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Review
. 2012 Jun;76(2):311-30.
doi: 10.1128/MMBR.05019-11.

Membrane Proteases in the Bacterial Protein Secretion and Quality Control Pathway

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Free PMC article
Review

Membrane Proteases in the Bacterial Protein Secretion and Quality Control Pathway

Ross E Dalbey et al. Microbiol Mol Biol Rev. .
Free PMC article

Abstract

Proteolytic cleavage of proteins that are permanently or transiently associated with the cytoplasmic membrane is crucially important for a wide range of essential processes in bacteria. This applies in particular to the secretion of proteins and to membrane protein quality control. Major progress has been made in elucidating the structure-function relationships of many of the responsible membrane proteases, including signal peptidases, signal peptide hydrolases, FtsH, the rhomboid protease GlpG, and the site 1 protease DegS. These enzymes employ very different mechanisms to cleave substrates at the cytoplasmic and extracytoplasmic membrane surfaces or within the plane of the membrane. This review highlights the different ways that bacterial membrane proteases degrade their substrates, with special emphasis on catalytic mechanisms and substrate delivery to the respective active sites.

Figures

Fig 1
Fig 1
Signal peptide cleavage of precursor proteins by signal peptidases. Signal peptidase I (SPI) employs a Ser-Lys catalytic dyad for signal peptide cleavage from secretory precursor proteins at the extracytoplasmic surface of the membrane. The Protein Data Bank (PDB) structure of the catalytic domain (accession number 1T7D) and the program JMol were used to generate the three-dimensional (3D) structure image of SPI. Signal peptidase II (SPII) is an aspartic acid protease that cleaves signal peptides from bacterial lipoprotein precursors just beneath the extracytoplasmic membrane surface. The lipoprotein precursor protein is diacylglyceride modified prior to SPII cleavage. Signal peptidase IV (SPIV) is an aspartic protease that cleaves signal peptides from prepilins and pseudopilins at the cytoplasmic surface of bacterial membranes. The eukaryotic ER signal peptidase complex (SPC) is composed of five subunits, of which SPC18 and SPC21 are catalytic. Transmembrane helices of signal peptidases are depicted as blue barrels, and substrate helices are depicted as red barrels. A zoomed-in view of the active site residues of SPI is shown. The locations of the N and C termini of the signal peptidases and their substrates are indicated.
Fig 2
Fig 2
Signal peptide substrates of different classes of signal peptidases. Schematic representations are shown for bacterial (Sec-type) signal peptides cleaved by SPI, twin-arginine (Tat) signal peptides cleaved by SPI, lipoprotein signal peptides cleaved by SPII, bacterial prepilin signal peptides cleaved by SPIV, and archaeal preflagellin signal peptides cleaved by the SPIV homologue FlaK. The N, H, C, and basic regions of the respective signal peptides, mature protein parts, and conserved SP recognition sites are indicated. The SP cleavage site is marked with a black arrowhead. N, N terminus; C, C terminus.
Fig 3
Fig 3
Structure of the preflagellin signal peptidase FlaK of Methanococcus maripaludis. The structure on the left shows a side view of FlaK, and the structure on the right represents a view from the cytoplasmic side, with the cytoplasmic domain removed. Each of the six α helices is colored differently. The two catalytic Asp residues are shown as ball-and-stick models, in black. The crystal structure data were obtained using PDB accession number 3S0X, and JMol was used to generate the 3D structure images.
Fig 4
Fig 4
Cleavage of signal peptides by signal peptide hydrolases. The eukaryotic signal peptide hydrolase/peptidase SPP is an aspartic acid protease with catalytic Asp residues located within the plane of the membrane. The RseP protease, which seems to function as a general bacterial signal peptide hydrolase, is a metalloprotease with an intramembrane catalytic site facing the cytoplasmic side of the membrane. The HEXXH motif that binds the catalytic Zn2+ ion is indicated. The bacterial signal peptide peptidase SPPA is a homotetramer with catalytic Ser-Lys dyads in domains that are juxtapositioned to the extracytoplasmic membrane surface. Transmembrane helices of SPP and RseP are depicted as blue barrels, and the substrate helix is depicted as a red barrel. Furthermore, the four subunits of the SPPA complex are depicted in blue, green, red, and yellow. A zoomed-in view of the active site residues of SPPA is shown. The PDB structure 3BF0 and the program JMol were used to generate the 3D structure image of SPPA. The proposed position of the SPPA N-terminal transmembrane segment for each monomer (which was missing from the construct used to the solve the structure) is shown schematically. The locations of the N and C termini of the signal peptide hydrolases and their substrates are indicated.
Fig 5
Fig 5
Structure of Methanocaldococcus jannaschii S2P. (Left) The top view of S2P (PDB accession number 3B4R) shows the presumed lateral gate formed by transmembrane helix 1 (TM1) on one side and TM5 and TM6 on the other side. S2P with an opened lateral gate is shown in blue, and S2P with a closed lateral gate is shown in green. The catalytic Zn2+ ion is depicted as a red ball. (Based on reference .) (Right) Side views of the molecular surface of S2P in the open and closed states. To illustrate the relative distance, TM1 and TM6 are shown in red. The rest of the molecule is shown in purple.
Fig 6
Fig 6
