Cryo-EM Structure of the Native Butyrylcholinesterase Tetramer Reveals a Dimer of Dimers Stabilized by a Superhelical Assembly
Total Page：16
File Type：pdf, Size：1020Kb
Cryo-EM structure of the native butyrylcholinesterase tetramer reveals a dimer of dimers stabilized by a superhelical assembly Miguel Ricardo Leunga,b, Laura S. van Bezouwena,c, Lawrence M. Schopferd, Joel L. Sussmane, Israel Silmanf, Oksana Lockridged, and Tzviya Zeev-Ben-Mordehaia,b,1 aCryo-Electron Microscopy, Bijvoet Center for Biomolecular Research, Utrecht University, 3584 CH Utrecht, The Netherlands; bThe Division of Structural Biology, Wellcome Centre for Human Genetics, The University of Oxford, OX3 7BN Oxford, United Kingdom; cCrystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Utrecht University, 3584 CH Utrecht, The Netherlands; dEppley Cancer Institute, University of Nebraska Medical Center, Omaha, NE 68198-6805; eDepartment of Structural Biology, Weizmann Institute of Science, 76100 Rehovot, Israel; and fDepartment of Neurobiology, Weizmann Institute of Science, 76100 Rehovot, Israel Edited by Arthur Karlin, College of Physicians and Surgeons, Columbia University, New York, NY, and approved November 14, 2018 (received for review October 2, 2018) The quaternary structures of the cholinesterases, acetylcholines- anchor in the central nervous system (12–14). BChE exists in plasma terase (AChE) and butyrylcholinesterase (BChE), are essential for mainly as highly glycosylated soluble tetramers (15). The use of their localization and function. Of practical importance, BChE is a human BChE (HuBChE) as a therapeutic agent for combating OP promising therapeutic candidate for intoxication by organophos- poisoning is contingent on its possessing a long circulatory half-life phate nerve agents and insecticides, and for detoxification of (16). Because its half-life depends on its ability to tetramerize, addictive substances. Efficacy of the recombinant enzyme hinges substantial effort has been invested in developing methods to on its having a long circulatory half-life; this, in turn, depends produce recombinant tetrameric HuBChE (17–20). strongly on its ability to tetramerize. Here, we used cryoelectron Massoulié and coworkers (21–24) identified the sequences in microscopy (cryo-EM) to determine the structure of the highly the AChE and ColQ polypeptides and, subsequently, in PRiMA. glycosylated native BChE tetramer purified from human plasma at ColQ and PRiMA are proteins that anchor the catalytic subunits of BIOPHYSICS AND 5.7 Å. Our structure reveals that the BChE tetramer is organized as AChE to the synaptic basal lamina and to the synaptic plasma COMPUTATIONAL BIOLOGY a staggered dimer of dimers. Tetramerization is mediated by as- membrane in the peripheral and central nervous systems, re- sembly of the C-terminal tryptophan amphiphilic tetramerization spectively (22, 24). In the catalytic subunit, the sequence is what (WAT) helices from each subunit as a superhelical assembly around they called a tryptophan amphiphilic tetramerization (WAT) a central lamellipodin-derived oligopeptide with a proline-rich at- sequence, close to the C terminus, because it contains three highly tachment domain (PRAD) sequence that adopts a polyproline II conserved Trp residues (21). In both ColQ and PRIMA, the sequence helical conformation and runs antiparallel. The catalytic domains was identified as what was denoted as a proline-rich attachment within a dimer are asymmetrically linked to the WAT/PRAD. In the resulting arrangement, the tetramerization domain is largely Significance shielded by the catalytic domains, which may contribute to the stability of the human BChE (HuBChE) tetramer. Our cryo-EM structure reveals the basis for assembly of the native tetramers Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) and has implications for the therapeutic applications of HuBChE. are specialized enzymes that hydrolyze choline esters. BChE This mode of tetramerization is seen only in the cholinesterases can inactivate the hunger hormone, ghrelin, thus implicating it but may provide a promising template for designing other pro- in regulation of obesity and behavior. Importantly, BChE is a teins with improved circulatory residence times. bioscavenger that can hydrolyze a wide range