Comparison of GlpG S201T structures in lipid and detergent environments. (a) An overlay of the two structures and (b) a graphical representation of the rmsd, calculated with Superpose in CCP4 28 suite, show the differences in the loop regions, particularly L1, L4 and L5. The graphical plot shows the main-chain rmsd along the length of the polypeptide. The average rmsd deviation is 0.664 Å for the C^α atoms. (c) A minor rearrangement in L1 as a result of crystal contact and to accommodate the lipids, which includes changes in the rotamer conformations of Lys132 and Phe135. (d) In the active site, the side chain of His254 shows a more prominent displacement in the GlpG S201T structure in a detergent environment.

Two-dimensional crystals of GlpG. (a) Crystalline membranes of N-terminally truncated GlpG WT negatively stained with 1.5% uranyl acetate. (b) High-magnification image of a crystalline membrane showing the crystal lattice. (c) Calculated Fourier transform of a single image of GlpG with a rectangular unit cell of 37.6/56.4 Å. Each square on the reciprocal lattice describes a Fourier component, with the size and number of the square reflecting its signal-to-noise ratio. 34 The largest boxes and the smallest numbers describe the most significant reflections. Concentric rings indicate the zero crossings of the contrast transfer function. Well-preserved and flat crystals show spots to ∼ 4 Å.

Lipid–protein interactions in GlpG. (a) Lipids PC509 and PC510 (yellow sticks) occupy the hydrophobic cavity created by the protrusion of L1. A number of contacts between the acyl chain and the hydrophobic side chains of TM1, TM3, and L1 (shown in stick representation and coloured green) are observed. Two-symmetry related lipids (cyan sticks) are found covering the H1 and H2 of L1. The present structure and the lipids around L1 explain the differential accessibility of the residues to cysteine-modifying reagents in a detergent or lipid environment observed by Wang et al. 36 (b) Three lipid molecules occupy the V-shaped gap created by L1 between TM1 and TM3. The polar interactions between the oxygen atoms of the glycerol backbone of lipids PC507 and PC508 (red dotted line) are with the indole nitrogen of Trp125 and hydroxyl of Tyr187, respectively. (c) The lipid molecules along TM1 are spaced apart to give a hydrophobic thickness of ∼ 32 Å. The acyl chains 513 and 514 extend beyond the disordered N-terminus in the present structure. PC503 occupies the groove formed by TM1 and TM2. PC511 and PC512 are probably part of same lipid molecule and likely to bend over TM1 and L1. The acyl chain of PC506 interacts with hydrophobic residues of TM1. (d) Lipid PC502 occupies the interface between TM2 and TM5. A hydrogen bond is formed between the oxygen atom of the phosphodiester group and the side-chain amide of Gln226, and numerous hydrophobic interactions between the acyl chain and the side chains of residues along TM2 and TM5 are observed. The acyl chain of PC502 extends beyond the midpoint of TM helices 2 and 5. The terminal methyl group is only 4.4 Å from the side chain of Phe245 in L5, indicating that the side chains of this loop are clearly within the hydrophobic region of the bilayer. The solid black bar denotes the length of TM5 (∼ 23 Å) measured from Arg227 to Phe242.

Detergent mimics substrate binding. (a) BNG514 is well ordered in the present structure as observed in the F_o − F_c difference map (magenta mesh) calculated before modelling of the detergent, contoured at 3σ. The protrusion of BNG514 causes minor displacement of L5 residues, in particular Phe245 and Met247. There are numerous hydrophobic as well polar contacts between BNG514 and residues from TM2, TM5, and L5. If modelled, the head group of BNG514 is only ∼ 4 Å from the threonine. (b) An overlay of GlpG WT and S201T structures showing partial displacement of L5. The side chain of Met247 is disordered in the GlpGS201T structure. The different conformations of L5 are functionally important because, in the WT structure, the two side chains of methionine point into the active site; for peptide hydrolysis, this loop needs to move away. With one of the methionine residues still occluding the GlpG S201T active site, it is possible that it reflects an intermediate conformation of L5. (c) Top view of the GlpG S201T molecule in surface representation (coloured as in Fig. 3b) to show the bending of detergent (yellow spheres) towards the active site. The active-site residues are coloured green (Thr201) and orange (His254). L5 is shown in cartoon representation, with side chains of Phe245 and Met249 highlighted in magenta. Three water molecules (cyan spheres) are found in a cavity, which was predicted as the substrate binding S1 site. 26 The side chain of Met249 points into the cavity.

