PDBsum entry 3b45

Membrane protein

PDB id: 3b45

Contents

Protein chain

180 a.a. *

Ligands

BNG ×17

Waters ×79

* Residue conservation analysis

PDB id:

3b45

Name:

Membrane protein

Title:

Crystal structure of glpg at 1.9a resolution

Structure:

Glpg. Chain: a. Fragment: core tm fragment, residues 91-270. Engineered: yes

Source:

Escherichia coli. Organism_taxid: 562. Gene: glpg. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

1.90Å R-factor: 0.215 R-free: 0.228

Authors:

Y.Wang,S.Maegawa,Y.Akiyama,Y.Ha

Key ref:

Y.Wang et al. (2007). The role of L1 loop in the mechanism of rhomboid intramembrane protease GlpG. J Mol Biol, 374, 1104-1113. PubMed id: 17976648 DOI: 10.1016/j.jmb.2007.10.014

Date:

23-Oct-07 Release date: 22-Jan-08

Protein chain

P09391  (GLPG_ECOLI) -  Rhomboid protease GlpG from Escherichia coli (strain K12)

Seq: 276 a.a.

Struc: 180 a.a.

Enzyme reactions

• Enzyme class: E.C.3.4.21.105 - rhomboid protease.

DOI no: 10.1016/j.jmb.2007.10.014

J Mol Biol 374:1104-1113 (2007)

PubMed id: 17976648

The role of L1 loop in the mechanism of rhomboid intramembrane protease GlpG.

Y.Wang, S.Maegawa, Y.Akiyama, Y.Ha.

ABSTRACT

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 proteolytic 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 A resolution, also clearly defined a 20-A-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.

Selected figure(s)

Figure 4.
Fig. 4. The crystal structure of W136A mutant. (a) Electron density, contoured at 1.5σ level, at the site of mutation. Water molecules are shown in red, and detergents in green. (b) The water molecules (numbered 1 to 6) substituting the indole ring of Trp136 are stabilized by a network of hydrogen bonds. (c) The structure of the wild type showing the interfacial location of Trp136. (d) After mutation, the protein surface is no longer compatible with its buried location.

Figure 5.
Fig. 5. The model of membrane-embedded GlpG. (a) The C^α trace of the protein is shown in yellow, externally bound water in red, and water inside the membrane protein in blue. The water molecules substituting for Trp136 in the mutant are shown in white. The two horizontal lines mark the boundaries of the hydrophobic core of the membrane around the protease. (b) A histogram of the number of water molecules observed across the bilayer in the same color scheme as in (a). (c) The location of various structural elements important for protease function within the lipid bilayer. Parts of L1 (marked by *) and TM helix S2 (**) are omitted to show the internal active site.

The above figures are reprinted from an Open Access publication published by Elsevier: J Mol Biol (2007, 374, 1104-1113) copyright 2007.

Figures were selected by an automated process.