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Published Version Biochimica et Biophysica Acta 1859 (2017) 1859–1871 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamem Crystallographic and biochemical characterization of the dimeric architecture of site-2 protease Magdalena Schacherl a,⁎,1, Monika Gompert a, Els Pardon b,c, Tobias Lamkemeyer d,2, Jan Steyaert b,c, Ulrich Baumann a a Institute of Biochemistry, University of Cologne, Otto-Fischer-Str. 14, 50674 Cologne, Germany b Structural Biology Brussels, Vrije Universiteit Brussel (VUB), 1050 Brussels, Belgium c Structural Biology Research Center, VIB, 1050 Brussels, Belgium d Cluster of Excellence in Cellular Stress Responses in Aging-associated Diseases (CECAD), University of Cologne, Cologne, Germany article info abstract Article history: Regulated intramembrane proteolysis by members of the site-2 protease family (S2P) is an essential signal trans- Received 22 June 2016 duction mechanism conserved from bacteria to humans. There is some evidence that extra-membranous Received in revised form 8 May 2017 domains, like PDZ and CBS domains, regulate the proteolytic activity of S2Ps and that some members act as di- Accepted 10 May 2017 mers. Here we report the crystal structure of the regulatory CBS domain pair of S2P from Archaeoglobus fulgidus, Available online 11 May 2017 AfS2P, in the apo and nucleotide-bound form in complex with a specific nanobody from llama. Cross-linking and SEC-MALS analyses show for the first time the dimeric architecture of AfS2P both in the membrane and in Keywords: fi Site-2 protease detergent micelles. The CBS domain pair dimer (CBS module) displays an unusual head-to-tail con guration CBS domain and nucleotide binding triggers no major conformational changes in the magnesium-free state. In solution, Intramembrane cleavage MgATP drives monomerization of the CBS module. We propose a model of the so far unknown architecture of Nanobody the transmembrane domain dimer and for a regulatory mechanism of AfS2P that involves the interaction of pos- Complex itively charged arginine residues located at the cytoplasmic face of the transmembrane domain with the nega- Dimer tively charged phosphate groups of ATP moieties bound to the CBS domain pairs. Binding of MgATP could promote opening of the CBS module to allow lateral access of the globular cytoplasmic part of the substrate. © 2017 Elsevier B.V. All rights reserved. 1. Introduction cleaving proteases (I-CLiPs) [3]. Thus, it differs radically, for example, from the ATP-dependent transmembrane proteases FtsH or Lon, Proteolysis is involved in the regulation of a variety of fundamental which cleave substrates outside of the lipid bilayer [4–6]. Contrary to cellular processes in living cells, e.g. antigen presentation, protein qual- proteolytic enzymes operating in an aqueous environment, I-CLiPs ity control, apoptosis and stress responses [1]. Thus, proteolysis itself face the mechanistic problem of delivering a water molecule within has to be tightly regulated and balanced with protein synthesis. Besides the hydrophobic membrane to the scissile peptide bond. Therefore, gat- the large variety of soluble proteases, there also exist membrane bound ing mechanisms allowing water and substrate polypeptide to access the proteases. One of them is the site-2 protease (S2P; MEROPS: M50 [2]), a active site have been discussed [7,8], allowing at the same time regula- Zn2+-metalloprotease that cleaves its substrates within the membrane tion of proteolytic activity. plane and thus belongs to the class of the so-called intramembrane Site-2 proteases are conserved in all kingdoms of life. In bacteria they are involved in the regulation of sporulation [9,10],inthetransductionof stress signals [11] or release of peptide pheromones [12].Inmammals Abbreviations: CBS, cystathionine-beta-synthase; CBSD, cystathionine-beta-synthase S2Ps regulate cholesterol synthesis [13] and activate the ER stress re- domain pair; S2P, site-2 protease; Nb, nanobody; CDR, complementarity-determining re- sponse [14]. S2Ps are also found in plants, fungi and protozoa and are gion; SEC-MALS, size exclusion chromatography with multiangle light scattering. ⁎ Corresponding author at: Institute of Biochemistry, University of Cologne, Otto- highly abundant in archaea. For the latter, substrates are mostly un- Fischer-Str. 12-14, 50674 Cologne, Germany. known. S2Ps cleave their α-helical substrates, which are mostly transcrip- E-mail addresses: [email protected], tion factors (e.g. SREBP or ATF-6), in the water-excluding environment of [email protected] (M. Schacherl). the membrane and must therefore unwind their α-helical substrates and 1 Present address: Center of Advanced European Studies and Research (CAESAR), deliver water to the active site. Surprisingly, the residues crucial for catal- Ludwig-Erhard-Allee 2, 53175 Bonn, Germany. 