Dynamics of SecY translocons with translocation-defective mutations

Abstract

The SecY/Sec61 translocon complex, located in the endoplasmic reticulum membrane of eukaryotes (Sec61) or the plasma membrane of prokaryotes (SecY), mediates the transmembrane secretion or insertion of nascent proteins. Mutations that permit the secretion of nascent proteins with defective signal sequences (Prl-phenotype), or interfere with the transmembrane orientation of newly synthesized protein segments, can affect protein topogenesis. The crystallographic structure of SecYEbeta from Methanococcus jannaschii revealed widespread distribution of mutations causing topogenesis defects, but not their molecular mechanisms. Based upon prolonged molecular dynamics simulations of wild-type M. jannaschii SecYEbeta and an extensive sequence-conservation analysis, we show that the closed state of the translocon is stabilized by hydrogen-bonding interactions of numerous highly conserved amino acids. Perturbations induced by mutation at various locations are rapidly relayed to the plug segment that seals the wild-type closed-state translocon, leading to displacement and increased hydration of the plug.

Figure 1

SecYEβ in a POPC lipid bilayer. Loops C2, C4, and C5 correspond to loops connecting, respectively, TM2/TM3, TM6/TM7, and TM8/TM9. (A, B) Cut away images of SecYEβ viewed along the membrane plane. Color scheme: SecY, green; SecE, light blue; Secβ, purple; gate helices TM2 and TM7, magenta; water oxygen atoms, pink; lipid phosphorous atoms, orange; lipid nitrogen, blue; lipid oxygen, red; and lipid carbon atoms, cyan. Sidechains of the hydrophobic pore are shown as brown surfaces (the probe radius used was 1.4 Å). In (B), only lipid phosphate groups of a ~50 Å-thick slab containing the membrane are shown. In all figures of the M. jannaschii translocon, the protein is oriented to align the z-axis parallel to the membrane normal. (C) The root-mean-squared deviation (rmsd; Å) of the SecYEβ C_α atoms relative to the starting crystallographic structure. For simplicity, the rmsd is shown at intervals of 10ps. (D,E) Overlap of 10 snapshots from the last 10ns of Sim1 taken at every 1ns (ending conformation, green; starting conformation, cyan). In (E), the hydrophobic ring at the end of Sim1 is shown as a green surface. Flexibility of the cytoplasmic loops and the C-terminus is largely responsible for rmsd values of SecY (green curve in panel D). The plug segment (p) of wild-type SecY (Sim1) samples a relatively restricted conformational space. (F) Location of amino acids whose mutations were investigated in Sim2 - Sim5. (G) Summary of the simulations performed (Sim1 - Sim5). The lengths of the simulations correspond to the unconstrained molecular dynamics trajectories. The average values (± s.d.) of the C_α rmsd of the SecY TM region in the last 10ns segments of Sim1-Sim5 are 1.9 ± 0.2, 1.7 ± 0.3, 1.6 ± 0.1, 2.0 ± 0.1, and 2.0 ± 0.1 Å for Sim1, Sim2, Sim3, Sim4, and Sim5, respectively (see Figure S1 for rmsd plots of the mutant simulations).

Figure 2

H-bonding clusters in wild-type SecY. As the H-bond criterion, we used a distance of less than 3.5 Å between the donor and acceptor heavy atoms. (A) H-bonding sidechains whose dynamics were investigated here are depicted in black, purple, or cyan for the SecY, SecE, and Secβ subunits of the translocon, respectively. (B) The H-bonding cluster EC-1 connects TM2, TM3, and TM7. (C) The crowded cytoplasmic cluster CP-6 that includes TM7. (D) H-bonding connectivity in wild-type SecY (for details see Figure S2, Table S1, and Table S2). Connections between two structural elements indicates that at least one H bond between them is sampled during Sim1. Note that Taura et al. (1994) denote the N- and C-terminus of SecY as C1 and C6, respectively. Examples of H bonding interactions in the T. maritima and T. thermophilus SecY translocons are described in Figures S3 and S4, respectively. Known mutation effects of H-bonding amino acids are given in Table S3.

