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 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.

Figure 1. Location of SecA domains and cysteine residues

A model of the NMR structure of E. coli SecA (Protein Data Bank entry 2VDA structure 1) (38) is shown in ribbon representation. The structure of SecA is colored according to domains: light blue for NBD-1, dark blue for NBD-2, yellow for PPXD, dark green for HSD, light green for HWD, and red for the structured portion of CTL. The SecA monocysteine residues used in this study are colored magenta and are labeled with their residue number. The two-helix finger is in the region that falls within the white oval, and the clamp region is identified with a white diamond (10).

Figure 2. ATPase activities of IAEDANS-labeled SecA proteins

The endogenous, membrane, and translocation ATPase activities of the purified monocysteine SecA mutant proteins were determined at 37 °C as described in Experimental Procedures. The data represent an average of at least three different experiments. WT indicates wild-type SecA, while the number indicates the position of the IAEDANS-labeled monocysteine substitution within SecA.

Figure 3. Equilibrium binding of the alkaline phosphatase signal peptide to SecA as determined by fluorescence anisotropy

Binding of IANBD-labeled SP22 (■)or SP2 (●) to SecA was assessed by fluorescence anisotropy (r). Signal peptides were maintained at a constant concentration of 1 μM, and wild-type SecA was titrated into the system from 0 to 40 μM. All experiments were performed in TKE buffer at 20 °C.

Figure 4. Stoichiometry of SecA–signal peptide binding as determined by fluorescence anisotropy

Binding of IANBD-labeled SP22 (■) to SecA was assessed as fluorescence anisotropy (r) and plotted to reveal the saturated molar ratio of SP22 to SecA protomer. IANBD-labeled SP22 was titrated from 0 to 20 μM into a solution of 14 μM wild-type SecA. All experiments were performed in TKE buffer at 20 °C.

Figure 5. Equilibrium binding of SP22 to monomeric SecA

Binding of IANBD-labeled SP22 to IAEDANS-labeled SecAΔ11-Cys-190 (■) was assessed by FRET. SecAΔ11-Cys-190 was maintained at a constant concentration of 0.05 μM, and SP22 was titrated into the system from 0 to 15 μM. All experiments were performed in TKE buffer at 20 °C.

Figure 6. Location of the SecA signal peptide-binding domain

(A) A model of the NMR structure of E. coli SecA (Protein Data Bank entry 2VDA structure 1) (38) is shown in a gray-colored ribbon representation. The SecA region in common for the SP22 and SP2 data sets is colored yellow, while those residues specific for SP22 and SP2 are colored dark and light green, respectively. The cysteine residues utilized for mapping are colored magenta. (B) Similar to panel A except the different SecA signal peptide-binding sites are color-coded for comparison to highlight overlapping and nonoverlapping regions: green, red, and magneta for nonoverlapping regions of our site compared to that determined by Musial-Siwek et al. (36) and Baud et al. (25), respectively; dark blue, orange, and light blue for single overlapping regions of our site with that determined by Gelis et al., Musial-Siwek et al., and Baud et al., respectively; purple and yellow for double overlapping regions of our site with that determined by both Gelis et al. and Musial-Siwek et al. or both Gelis et al. and Baud et al., respectively. (C) Comparison of the SecA-bound KRR-LamB signal peptide structure (Protein Data Bank entry 2VDA SP structure 1) with our FRET-mapped signal peptide-binding domain, which is colored green. The KRR-LamB signal peptide is colored blue, and its amino terminus is located at the bottom of the figure. (D) A model of the cocrystal structure of the Thermotoga maritima SecA–SecYEG complex (Protein Data Bank entry 3din) (10) viewed from the side. Most of SecA is shown in a gray-colored ribbon representation, SecY as a gray solid surface structure, SecE as a light blue solid surface structure, and SecG as a dark blue solid surface structure. The FRET-mapped SecA signal peptide-binding domain is colored dark green, except for those regions that fall within the two-helix finger, which are colored light green for the sake of clarity. SecA residues 668–741 are not shown for the sake of clarity of presentation of the two-helix finger. (E) Similar to panel D except viewed from the cytoplasm.