A large conformational change of the translocation ATPase SecA - PubMed (original) (raw)

A large conformational change of the translocation ATPase SecA

Andrew R Osborne et al. Proc Natl Acad Sci U S A. 2004.

Abstract

The ATPase SecA mediates the posttranslational translocation of a wide range of polypeptide substrates through the SecY channel in the cytoplasmic membrane of bacteria. We have determined the crystal structure of a monomeric form of Bacillus subtilis SecA at a 2.2-A resolution. A comparison with the previously determined structures of SecA reveals a nucleotide-independent, large conformational change that opens a deep groove similar to that in other proteins that interact with diverse polypeptides. We propose that the open form of SecA represents an activated state.

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Figures

Fig. 1.

Fig. 1.

Structure of monomeric B. subtilis SecA. Monomeric B. subtilis SecA is presented as a ribbon diagram. NBF1 is shown in yellow, NBF2 is shown in blue, the PPXD is shown in orange, the HSD is shown in green, and the HWD is shown in cyan. ADP is shown in a ball-and-stick representation. The images were prepared by using

molscript

(40),

raster

3

d

(41), or

spock

(available at

http://mackerel.tamu.edu/spock

).

Fig. 2.

Fig. 2.

Contacts in different SecA crystal forms. (a) Ribbon diagram of the previously determined B. subtilis SecA structure, which is likely to be the physiological dimer (16). (b) The largest contact in the crystal lattice of monomeric B. subtilis SecA. (c) The proposed M. tuberculosis SecA dimer (17). Residues 757–768 in the HSD and the N terminus of SecA that are important for dimerization of E. coli SecA are shown in red and yellow, respectively.

Fig. 3.

Fig. 3.

Domain movements in monomeric SecA. Ribbon diagram of monomeric B. subtilis SecA in the open conformation (a) and of a single subunit of dimeric B. subtilis SecA in the closed conformation (b). Color codes are as described for Fig. 1. The first and last helices in the PPXD are represented as cylinders to better visualize the transition between the conformations. The arrows in a indicate the movements that are required to convert the open conformation to the closed conformation. The side chains of residues 232 and 773 are shown in red in stick representation. Corresponding E. coli SecA residue numbers are given in parentheses. These residues were mutated to cysteines in E. coli SecA, and the accessibility of residue 824 to a modification reagent was used to probe the transition from the closed to the open conformation.

Fig. 4.

Fig. 4.

Potential ligand-binding sites in monomeric B. subtilis SecA. (a) A surface representation of SecA showing the surface grooves 1 and 2 in red and blue, respectively. (b) Surface representation of calmodulin with bound peptide shown in green as an α-carbon trace (25). (c) Surface representation of Hsp70, with the C terminus occupying the peptide-binding groove shown in green as an α-carbon trace (26). (d and e) Surface representations of groove 1, showing the location of pockets H and P, respectively. The surface is rendered transparent, and the underlying peptide backbone is color-coded as described for Fig. 1.

Fig. 5.

Fig. 5.

E. coli SecA can adopt an open conformation in solution. (a) SecA containing single cysteines at positions 824 or 234, or a mutant lacking cysteines (no cys), were labeled with maleimide fluorescein in the presence of the indicated additions. When nucleotide (0.25 mM) was added, 0.5 mM MgCl2 was also included. The samples were separated by SDS/PAGE and visualized under UV light. In the lane labeled quench+SDS, the quenching reagent, either DTT or glycine, was added before labeling or crosslinking. (b) In parallel, samples were crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, separated by SDS/PAGE, and stained with Coomassie blue. The position of dimer crosslinks is indicated. (c) Modification reactions with SecA containing a cysteine at position 824 were performed as in a in the presence of 1-myristoyl-2-hydroxy-_sn_-glycero-3-[phospho-rac-(1-glycerol)] (MLPG, 0.1 mM); 1,2-diheptanoyl-_sn_-glycero-3-phosphocholine (DHPC, 4.2 mM); dodecyl maltoside (DDM, 0.6 mM); decyl maltoside (DM, 3.6 mM); octyl maltoside (OM, 39 mM); octyl glucoside (OG, 36.4 mM); CYMAL4 (15.2 mM); CYMAL6 (1.12 mM); lauryldimethylamine-_N_-oxide (LDAO, 2 mM); digitonin (1%); or SDS (0.5%). (d) In parallel, samples were crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

Fig. 6.

Fig. 6.

The ATPase site and mutations affecting SecA function. (a) The Walker A motifs of ADP-bound SecA and ATP-bound PcrA are superimposed. The Walker A and B motifs and motif 6 of monomeric B. subtilis SecA are shown in color, and the equivalent motifs in PcrA are shown in gray (18). Arginine residues in motif 6 are shown in stick representation, and a magnesium ion bound to SecA is shown as a green sphere. The ATP bound to PcrA and the ADP bound to SecA are shown in a stick representation in gray and light blue, respectively. The phosphorus atom of the γ-phosphate of ATP bound to PcrA is shown in red. (b) A view of the SecA ATPase site showing that the Walker B motif (red) is connected to the PPXD via a β-strand (gray). The β-strand (blue) that leads from the PPXD back to NBF1 is connected to the strand between the Walker A (black) and B motifs. NBF2 is omitted for clarity, and the remaining domains are color-coded as described for Fig. 1. The aspartate residue in the Walker B motif and ADP are shown in stick representation. (c) SecA mutations that suppress mutations in SecY channel components are shown in yellow, and PrlD mutations that suppress signal sequence mutations are shown in cyan on a backbone representation of monomeric B. subtilis SecA. Residues identified in both screens are shown in green. ADP is shown as a stick representation.

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