurements dem...
Biophysical Journal, 1994
We have used tryptophan fluorescence spectroscopy to characterize the binding affinities of an Escherichia coli LamB signal peptide family for lipid vesicles. These peptides harbor charged residue substitutions in the hydrophobic core region. Titrations of peptides with vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2oleoyl-sn-3-phosphoglycerol (65:35 mol%), in conjunction with evaluation of peptide dissociation rates from these vesicles, were used to determine binding parameters quantitatively. We find that under low ionic strength conditions, point mutations introducing negatively charged aspartate residues substantially reduce peptide affinity relative to the wild-type peptide. However, the difference between wild-type and mutant peptide affinities was much lower under approximately physiological ionic strength. In addition, the lipid affinities of model surface-binding and transmembrane peptides were determined. These comparative studies with signal and model peptides permitted semi-quantitative deconvolution of signal peptide binding into electrostatic and hydrophobic components. We find that both interactions contribute significantly to binding, although the theoretically available hydrophobic free energy is largely offset by unfavorable polar-group effects. The implications of these results for understanding the potential roles of the signal sequence in protein translocation are discussed.