AFM observation of protein translocation mediated by one unit of SecYEG-SecA complex

Abstract

Protein translocation across cellular membranes is an essential and nano-scale dynamic process. In the bacterial cytoplasmic membrane, the core proteins in this process are a membrane protein complex, SecYEG, corresponding to the eukaryotic Sec61 complex, and a cytoplasmic protein, SecA ATPase. Despite more than three decades of extensive research on Sec proteins, from genetic experiments to cutting-edge single-molecule analyses, no study has visually demonstrated protein translocation. Here, we visualize the translocation, via one unit of a SecYEG-SecA-embedded nanodisc, of an unfolded substrate protein by high-speed atomic force microscopy (HS-AFM). Additionally, the uniform unidirectional distribution of nanodiscs on a mica substrate enables the HS-AFM image data analysis, revealing dynamic structural changes in the polypeptide-crosslinking domain of SecA between wide-open and closed states depending on nucleotides. The nanodisc-AFM approach will allow us to execute detailed analyses of Sec proteins as well as visualize nano-scale events of other membrane proteins.

Fig. 1. Specific side-on orientation of SecYAEG-ND and SecYA(ΔPPXD)EG-ND observed by HS-AFM.

a Typical HS-AFM images of SecYAEG-ND (upper panels) and SecYA(ΔPPXD)EG-ND (lower panels) on a mica surface. The AFM image height (nm) is shown by the color bar. Representative data from more than 10 independent experiments with similar results are shown. b Cross-sectional height profiles for SecA regions along the red lines shown in (a). c Schematic representation of the structural models of SecYAEG-ND (upper) and SecYA(ΔPPXD)EG-ND (lower) at 80° in (f), accompanied by conical AFM probes utilized in the generation of simulated AFM images. PPXD is highlighted in yellow, the rest of SecA in blue, and SecYEG in magenta. d Simulated AFM images of SecYAEG-ND (upper) in wide-open form and SecYA(ΔPPXD)EG-ND (lower) in (c). The simulated AFM image height (nm) is shown by the color bar. e Cross-sectional height profiles for SecA regions along the red lines shown in (d). f Averaged image correlation scores as a function of rotation angles between the actual HS-AFM and the simulated AFM images. Black arrowhead indicates the angle with the highest correlation score. Source data of the graphs are provided as a Source Data file.

Fig. 2. Visualization of protein translocation mediated by one unit of SecYEG-SecA complex.

a Schematic model and HS-AFM image of proOmpA-sfGFP on a mica surface (upper) and histogram of the end-to-end distance, extracted from snapshots of the HS-AFM movies, between sfGFP and signal sequence (lower). The solid line represents a fitting curve following a Gaussian distribution and X₀ is the center value of the distribution with standard deviation (n = 55). The AFM image height (nm) is shown by the color bar. A representative image from more than 20 independent experiments with similar results are shown. b Schematic model and HS-AFM image of proOmpA-sfGFP bound to SecYAEG-ND (upper) and histogram of the end-to-end distance, extracted from snapshots of the HS-AFM movies, between sfGFP and SecYAEG-ND (lower). The solid line represents a fitting curve following a Gaussian distribution and X₀ is the center value of the distribution with standard deviation (n = 89). The AFM image height (nm) is shown by the color bar. A representative image from more than 10 independent experiments with similar results is shown. c Clipped HS-AFM images of proOmpA bound to SecYAEG-ND in the presence of 5 mM ATP. Time 0 s in the initial image indicates an arbitrarily chosen time point of the HS-AFM observation. The AFM image height (nm) is shown by the color bars. The proOmpA and sfGFP regions are given a reddish and greenish tint, respectively. The red arrowhead in the 10.2 s panel indicates the substrate that has inserted into the SecA side. In the 15.4 s image, the red arrowhead highlights the translocated proOmpA extending from the SecYEG-ND side. These images represent snapshots from the highest-resolution movie obtained across numerous experiments. Source data of the graphs are provided as a Source Data file.

Fig. 3. High–Low conformational dynamics of SecA region during protein translocation.

a Real-time HS-AFM images of SecYAEG-ND complexed with proOmpA-sfGFP. Arrows indicate regions of height variation upon exposure to 5 mM ATP. Height profiles of the SecA region in the High (b) and Low (c) states, traced along the red lines. Height profiles of the SecA region in the wide-open (d) and closed (e) forms of the simulated AFM images generated from the predicted structures at an 80° angle, which is defined in Fig. 1f, traced along the red lines. Representations of the SecYAEG-ND in wide-open (f) and closed (g) forms predicted by MD simulation in two different orientations. PPXD is highlighted in yellow, the rest of SecA in blue, and SecYEG in magenta. The color bars show the AFM or simulated AFM image height (nm). Representative data from more than 5 independent experiments with similar results are shown. Source data of the graphs are provided as a Source Data file.

Fig. 4. SecA dynamics derived from PPXD movement depending on nucleotide states in protein translocation.

a Distribution of the High and Low forms in different nucleotide conditions, as counted from HS-AFM snapshot images. SecA can transition to the Low form upon interacting with its substrate, as opposed to being in the High state in the resting condition. The number of particle images for each state is as follows: Apo (n = 100), 5 mM ATP (n = 284), 1 mM AMP-PNP (n = 140), 5 mM ADP-AlF₃ (n = 174), 5 mM ADP (n = 134), 5 mM ATP, and 50 mM NaH₂PO₄ (n = 134). b Schematic representation of SecA conformational dynamics during the ATP hydrolysis cycle. The diagram illustrates SecA conformational transitions between High (wide-open) and Low (closed) conformations in response to nucleotide state changes. Upon ATP binding, SecA adopts the High conformation, which transitions to the Low conformation after ATP hydrolysis. Following phosphate release, SecA returns to the High conformation.