An active-site mutant of GlpG. (a) A rhomboid activity assay showing the cleavage of an artificial substrate (LacY TM2) expressed as a fusion protein to maltose-binding protein and thioredoxin. The cleavage at a specific site in LacY TM2 of the full-length (FL) substrate results in two products. The product (P1) with MBP migrates at ∼ 55 kDa, and a second product (P2) with thioredoxin migrates at ∼ 24 kDa. The mutation of serine to threonine at the active site results in an inactive enzyme with no cleavage of the substrate. Lanes marked C denote the control for substrate (sub) or enzyme (E). Substrate was used at a concentration of ∼ 2 μM, and enzymes were used at concentrations of 5 or 10 μM. A large excess of enzyme was used to test whether the S201T mutant had any residual activity. (b) An overlay of the structures of GlpG WT and S201T, determined in the trigonal crystal form in a detergent environment. The rmsd for the C^α atoms between these structures excluding L5 (residues 245–249) is 0.15 Å. (c) The active site of GlpG S201T: Replacement of active-site serine by threonine results in the displacement of the active-site histidine, such that it is within hydrogen-bonding distance of the side chain of Asn251. In the WT structure, a water molecule mediates this interaction. The hydroxyl of Thr201 points towards the oxyanion hole, which is characterised by the main-chain amides of residues Leu200 and Ser201, the imidazole nitrogen of His150, and the side-chain amide of Asn154. These residues are thought to stabilise the negative charge on the carbonyl oxygen formed during proteolysis. The hydroxyl of Thr201 is within hydrogen-bonding distance to the side chains of His150 (3.3 Å) and Asn154 (3.12 Å). This and the fact that it points away from the active-site histidine explain why this mutant is inactive. (d) The overlay of the active sites of WT and S201T shows clearly the extent of histidine side-chain movement.

Structure of GlpG in a lipid environment. (a) Type I crystals of GlpG showing an alternating up and down orientation of GlpG molecules (shown in cartoon representation) in the same layer with a shift along the a-axis. Within each layer, lipid molecules (green sticks) along with TM1 and TM5 mediate the lateral interaction between protein molecules. Two membranous layers are held together by interactions (marked with dotted circle) between residues of symmetry-related molecules in L1 and L4. The polar contacts mediated by side chains of charged residues (shown in stick representation) play a major role in this interaction. The interaction between E118, D128, and R227 is shown separately as a magnified view with the lipids removed. The molecules of GlpG are colour coded according to the function of each structural element. TM helices 4 and 6 harbouring the active-site residues are shown in light blue; TM helices 2 and 5 that form the interface for substrate binding are in yellow; L1 is in pink; the extended loop, L3 upstream of the active-site serine that probably binds substrate, is in cyan; and TM helices 1 and 3 are in grey. L5 that covers the active site is in orange. (b) Two views of a surface representation of GlpG in a lipid environment. An almost complete bilayer around a GlpG molecule is depicted. The protein molecule is colour coded according to the biochemical properties of the amino acids: positive and negatively charged residues are shown in blue and red, polar residues are in light blue; and the rest in grey. The carbon atoms of lipids from the same molecule are shown as green spheres, and those from symmetry-related molecules are shown as yellow spheres. Water molecules are shown as blue spheres. The solid grey bars denote the position of the phosphodiester groups of the lipid molecules and mark the hydrophobic boundary.

