The HtrA Family of Proteases Tim Clausen, Chris Southan, Michael Ehrmann  Molecular Cell  Volume 10, Issue 3, Pages 443-455 (September 2002) DOI: 10.1016/S1097-2765(02)00658-5

Figure 1 Domain Organization and Phylogram of Selected HtrA Family Members (A) The positions of signal peptides (SS) and transmembrane segments (TM) are taken from database entries. Most of the domain annotations are derived from InterPro analysis (Apweiler et al., 2001). The domain or family identifiers used are IPR001254 trypsin family, IPR001940 S2C (HtrA) serine protease family, IPR001478 PDZ domain, IPR000867 insulin growth factor binding domain (IGFBP), and IPR002350 Kazal protease inhibitor (KI) domain. The trypsin and PDZ total numbers include those detected by sequence similarity. Note that the exact position of the individual domains cannot be taken from the diagram, only their relative position. SwissProt/TREMBL accession numbers are DegS (P31137), DegP (P09376), DegQ (P39099), HtrA Synechocystis (P73354), HhoA (P72780), HhoB (P73940), hHtrA1 (Q92743), hHtrA2 (O43464), hHtrA3 (P83110), hHtrA4 (P83105), YNM3 (P53920), S. pombae (Q9P7S1), A. thaliana (Q8RY22), DegP1 (O22609), and D. melanogaster (Q9VFJ3). The partial Zebrafish (D. rerio) sequence was assembled from unfinished geneomic sequence AL773584, and YNM3-like C. albicans sequence was obtained from http://www-sequence.stanford.edu/group/candida/. (B) The phylogram was produced by Clustal W (Higgins et al., 1994). Molecular Cell 2002 10, 443-455DOI: (10.1016/S1097-2765(02)00658-5)

Figure 2 Fold of the Individual HtrA Domains (A) Protease domain. The protease domains of Streptomyces griseus Trypsin (left), DegP (middle), and hHtrA2 (right) are illustrated. β strands are colored green, helices yellow, and the mechanistically important loops LA, L1, L2, and L3 are colored blue, magenta, red, and lilac, respectively. The L3 loops of both HtrA structures are highly flexible and not defined by electron density. Residues of the catalytic triad are given in a ball-and-stick representation. The upper panel shows the single uncomplexed protease domains, the lower panel the “complexed” forms, i.e., the peptide-bound form of trypsin and the occluded forms of DegP and hHtrA2. In DegP, the active site is buried by interaction of loops L1 and L2 with loop LA* that originates from a different subunit. In hHtrA2, a segment of the PDZ domain (orange) partially blocks the entrance to the proteolytic site. The Protein Data Bank entry codes are 1SGT (Streptomyces griseus Trypsin), 1KY9 (DegP), and 1LCY (hHtrA2). (B) PDZ domain. β strands are colored orange, helices yellow, and the extra segment of the HtrA PDZ red. Molecular Cell 2002 10, 443-455DOI: (10.1016/S1097-2765(02)00658-5)

Figure 3 Overall Architecture of HtrA (A) Molecular surface of one trimeric funnel that represents the functional unit of HtrA proteins. The location of different DegP domains (protease in green, PDZ1 in yellow, and PDZ2 in orange) and the proteolytic sites (blue) are indicated. (B) Molecular surface of the DegP hexamer colored by the thermal motion factors (blue, rigid portions; red, flexible portions). The protease domains build up a solid molecular cage with the PDZ domains acting as tentacular arms controlling the lateral access to the inner cavity. Molecular Cell 2002 10, 443-455DOI: (10.1016/S1097-2765(02)00658-5)

Figure 4 Sequence Alignment of E. coli DegP and DegQ with Human HtrA1 and HtrA2 Conserved residues are shown in red, homologous in yellow. Secondary structure elements, residues of the catalytic triad (His105, Asp135, and Ser210 in cyan), and mechanistically important loops (LA, L1, L2, L3) are indicated. Molecular Cell 2002 10, 443-455DOI: (10.1016/S1097-2765(02)00658-5)

Figure 5 Substrate Discrimination by HtrA (A) Left: To demonstrate the molecular sieve character of DegP, a half cut presentation of the hexamer was prepared. Cut regions are shown in dark gray. The height of the internal tunnel restricts access to single secondary structure elements as shown by the modeled polyalanine helix (orange). Right: The hydrophobic docking platforms in the inner cavity of DegP. Structure of the two Phe-clusters that form the ceilings of the internal cavity. These platforms are highly mobile, as shown by the mapped thermal motion factors. The Phe triplet of one monomer is labeled. All three phenylalanines are part of the regulatory loop LA. (B) Molecular surface of the hHtrA2 trimer with the protease domains hold in green, the PDZ domain in yellow, and the active site in blue. The PDZ domain is packed onto the protease domain such that a narrow canyon extends toward the proteolytic sites (highlighted by the red circle). Molecular Cell 2002 10, 443-455DOI: (10.1016/S1097-2765(02)00658-5)

Figure 6 The Malformed Oxyanion Hole of HtrA Superposition of the L1 loops from Streptomyces griseus trypsin (yellow), DegP (green), and hHtrA2 (cyan). The oxyanion hole is formed by the amide nitrogens of the active site serine and its –2 residue. In order to form the NH cradle, the backbone of these residues obtains a characteristic turn structure as seen for trypsin. While the conformation of this segment is completely distorted in DegP, HtrA2 is strongly kinked such that the carbonyl of the –3 residue occupies the oxyanion hole. Molecular Cell 2002 10, 443-455DOI: (10.1016/S1097-2765(02)00658-5)

Figure 7 Regulation of the degP Promoter in E. coli Two signal transduction pathways (Cpx and RpoE) are involved in regulating degP transcription. Misfolded polypeptides are sensed by either CpxP or RseB, which interact with CpxA or RseA, respectively. After detection of folding problems, CpxA activates CpxR, which then induces the DegP promoter while RseA must be degraded by DegS before RpoE can function as a σ factor. CM is the cytoplasmic membrane. Molecular Cell 2002 10, 443-455DOI: (10.1016/S1097-2765(02)00658-5)