Cutting Proteins within Lipid Bilayers: Rhomboid Structure and Mechanism  Marius K. Lemberg, Matthew Freeman  Molecular Cell  Volume 28, Issue 6, Pages 930-940 (December 2007) DOI: 10.1016/j.molcel.2007.12.003 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Molecular Structure of the E. coli Rhomboid GlpG Ribbon diagram of E. coli GlpG, taken from coordinates of Wang et al. (2006) (PDB code 2IC8). See text for description of overall architecture. The loop connecting TM helices 1 and 2 (labeled L1, bottom panel, left) forms a novel protein fold extending sidewise into the plane of the membrane. For clarity, no secondary structure is indicated for the two non-TM helices of L1 in the front and back view (top panel). In order to show clearly the hydrophilic indentation and the proposed gate and cap structures, L1 and L5 are not shown in the surface representation (bottom panel, right). The white asterisk indicates the position of the catalytic center. Molecular Cell 2007 28, 930-940DOI: (10.1016/j.molcel.2007.12.003) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Rhomboid Active Site Highly conserved residues are shown in stick representation. The active-site structure illustrates the serine protease catalytic dyad (between S201 and H254 of E. coli GlpG). Conserved residues in TM helix 2 are not in hydrogen bonding distance and may fulfill other functions, such as substrate binding. Note that the tyrosine (Y205), which has been proposed to help stabilize the position of the catalytic histidine (H254), is not strictly conserved, so that in some rhomboids the histidine may be stabilized by other contacts. Ribbon representation as in Figure 1, back view. Molecular Cell 2007 28, 930-940DOI: (10.1016/j.molcel.2007.12.003) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Alternative Conformations for the Putative Gating Helix 5 The GlpG structure reported by Wang et al. (2006) (shown in red; structure as in Figure 1) was superimposed on the altered conformation reported by Wu et al. (2006) (shown in yellow; PDB code 2NRF, molecule A). The outward bending of TM helix 5 (H5) may act as a gate, shielding the active site from the lipid environment in the closed state (red), and opening (yellow) to accommodate entering substrate (Wu et al., 2006). Molecular Cell 2007 28, 930-940DOI: (10.1016/j.molcel.2007.12.003) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Model for Rhomboid Structure Function Relationship A front view, in which the positions of highly conserved residues are indicated, is shown. For clarity, the putative lateral gate L1 is moved within the plane of the membrane. Red arrows indicate this and other suggested movements, potentially providing the gate for substrate entry and a surface cap structure (GlpG regions with apparent high structural plasticity are highlighted in pale blue). Two proposed substrate entry routes are indicated by green arrows (see text for details). Molecular Cell 2007 28, 930-940DOI: (10.1016/j.molcel.2007.12.003) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Rhomboid Protease Consensus Conservation between rhomboids from multiple species that have been used in mutagenesis experiments is superimposed on the E. coli GlpG structure. Regions of highly conserved residues are indicated in red (>90% identity), orange is used for 80%–89% identity, and yellow is used for 50%–79% identity (see Lemberg and Freeman [2007] and references therein for details). The active-site consensus is GxSx in TM domain 4 (H4) and a single histidine (H) in TM domain 6 (H6). Small residues such as glycines (G) and alanine (A) in H4 and H6 allow tight helix packing that brings the catalytic serine and histidine in hydrogen bonding distance (Ben-Shem et al., 2007). The function of the conserved residues in TM domain 2 (H2) and the L1 loop is not yet clear. Note that the tryptophan-arginine motif (WR) of L1 is absent in mitochondrial PARL-type rhomboids but is strikingly conserved in the secretase-type and prokaryotic rhomboids. Residues implicated in the putative active-site gating mechanism centered around TM domain 5 and L5, such as F245 in E. coli GlpG, are not conserved (see text for details). Molecular Cell 2007 28, 930-940DOI: (10.1016/j.molcel.2007.12.003) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 Active-Site and Substrate Topology Secretase and mitochondrial PARL-type rhomboids have active sites with opposite orientations. Comparative analysis of rhomboids showed variations of the basic six-TM-domain rhomboid core (Koonin et al., 2003; Lemberg and Freeman, 2007): most eukaryotic rhomboids, such as Drosophila Rhomboid-1, have an extra TM domain fused to the C terminus (indicated in red); mitochondrial PARL-type rhomboids, however, have an extra TM domain fused to the N terminus, thereby changing the orientation of the catalytic residues. The catalytic GASG and histidine of secretase rhomboids reside in TM helices 4 and 6, which both have out-to-in orientation (indicated by white arrowheads). In contrast, these catalytic motifs in PARLs are in the in-to-out helices 5 and 7. Intriguingly, there is a corresponding inversion of substrate orientation: PARL substrates have an Nin/Cout topology, but secretase rhomboids cleave type I membrane proteins (Nout/Cin). Upon Star-dependent transport to the Golgi compartment, cleavage by Drosophila Rhomboid-1 releases the N-terminal portion of the membrane-tethered growth factor Spitz, thereby allowing its secretion to trigger EGFR signaling (Lee et al., 2001). In contrast, the mitochondrial S. cerevisiae PARL (Pcp1/Rbd1) cleaves its substrate Mgm1 to release the C-terminal portion into the intermembrane space (IMS) (Herlan et al., 2003; McQuibban et al., 2003). Mgm1 processing is controlled by the ATP-dependent integration of the scissile TM domain via the TIM23 translocase (Herlan et al., 2004). Molecular Cell 2007 28, 930-940DOI: (10.1016/j.molcel.2007.12.003) Copyright © 2007 Elsevier Inc. Terms and Conditions