A comparison of maps obtained by electron and X-ray crystallography. (a) A projection map was calculated by merging five images of 2D crystals to 8 Å in p22₁2₁ symmetry by including spots to an IQmax of 4. Note that space groups p22₁2₁ and P2₁2₁2₁ are indistinguishable in projection. The phases were averaged and rounded to 0° or 180°. Continuous lines indicate density above the mean, while negative contours are shown as dotted lines. An isotropic temperature factor (B = − 200) was applied to compensate (sharpen) for the resolution-dependent degradation of image amplitudes. The map was scaled to a maximum peak density of 250 and contoured in steps of 21. (b) A projection map of GlpG calculated from the PDB coordinates of GlpG S201T in the orthorhombic crystal form truncated to 8 Å. No temperature factor was applied, but the map was scaled and contoured as described for 2D crystals. For a direct comparison of the projection map with the packing in 3D crystals, please refer to Supplementary Fig. 3a. A unit cell is displayed with the a-axis vertical and the b-axis horizontal in both figures. It should be noted that the symmetry elements depicted in Fig. 5a are for the 3D space group P2₁2₁2₁ and not for the 2D space group p22₁2₁ used to merge the images. It was obvious that these 2D crystals were thin 3D crystals rather than a single layer, and the fact that they have a similar packing to the 3D crystal obtained by vapour diffusion prompted me to use the same depiction of symmetry elements. The reason for the formation of stacked 2D arrays can be appreciated from the polar interaction observed between two layers mediated by charged residues between L1 and L4 (Fig. 3a).

Substitution of lipids by detergent. (a) Surface representations showing two views of GlpG S201T in the trigonal crystal form with a detergent environment. The protein molecule is coloured as in Fig. 3b. Some detergent molecules (carbon atoms as green spheres) are observed in analogous positions to lipid molecules. Water molecules (blue spheres) and the solid grey bars mark the membrane boundary. The arginine (R92) residue at the N-terminus of the protein is marked to show the region that is disordered in the structure determined in a lipid environment. (b and c) Interactions of selected detergent molecules with the protein. Detergent molecules are shown as yellow stick representations; the side chains of amino acids interacting with detergent are shown in green; and the backbone of the protein molecule is in grey. Panel (b) shows four detergent molecules found interacting with residues in L1. The hydrophobic cavity created by L1 is filled with two of these molecules but in contrast to the position of the lipid (Fig. 4a); the head groups of these detergents are in direct contact to some of the residues. Panel (c) shows the detergent molecules BNG508 and BNG514 at the TM2/TM5 interface stretching from the cytoplasmic end of TM2 to L5 at the periplasmic end. Together, these two detergents occupy the same position as the sn1 acyl chain of the lipid molecule PC502 (shown as transparent green sticks), while BNG513 substitutes for the sn2 acyl chain.

Three experimentally determined structures that provide a sequential picture, and therefore a model, for substrate binding and catalysis in rhomboid protease. The structural changes required for substrate binding and catalysis can be followed by changes in loop 5 (backbone coloured in orange) and the side chains (shown as green sticks) within it. The active-site residues Ser/Thr201 and His254 are shown as green sticks. Substrate is likely to partition into the enzyme through TM helices 2 and 5 (shown in yellow). The rest of the molecule is shown in grey. (a) The closed state of GlpG (PDB code 3B45 or 2XOV), as defined by the inward pointing of side chains of Met247 and Met249 in loop 5, prevents binding at the active site. A partially ordered detergent molecule (cyan sticks and labelled BNG) is found at the TM2/TM5 interface. (b) A partially open state of GlpG (PDB code 2XTU) is represented by the protrusion of a detergent molecule (cyan sticks) with a displacement of residues 245–247 in loop 5. The side chain of Met249 still points into the active site, thus indicating that this needs to make way for binding at the active site. (c) The open state with an inhibitor (cyan) bound covalently to the active residues (PDB code 2XOW) shows that both Met247 and Met249 are lifted away from the active site. This is accompanied by a small displacement of TM5.