Figure 3

Conservation of H bonding in the EC-1 cluster. In the table, ‘Posn’ gives the amino acid in the M. jannaschii sequence. ‘Consensus’ indicates the amino acid present in at least 75% of the sequences, compared to the sequence of the M. jannaschii SecY (‘SECY_METJA’). ’Gap’ indicates insertion/deletion mutations. The numbers indicate how many sequences have a specific amino acid at each of the positions investigated. For example, in archaea T80 is found as Thr in 53 sequences, and replaced with Ser, Met, or Ile in 1, 1, and 8 sequences, respectively. Sequence alignments for several pertinent SecY/Sec61p sequences are shown in Figure S5. Conservation of H-bonding residues for other H-bond clusters are shown in Figures S6-S11. Complete sequence alignments for archaea are shown in Figure S12. The conservation analysis was generated according to the Kyte and Doolittle scheme (Kyte and Doolittle, 1982), in which amino acids are colored from red (most hydrophobic) to blue (most hydrophilic). We analyzed the conservation of H-bonding amino acids in archaea, bacteria, and eukarya (78, 865, and 187 sequences, respectively). The initial list of SecY sequences was compiled using the Pfam database (Finn et al., 2008). The complete alignments for all sequences are available on our web site: http://blanco.biomol.uci.edu/download/Bondar_SecYE_align.zip.

Figure 4

Selected H-bonding amino acids of the TM2/TM7 gate helices and of the plug in the closed state of the M. jannaschii and in the open structure of the SecA-bound SecY from T. maritima. (A) Snapshot at the end of Sim1 (wild-type M. jannaschii translocon). (B) The translocon from T. maritima. In the crystal structure of the closed state of the M. jannaschii translocon (Van den Berg et al., 2004) and in Sim1 (wild-type), helices TM2, TM3, and TM7 are interconnected via H bonding (panel A, and Figure 2C). In the open structure of the SecA-bound SecY (Zimmer et al., 2008), TM2 and TM3 H bond via T83 and Q131, which correspond to M. jannaschii T80 and E122, respectively. In both translocons, TM4 can H bond to the extracellular tip of TM2 to the plug via a Ser/Thr amino acid.

Figure 5

Mutations perturb the structure and interactions of the plug. TM2, TM3, TM7, TM10 the hydrophobic pore, and the plug are depicted for the wild-type (panel A; Sim1), the L406K mutant (panel B; Sim4), and the in silico mutant T72V/T80V/R104A (panel C; Sim3). Amino acids of the hydrophobic ring are shown as pink van der Waals spheres, and other selected amino acids as blue. (D–F) Time series of the distance between the C_α atoms of S65 and L73 (panel D), S65 and T63 (panel E), and between A64 and I174 (panel F), illustrate the perturbation of the plug in the mutants. The average C_α distances for S65:L73, S65:T63, and A64:I174 in Sim1 are, respectively, 6.1±0.2 Å, 5.5±0.1 Å, and 5.0±0.3 Å, as compared to 5.5 Å, 5.1 Å and, 4.8 Å in the crystal structure of (Van den Berg et al., 2004). The average S65:T63 distances in the K250E (Sim2) and E336R (Sim5) mutants are 5.6±0.2 Å and 5.4±0.2 Å, respectively. (G) Close view of T63, S65, and L73 in L406K (Sim4). T63 and S65 are also depicted in Figure S2J for the wild type (Sim1).

Figure 6

Coupling of TM segments, plug, and hydrophobic-pore amino acids. (A) Overlap of snapshots from Sim1 (wild-type), Sim2 (K250E) and Sim4 (L406K) depicted in green, purple, and orange, respectively. The sidechains of the pore amino acids in Sim1 are shown as a green surface, except for L406 which is colored cyan. The sidechain and C_α atom of A64 are shown as black van der Waals spheres, and other C_α atoms in Sim1 as silver or green van der Waals spheres. H atoms are not shown. (B) The change in the plug location and hydration (see Figures 5 and 7) is associated with changes in the dynamics of R66. The normalized number density of the guanidinium group of R66 along the membrane normal is depicted in green, purple, cyan, orange, and gray for the wild type (Sim1), K250E (Sim2), T72V/T80V/R104A (Sim3), L406K (Sim4), and E336R (Sim5), respectively. Note that relative to the wild type, in Sim3-Sim5 the distribution of the R66 guanidinium group location broadens, and in Sim2 R66 samples preferentially a more cytoplasmic orientation.

