doi:10.1016/j.jmb.2004.10.050 J. Mol. Biol. (2005) 345, 211–227 Proprotein Convertase Models based on the Crystal Structures of Furin and Kexin: Explanation of their Specificity Stefan Henrich1, Iris Lindberg2, Wolfram Bode1* and Manuel E. Than1* 1Max-Planck-Institut fu¨r In eukaryotes, many secreted proteins and peptide hormones are excised Biochemie, Abteilung fu¨r from larger precursors by calcium-dependent serine proteinases, the Strukturforschung, Am proprotein/prohormone convertases (PCs). These PCs cleave their protein Klopferspitz 18, 82152 substrates very specifically following multiple basic residues. The seven Martinsried, Germany mammalian PCs and their yeast orthologue kexin are multi-domain proteinases consisting of a subtilisin-related catalytic domain, a conserved 2Dept. of Biochemistry & P-domain and a variable, often cysteine-rich domain, which in some PCs is Molecular Biology, Louisiana followed by an additional C-terminal trans-membrane domain and a short State University Health cytoplasmic domain. The recently published crystal structures of the Sciences Center, New Orleans soluble mouse furin and yeast kexin ectodomains have revealed the LA 70112, USA relative arrangement of catalytic and P domains, the exact domain fold and the detailed architecture of the substrate binding clefts. Based on these experimental structures, we now have modelled the structures of the other human/mouse PCs. According to topology and to structure-based sequence comparisons, these other PCs closely resemble furin, with PC4, PACE4 and PC5/6 being more similar, and PC1/3, PC2 and PC7 being less similar to furin. Except for PC1 and PC2, this order of similarity is valid for the catalytic as well as for the P domains, and is almost reversed using kexin as a reference molecule. A similar order results from the number and clustering of negative charges lining the non-prime subsites, explaining the gradually decreasing requirement for basic residues N-terminal to substrate cleavage sites. The preference of the different PCs for distinct substrates seems to be governed by overall charge compensation and matching of the detailed charge distribution pattern. q 2004 Elsevier Ltd. All rights reserved. Keywords: proprotein convertases (PCs); furin; kexin; substrate specificity; *Corresponding authors homology modelling Introduction calcium-dependent serine endopeptidases, which very specifically cleave C-terminal to multiple basic Many secreted proteins and peptides essential for residues. Because of their subtilisin-like catalytic the regulation of biological activity are initially domain,7,8 these proteinases have been called synthesized as inactive precursor proteins, and are subtilisin-like proprotein convertases (SPCs) or subsequently proteolytically converted in the proprotein convertases (PCs). Besides a number of secretory pathway to the mature active forms.1–6 non-mammalian enzymes, such as the yeast process- 9–12 Most of the proteolytic enzymes involved are ing kexin/Kex2 proteinase, this PC family (being part of the MEROPS family S8 of clan SB)13 contains seven closely related mammalian enzymes, namely Present address: S. Henrich, EML Research gGmbH, furin/SPC1/PACE, PC2/SPC2, PC1/PC3/SPC3, Schloss-Wolfsbrunnenweg 33, 69118 Heidelberg, PACE4/SPC4, PC4/SPC5, PC5/PC6/SPC6, and Germany PC7/PC8/LPC. The more distantly related endo- Abbreviations used: PC, proprotein convertase; CMK, chloromethylketone; PDB, protein data bank; TGN, trans proteinases SKI-1/S1P and NARC-1, in contrast, are Golgi network; VRs, variable regions; SCRs, structurally predicted to exhibit a more pyrolisin-like and conserved regions. proteinase K-like fold, respectively, and to cleave 14 E-mail addresses of the corresponding authors: precursors C-terminal to non-basic amino acids. [email protected]; [email protected] The pro-PCs are multi-domain proteins, which all 0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. 212 Models and Specificity of Proprotein Convertases Figure 1. Stereo plots of the ectodomains of furin and the other modelled PCs, shown in standard orientation, i.e. the active-site cleft horizontally extends across the catalytic domain surface with a substrate polypeptide chain running from left (N terminus) to right (C terminus). (a) Stereo ribbon plot of the experimental furin ectodomain.61 The b-strands are shown in red (catalytic domain) and rainbow colours (P domain), and the helices of the catalytic domain are given in yellow. The active-site residues (dark grey) and the decanoyl-Arg-Val-Lys-Arg-CMK inhibitor residues (light-coloured ball-and-stick model) are given with all non-hydrogen atoms, the three disulfide bridges as two yellow balls each, and the two bound calcium ions as pink spheres. (b) Stereo ribbon plot superposition of the six modelled PCs (PC1 gold, PC2 dark green, PC4 pink, PC5 light green, PC7 brown, PACE4 grey) and kexin (blue, PDB105 entry 1OT5),62 superimposed with furin (thick red rope, PDB105 entry 1P8J).61 The bound decanoyl-Arg-Val-Lys-Arg-CMK inhibitor (stick