Mapping of the Signal Peptide-Binding Domain of Escherichia coli SecA Using Förster Resonance Energy Transfer

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 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 Figure S1), binding affinity of unlabeled SP2 and IANBD-labeled SP2 with IAEDANS-labeled SecA-Cys-256 ( Figure S2) and mapping of the FRET-determined signal peptide-binding site on the SecA crystal structures of B. subtilis, E. coli, T. thermophilus, and Mycobacterium tuberculosis ( Figure S3). This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 April 7. Recently, a model for preprotein translocation has been proposed on the basis of a SecASecYEG cocrystal structure (10) and disulfide cross-linking studies (11). In this model, SecA captures the preprotein in a clamp formed by nucleotide-binding domain 2 (NBD-2) 1 , the preprotein binding domain (PPXD), and the h...

Conserved SecA Signal Peptide-Binding Site Revealed by Engineered Protein Chimeras and Förster Resonance Energy Transfer

“…Thus, the parallel-binding model is supported by the relative equivalence of the distances measured between either end of the signal sequence and Trp 724, the agreement between our FRET-measured distances and the distances predicted from the parallel-binding model, and the agreement between the distances measured for the exogenous peptides and the chimeras. Combined with our previous studies where 13 distinct distances between SecA and a bound PhoA signal peptide were determined and pointed to a parallel-binding model, 22 these new results indicate that both signal peptides appear to bind SecA at a conserved signal peptide-binding site and in a similar parallel orientation.…”

“…18,22 By comparing the relevant bsSecA-signal peptide chimera with its identical bsSecA homologue lacking a signal peptide, we were able to discern the degree of inhibition caused by self-binding of the attached signal peptide. The results of this analysis are given in Table 1, and some representative data are shown in Figure 3.…”

“…30 In fact, Hunt et al 30 originally suggested that this region was the mostly likely location for the SecA signal peptide-binding site: a hypothesis supported by our previous FRET mapping and mutagenesis work. 22,40 Thus, this region of SecA could be utilized to mimic a signal peptide-bound SecA complex with the parallel orientation. First, the PhoA signal peptide was mapped onto the relevant 21-residue region of the longer KRRLamB signal peptide from the NMR structure, centering it on the critical hydrophobic core region, which was of similar length (13 and 12 residues, respectively).…”

“…Furthermore, probes placed within the hydrophobic core region of the signal peptide have previously been shown to disrupt signal peptide binding to SecA protein. 22 …”

Mapping of the SecA·SecY and SecA·SecG Interfaces by Site-directed in Vivo Photocross-linking

“…SecG has been shown to enhance the SecA membrane insertion/de-insertion cycle through its coupled topology inversion, although this latter proposal has been challenged by the normal translocation behavior of a topologically fixed form of SecG (12)(13)(14)(15)(16). Although significant progress has been made recently in defining those regions of SecA that are responsible for SecB and signal peptide recognition, elucidation of those regions of SecA that are responsible for SecYEG binding and activation has remained a more difficult task (17)(18)(19)(20).…”