Figure 7

Increased hydration in the extracellular half of the translocon upon displacement of the plug. The wild-type translocon (panel A; Sim1) is compared to the L406K mutant (panel B; Sim4). Color code used for van der Waals spheres: sidechains of the amino acids forming the hydrophobic pore - mauve, except for L406 which is depicted in violet; sidechains of S65 and A64 - green; water oxygen atoms – yellow; lipid nitrogen, oxygen, phosphorous, and carbon atoms are depicted in blue, red, orange, and black, respectively. For simplicity, hydrogen atoms are not shown. (C) Close-up view of the hydrophobic pore in the wild-type (Sim1; green) and L406K (orange; Sim4). Sidechains of pore ring amino acids and A64 are shown as van der Waals spheres; water molecules within 6 Å of A64 and within 6 Å of L/K406 are shown as cyan and purple spheres, respectively. (D) Histograms of the number of water molecules within 6 Å of A64 in the wild-type (Sim1; green), K250E (Sim2; purple), L406K (orange, Sim4), and E336R (Sim5). The histograms were computed with a bin size of 1.

Figure 8

Coupling between the SecYE translocon and the lipid membrane. The wild-type translocon structure (green, Sim1; panels A and C) is compared to E336R (gray, Sim4; panel B) and K250E (pink, Sim2; panel D). (A) Snapshot from the end of Sim1 showing the SecE:lipid and SecE:SecY interactions. Charged amino acids on the cytoplasmic side of SecE, E336, and SecY amino acids that H bond to SecE on the cytoplasmic side are depicted as bonds with carbon in yellow, oxygen red, and nitrogen in blue. Selected SecE amino acids are labeled with italics. Lipid headgroups (shown as van der Waals spheres) located within 3.5 Å from cytoplasmic charged amino acids of SecE are depicted with the carbon atoms in black, oxygen red, nitrogen blue, and phosphorous atoms in orange; all other lipid heavy atoms are in cyan. Note that E335 does not interact closely with a lipid headgroup. (B) In the E336R mutant, R336 H bonds to a lipid headgroup. (C,D) Lipid and protein interactions of the N terminus in the wild type (panel C; Sim1) and K250E (panel D; Sim2). In the wild-type and in Sim3-Sim5, K10 snorkels towards the cytoplasmic side and salt-bridges with E9; in Sim2, K10 interacts with a solvated lipid headgroup. Water molecules within 9 Å of the Nζ atom of K10 are shown as green van der Waals spheres. The alkyl chains of a POPC lipid molecule whose headgroup interacts with K10 is depicted in brown.

Figure 9

Conserved H bonds and the relay of structural perturbations in the translocon. (A, B) Conservation of H-bonding amino acids (panel A) and of the H-bonding property (panel B). H-bonding amino acids whose dynamics were studied here are depicted as bonds colored in black, purple, cyan, or blue if they are conserved (at least 40%) in all organisms, in archaea and bacteria (cyan), archaea and eukarya (purple) or only in archaea (blue). See Figures S6–S8 for the detailed frequency analysis. (C) The many H bonds of SecY likely contribute to the fast relay of structural perturbation to the plug. H-bonding amino acids of SecY are colored in yellow, SecE – purple, and Secβ – blue. For simplicity, only helices TM2, TM3, TM7, and the plug are shown for the wild-type (yellow; Sim1) and the T72V/T80V/R104A mutant (cyan; Sim3). The hydrophobic pore amino acids of the wild type structure are shown as yellow surface (probe radius is 1.4 Å). The sidechains of A64, S65, and L73 are shown as van der Waals spheres depicted in orange for the wild type, and cyan for T72V/T80V/R104A. Notice the displacement of the plug in T72V/T80V/R104A relative to the wild type as indicated by A64 and S65. Lipid heavy atoms (cutaway view) are shown with carbon in black, phosphorous lime, oxygen violet, and nitrogen atoms in blue. Water oxygen atoms (cutaway view) are shown as blue dots. H atoms are not depicted. (D) Cartoon illustrating the displacement of the plug and enhanced solvation of the plug in the mutants.