model) and the bound calcium ions Ca1 and Ca2 of furin (grey spheres) are shown in addition. Only a few residues have been labelled for orientation using the furin nomenclature. (c) Solid surface representation of furin, colored according to surface conservation in the PC family.61 The colours indicate sequentially and structurally conserved (red), sequentially conserved but structurally deviating (orange), structurally conserved (yellow), structurally deviating (green), and Models and Specificity of Proprotein Convertases 213 contain a pro-domain acting as an intra-molecular hand, PC2 and PC1/3 also efficiently cleave at sites chaperone in folding,15–18 an about 340 residue lacking additional basic residues beyond P1 and P2, subtilisin-like catalytic domain, an about 130 as does kexin.21,30–32 Distinct PCs often process residue P-domain important for proteolytic activity, their substrates differentially, as exemplified by the and a quite variable segment, which in furin, different cleavage products of pro-insulin and pro- PACE4 and PC5 (and less obviously in PC4 and glucagon upon hydrolysis by PC1/3 and PC2, PC7) contains a number of Cys residues and is thus respectively.1,4 often referred to in the literature as the Cys-rich Only a very few endogenous protein inhibitors domain. Full-length furin, the PC5/6B isoform, PC7 have been identified that are directed against the and kexin exhibit an additional trans-membrane PCs (see, e.g. Rockwell et al.21). Furin can be rapidly and a C-terminal cytoplasmic domain, i.e. are trans- inactivated by the cytosolic serpin inhibitor PI8, a membrane proteinases. PC1/3, PC2 and PC4 are fraction of which under physiological conditions soluble proteinases, lacking these latter domains might enter the secretory pathway to interact with but terminating in long Ser/Thr-rich tails instead. furin.33 By replacing the P1 and the P4 residue in PC1/3 and PC2 are predominantly expressed in the reactive-site loop by Arg residues, the serpin neuroendocrine cells, and PC4 is only found in a1-PI has been re-engineered into a1-Antitrypsin testicular and ovarian germ cells, while the other Portland/a1-PDX, which quite selectively forms mammalian PCs are more broadly or even ubiqui- SDS-stable complexes with furin, and reacts more tously distributed. The mammalian PCs are often weakly with PC6B, PC1/334,35 and PACE4.36 The simultaneously expressed in various tissues, where substrate-like binding inhibitors turkey ovomucoid they are mainly localized in the trans-Golgi network third domain37 and eglin c38,39 have been mutated to (TGN), but are also found in other compartments become potent inhibitors of furin and kexin. Very of the secretory pathways and on the cell surface. recently, a Drosophila Spn4 gene product has been Kexin is required for the endogenous generation identified as a potent furin-directed serpin.40,41 of mature a-factor and killer toxin,9,10,12,19 while the The isolated PC pro-domains and, less effectively, mammalian PCs are involved in the processing of smaller constructs containing the multiple basic the precursors of virtually all neuropeptides and “primary activation cleavage segment” (Lys-Arg- peptide hormones, growth and differentiation Arg-Thr-Lys-Arg107f-Y-Asp108f-Val in furin) can factors, cell-surface receptors, extracellular metallo- block the PC active sites, with some preference for proteinases, coagulation factors and other serum their endogenous PCs.18,42–46 The binding protein proteins, adhesion molecules, plasma proteins, 7B2, which is co-expressed with proPC2 and bacterial toxins such as the anthrax toxin protective facilitates maturation to an active enzyme species, antigen20 or diphtheria toxin, and viral coat proteins contains an inhibitory sequence at the C termi- such as from lethal Ebola viruses, HIV-1, or nus.47–50 In addition, a number of synthetic inhibi- cytomegalovirus.2,3,5,6 tors have been developed. Most of these exhibit The role of the PCs is to cleave these substrates polybasic peptidyl moieties mimicking substrate or accurately, even in the presence of an excess of PC pro-domain sequences,51–53 often linked to an similar other cleavage sites.21 In most substrates, the active-site directed group such as a chloromethyl- PCs cleave C-terminal to paired basic residues, with ketone (CMK), ketomethylene, phosphonate or the first so-called P2 residue often being a Lys, and boronic acid group.21,29,54–56 Oligopeptides consist- 52 the second P1 residue strongly or even strictly ing of up to ten L-Arg or D-Arg residues strongly restrained to an Arg residue (using the nomencla- block furin activity, but inhibit the other mamma- ture of Schechter and Berger,22 with P1, P2 etc. and lian PCs much more weakly, allowing some P10,P20 etc. defining the peptide substrate residues discrimination.57 N and C-terminal to the scissile peptide bond, In order to assign the experimental observations respectively,andS1,S2etc.andS10,S20 etc. available to the structural properties of the different representing the opposing proteinase subsites).