Structures of bacterial FtsH and the homologous m-AAA protease in the inner mitochondrial membrane. (A) Structures of the cytoplasmic domains of apo-FtsH (left) and ADP-FtsH (right). The AAA domains are shown in cyan, and protease domains are shown in green. To give a clear inside view of the hexamer chamber, only 3 subunits, forming half of the holoenzyme, are shown. Amino acids 450 to 460 are proposed to form an active site switch (highlighted in yellow). These residues change from a β-sheet conformation in the apoprotein to an α-helical conformation in the ADP-bound form, which closes the proteolytic site of the corresponding subunit. The inward movement of this AAA domain opens the substrate tunnel, and the substrate polypeptide chain (red) is pulled through the chamber toward the open proteolytic site of the adjacent subunit. Phe234 at the substrate binding pore is shown in gray. The proposed positions of the N-terminal transmembrane segments of each monomer (which were missing from the construct used to the solve the structure) are shown schematically. Bound ADP is shown as a ball-and-stick model. The Zn2+ ion at the proteolytic site is shown as an enlarged purple sphere. The apo-FtsH structure was obtained from PDB accession number 3KDS, and that of ADP-FtsH was obtained from PDB accession number 2CEA. JMol was used to generate the 3D structure images. (B) Proposed mode of action of the mitochondrial m-AAA protease based on the cryo-electron microscopy (cryo-EM) structure. The structure data were obtained from EMDataBank (accession number 1712), and the Astex Viewer was used to generate the image. From left to right, the model depicts subsequent stages in substrate degradation. First, an unfolded terminal peptide of the substrate binds the surface of the AAA domain at the initial contact site (purple arrow). Subsequently, the unfolded peptide is transferred to the secondary binding site (black arrow), where it enters the AAA ring through the center pore. Lastly, the substrate is degraded in the chamber of the protease domain, and the resulting degradation products are released from the side pores (orange arrow).
Fig 7
Fig 7
Secondary structure of presenilin and 3D structure of GlpG. (A) The aspartic acid protease presenilin is synthesized as a membrane protein with nine transmembrane helices. It is cleaved upon activation, which results in an N-terminal moiety with six transmembrane regions and a C-terminal moiety with three transmembrane regions. Each of these moieties contains one catalytic aspartic acid residue. GlpG (PDB accession number 2IC8) employs active site Ser and His residues in intramembrane proteolysis. Transmembrane helices are depicted as blue barrels for presenilin and as a 3D ribbon structure for GlpG. Substrate helices are depicted as red barrels. The locations of the N and C termini of presenilin, GlpG, and their substrates are indicated. (B) Side views of the rhomboid protease GlpG (PDB accession number 2NRF) show the lateral gate formed by transmembrane helices TM2 (α2) and TM5 (α5). α2 and α5 are highlighted in green. The water channel is marked as a transparent blue cone. JMol was used to generate the 3D structure images.
Fig 8
Fig 8
Trimeric structure of the sheddase DegS. (A) Side view of DegS. (B) View from the periplasmic side. The protease domains in the center surround a funnel-like exposed surface where the proteolytic sites are located (outlined by a black dotted line). The PDZ domains that contain the peptide-binding grooves (gray shaded areas in panel B) are located on the outside of the DegS trimer. They are connected to the protease domains at their C termini. A peptide that is bound to the PDZ domain in one of the subunits is colored red. The structure data were obtained from PDB accession number 1SOZ, and JMol was used to generate the images. (C) Monomer structures of DegS in the peptide-free inactive (left), intermediate (middle), and peptide-bound active (right) states. The L1, L2, L3, and LD loops are highlighted in violet. In the intermediate form, without a substrate, L3 retreats from the substrate docking position (active form) to the inactive conformation, while L1, L2, and LD, located at the active site center, still adopt the active conformation. The active sites are shown in enlarged insets for all structures. The catalytic residues Ser201, His96, and Asp126 are shown in black. Residues forming the S1 pocket are shown in orange. These residues are reorganized upon substrate binding (compare the inactive and active forms). N94 (yellow) and H198 (green), which interfere with the catalytic triad and destabilize the oxyanion hole, as shown in the inactive form, move away in the active form. The stress signaling peptide binding site in the PDZ domain for DegS in the proteolytic active state is also shown in an enlarged inset. The peptide is colored red, and residues forming the hydrophobic pocket surrounding the Phe0 position are colored blue. Structure data were obtained from PDB, using accession numbers 1SOT (inactive state), 1VCW (intermediate state), and 1SOZ (active state). JMol was used to construct the images.
Fig 9
Fig 9
The sheddases BACE and ADAM17 cleave substrates at the extracellular membrane surface. Sheddases belonging to the BACE (A) and ADAM (B) families cleave membrane proteins at aqueous juxtamembrane positions. This results in the release of ectodomains from the substrate proteins into the extracellular space. The catalytic domains of both BACE (PDB accession number 1W50) and ADAM (PDB accession number 1BKC) are shown with zoomed-in views of the active site regions. JMol was used to generate the 3D structure images.
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