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Title: Structrural Analysis of a Rhomboid Family Intramembrane Protease Reveals a Gating Mechanism for Substrate Entry

Abstract

Intramembrane proteolysis regulates diverse biological processes. Cleavage of substrate peptide bonds within the membrane bilayer is catalyzed by integral membrane proteases. Here we report the crystal structure of the transmembrane core domain of GlpG, a rhomboid-family intramembrane serine protease from Escherichia coli. The protein contains six transmembrane helices, with the catalytic Ser201 located at the N terminus of helix {alpha}4 approximately 10 Angstroms below the membrane surface. Access to water molecules is provided by a central cavity that opens to the extracellular region and converges on Ser201. One of the two GlpG molecules in the asymmetric unit has an open conformation at the active site, with the transmembrane helix {alpha}5 bent away from the rest of the molecule. Structural analysis suggests that substrate entry to the active site is probably gated by the movement of helix {alpha}5.

Authors:
; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
930653
Report Number(s):
BNL-81121-2008-JA
TRN: US200901%%164
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Nature Structural and Molecular Biology; Journal Volume: 13; Journal Issue: 12
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; CLEAVAGE; CRYSTAL STRUCTURE; ESCHERICHIA COLI; MEMBRANES; PEPTIDES; PROTEINS; PROTEOLYSIS; SERINE; SUBSTRATES; WATER; national synchrotron light source

Citation Formats

Wu,Z., Yan, N., Feng, L., Oberstein, A., Yan, H., Baker, R., Gu, L., Jeffrey, P., Urban, S., and Shi, Y. Structrural Analysis of a Rhomboid Family Intramembrane Protease Reveals a Gating Mechanism for Substrate Entry. United States: N. p., 2007. Web.
Wu,Z., Yan, N., Feng, L., Oberstein, A., Yan, H., Baker, R., Gu, L., Jeffrey, P., Urban, S., & Shi, Y. Structrural Analysis of a Rhomboid Family Intramembrane Protease Reveals a Gating Mechanism for Substrate Entry. United States.
Wu,Z., Yan, N., Feng, L., Oberstein, A., Yan, H., Baker, R., Gu, L., Jeffrey, P., Urban, S., and Shi, Y. Mon . "Structrural Analysis of a Rhomboid Family Intramembrane Protease Reveals a Gating Mechanism for Substrate Entry". United States. doi:.
@article{osti_930653,
title = {Structrural Analysis of a Rhomboid Family Intramembrane Protease Reveals a Gating Mechanism for Substrate Entry},
author = {Wu,Z. and Yan, N. and Feng, L. and Oberstein, A. and Yan, H. and Baker, R. and Gu, L. and Jeffrey, P. and Urban, S. and Shi, Y.},
abstractNote = {Intramembrane proteolysis regulates diverse biological processes. Cleavage of substrate peptide bonds within the membrane bilayer is catalyzed by integral membrane proteases. Here we report the crystal structure of the transmembrane core domain of GlpG, a rhomboid-family intramembrane serine protease from Escherichia coli. The protein contains six transmembrane helices, with the catalytic Ser201 located at the N terminus of helix {alpha}4 approximately 10 Angstroms below the membrane surface. Access to water molecules is provided by a central cavity that opens to the extracellular region and converges on Ser201. One of the two GlpG molecules in the asymmetric unit has an open conformation at the active site, with the transmembrane helix {alpha}5 bent away from the rest of the molecule. Structural analysis suggests that substrate entry to the active site is probably gated by the movement of helix {alpha}5.},
doi = {},
journal = {Nature Structural and Molecular Biology},
number = 12,
volume = 13,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
  • Escherichia coli GlpG is an integral membrane protein that belongs to the widespread rhomboid protease family. Rhomboid proteases, like site-2 protease (S2P) and {gamma}-secretase, are unique in that they cleave the transmembrane domain of other membrane proteins. Here we describe the 2.1 {angstrom} resolution crystal structure of the GlpG core domain. This structure contains six transmembrane segments. Residues previously shown to be involved in catalysis, including a Ser-His dyad, and several water molecules are found at the protein interior at a depth below the membrane surface. This putative active site is accessible by substrate through a large 'V-shaped' opening thatmore » faces laterally towards the lipid, but is blocked by a half-submerged loop structure. These observations indicate that, in intramembrane proteolysis, the scission of peptide bonds takes place within the hydrophobic environment of the membrane bilayer. The crystal structure also suggests a gating mechanism for GlpG that controls substrate access to its hydrophilic active site.« less
  • Intramembrane proteases are important enzymes in biology. The recently solved crystal structures of rhomboid protease GlpG have provided useful insights into the mechanism of these membrane proteins. Besides revealing an internal water-filled cavity that harbored the Ser-His catalytic dyad, the crystal structure identified a novel structural domain (L1 loop) that lies on the side of the transmembrane helices. Here, using site-directed mutagenesis, we confirmed that the L1 loop is partially embedded in the membrane, and showed that alanine substitution of a highly preferred tryptophan (Trp136) at the distal tip of the L1 loop near the lipid:water interface reduced GlpG proteolyticmore » activity. Crystallographic analysis showed that W136A mutation did not modify the structure of the protease. Instead, the polarity for a small and lipid-exposed protein surface at the site of the mutation has changed. The crystal structure, now refined at 1.7 Angstroms resolution, also clearly defined a 20-Angstroms-wide hydrophobic belt around the protease, which likely corresponded to the thickness of the compressed membrane bilayer around the protein. This improved structural model predicts that all critical elements of the catalysis, including the catalytic serine and the L5 cap, need to be positioned within a few angstroms of the membrane surface, and may explain why the protease activity is sensitive to changes in the protein:lipid interaction. Based on these findings, we propose a model where the end of the substrate transmembrane helix first partitions out of the hydrophobic core region of the membrane before it bends into the protease active site for cleavage.« less
  • Regulated intramembrane proteolysis by members of the site-2 protease (S2P) family is an important signaling mechanism conserved from bacteria to humans. Here we report the crystal structure of the transmembrane core domain of an S2P metalloprotease from Methanocaldococcus jannaschii. The protease consists of six transmembrane segments, with the catalytic zinc atom coordinated by two histidine residues and one aspartate residue {approx}14 angstroms into the lipid membrane surface. The protease exhibits two distinct conformations in the crystals. In the closed conformation, the active site is surrounded by transmembrane helices and is impermeable to substrate peptide; water molecules gain access to zincmore » through a polar, central channel that opens to the cytosolic side. In the open conformation, transmembrane helices {alpha}1 and {alpha}6 separate from each other by 10 to 12 angstroms, exposing the active site to substrate entry. The structure reveals how zinc embedded in an integral membrane protein can catalyze peptide cleavage.« less
  • The X-ray structure of protease-cleaved E. coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. colimore » α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.« less