2 Present address: Federal Institute for Drugs and Medical Devices, Kurt-Georg- ysis were found to be the same as in soluble proteases, contained in the Kiesinger-Allee 3, 53175 Bonn, Germany. HEXXH and the NPDG motives (with the three zinc-ligands in bold and http://dx.doi.org/10.1016/j.bbamem.2017.05.006 0005-2736/© 2017 Elsevier B.V. All rights reserved. 1860 M. Schacherl et al. / Biochimica et Biophysica Acta 1859 (2017) 1859–1871 the catalytic base in italics) [15]. The crystal structure of the transmem- 2. Material and methods brane domain of an archaeal site-2 protease from Methanocaldococcus janaschii (MjS2P) shows six transmembrane helices (TMH), with TMHs 2.1. Materials α1andα5–α6 are considered to constitute the gate domain [8].Theac- tive site residues are located on TMHs α2andα4, respectively. In the crys- All detergents were purchased from Anatrace and were of tal structure of MjS2P two different states, open and closed, were Anagrade® quality. All other chemicals were purchased from Sigma- observed. In the open state TMH α1andα6 (gate domain) are thought Aldrich, AppliChem or Merck, if not stated otherwise. Enzymes for clon- to move away from each other by 10–12 Å to allow lateral access of the ing were from New England Biolabs and DNA purification kits from substrate helix. From a structural point of view, the open structure Sigma-Aldrich. could represent the catalytically competent state, and in analogy the closed state would be the inactive one. 2.2. Methods S2P take their name from their function: they are setting a cut at site- 2 of a substrate in a cascade that requires prior cleavage at site-1 [16]. 2.2.1. Cloning and protein expression of AfS2P constructs The latter is accomplished by the site-1 protease (S1P), a serine protease Using genomic DNA from Archaeoglobus fulgidus (DSM 4304, DSMZ, located in the periplasm of bacteria or the ER-lumen of eukarya. S1P Braunschweig, Germany) the af0332 gene was PCR amplified and cleaves a substrate just outside the membrane plane and prepares it subcloned into pET-based vectors (used oligonucleotides, restriction for the S2P that can only cleave the shed substrate [17]. enzymes and vectors in Table S1). In this manner, constructs for the There are three subtypes of S2Ps differing in membrane topology full-length protein (AfS2P) and the CBS domain (AfCBSD) only were and domain architecture. Most of the archaeal S2Ps display six trans- generated. membrane helices and contain a CBS (cystathionine-β-synthase) do- Expression was performed with the BL21 (DE3) derivative strains main pair facing the cytoplasm. Most bacterial and mammalian S2Ps C43 (DE3) for full-length protein and BL21 (DE3) pLysS (DE3) for the contain one or several extracytoplasmic PDZ domains and possess four AfCBS domain. A single colony of freshly transformed E. coli cells was in- or more transmembrane helices. Some S2Ps display no additional do- oculated in 50 ml lysogeny broth (LB) with appropriate antibiotics mains beside the transmembrane domain (TMD). Regarding the impor- (Tables S1 and S2) per liter of culture and grown in an orbital shaker tant role S2Ps play, their activity must be strictly controlled. Regulation overnight (~16 h) at 30 °C and 220 rpm. The overnight culture was in- may be exerted via adaptor proteins analogous to the ATP-driven prote- oculated into fresh LB to an optical density at 600 nm (OD600) of 0.1 ases [18] or via the PDZ-domains in the relevant S2Ps, e.g. Rip1 [19].For and further incubated at 37 °C to an OD600 of 0.6. Gene expression those S2Ps that possess CBS domains, a regulatory role of the latter ap- was induced with 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) pears plausible. CBS domains are known to bind nucleotides like ATP, followed by incubation at 20 °C overnight. Cells were harvested by cen- AMP or S-adenosyl-derivatives and are implicated in protein regulation trifugation (7000 ×g, 4 °C, 20 min), washed with buffer (20 mM Tris [20]. For Bacillus subtilis S2P SpoIVFB, which contains such a CBS do- pH 8.0, 300 mM NaCl), and after a second centrifugation step cell pellets main, a direct activation of proteolytic activity by ATP was demonstrat- were stored at −80 °C until further use. ed [21]. However, it is not clear how exactly ATP influences the conformation of SpoIVFB and its binding to the substrate pro-σK. Bio- 2.2.2. Selection, cloning and expression of llama nanobodies chemical data suggest that ATP-binding influences the oligomerization Llama immunization was performed according to previously de- of SpoIVFB and this allows pro-σK to gain access to its active site [21]. scribed protocols [25,26]. In summary, a llama was injected several The estimated oligomeric state of SpoIVFB after solubilization from times with in total 2 mg of dimeric AfS2P-His in DDM over 6 weeks. membranes is a tetramer. For all other S2P it is unclear, if they act as Phage display library construction and selection have been performed monomers or if they oligomerize analogous to other intramembrane following procedures described by Conrath et al.
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