of organo- phosphates, thus being tested for treatment of soldiers or ci- butyrylcholinesterase | cryoelectron microscopy | acetylcholinesterase | vilians exposed to deadly organophosphate nerve agents. bioscavenger | superhelical assembly HuBChE, and engineered variants, are also being studied for treatment for cocaine and heroin addictions. All these appli- cations require use of tetrameric HuBChE, since effective he cholinesterases (ChEs) are a specialized family of enzymes treatment requires the long circulatory residence time dis- Tthat hydrolyze choline-based esters, such as the neurotrans- played by the tetramer. Knowledge of the structure of the mitter acetylcholine. Acetylcholinesterase (AChE) functions HuBChE tetramer, and of its sugar-coated surface, should be primarily to terminate transmission at cholinergic synapses by relevant to the development of other proteins for therapeutic rapid hydrolysis of acetylcholine (1). Butyrylcholinesterase (BChE), purposes. its sister enzyme, is rather enigmatic. It is abundant in plasma and was initially believed to be an evolutionary vestige (2). It has been Author contributions: M.R.L., J.L.S., I.S., O.L., and T.Z.-B.-M. designed research; M.R.L., suggested that it functions primarily as a detoxification enzyme, L.S.v.B., and T.Z.-B.-M. performed research; L.M.S. contributed new reagents/analytic tools; M.R.L., L.S.v.B., J.L.S., I.S., O.L., and T.Z.-B.-M. analyzed data; and M.R.L., J.L.S., thereby protecting AChE from inhibition (3, 4). It has recently been I.S., O.L., and T.Z.-B.-M. wrote the paper. shown, however, that it inactivates the hunger hormone, ghrelin, The authors declare no conflict of interest. thus reducing obesity, as well as aggression, in male mice (5, 6). This article is a PNAS Direct Submission. Because of its ability to hydrolyze organophosphates (OPs), such as Published under the PNAS license. nerve agents and pesticides (3), and drugs such as cocaine (7, 8) and – Data deposition: The cryo-EM map of BChE and the coordinates of the model have been heroin (9 11), BChE is a promising therapeutic agent with multiple deposited in the Electron Microscopy Data Bank (EMDB) (accession no. EMD-4400)atthe applications. Protein Data Bank in Europe (PDBe), www.ebi.ac.uk/pdbe/emdb/ (PDB ID code 6I2T). Both AChE and BChE exist in various oligomeric forms, in- 1To whom correspondence should be addressed. Email: [email protected]. cluding monomers, dimers, and tetramers (12). AChE tetramers This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. are anchored at cholinergic synapses through association with either a 1073/pnas.1817009115/-/DCSupplemental. collagen tail, in the peripheral nervous system, or a transmembrane www.pnas.org/cgi/doi/10.1073/pnas.1817009115 PNAS Latest Articles | 1of6 Downloaded by guest on September 23, 2021 domain (PRAD) (22, 24). Synthetic oligopeptides were prepared, Table 1. Cryo-EM collection, processing, refinement, and corresponding to the WAT and PRAD sequences in mammalian validation statistics AChE and ColQ, respectively (25). When mixed in a 4:1 ratio, they Human butyrylcholinesterase produced well-diffracting crystals from which a structure was obtained tetramer in which four WATs are seen to assemble in a superhelical, or coiled- coil, structure around a central polyproline II (PPII) helix. This PPII Data collection helix, formed by the PRAD, runs antiparallel to the WAT helices Microscope Talos Arctica (25). The WATs are tightly associated with the PRAD via both hy- Accelerating voltage, kV 200 drophobic stacking and hydrogen bonds. On the basis of the WAT/ Nominal magnification 130,000× PRAD, a model for the structure of the synaptic A12 form of AChE Pixel size, Å 1.0285 was proposed (25). Detector Gatan K2 Summit Mass spectrometry of HuBChE assemblies revealed the pres- Exposure time (Frame length), s 6.3 (0.3) − ence of proline-rich peptides that are 10–39 residues long. Of Total electron exposure, e /Å2 49.5 these, ∼70% are derived from lamellipodin and the remaining Total movies collected 4,518 ∼30% from up to 20 other proteins (26). Lamellipodin is a Data processing pleckstrin homology (PH) domain-containing protein that reg- Software used Relion 2.1, Relion 3.0 beta, ulates the formation of actin-based cell protrusions by interact- cryoSPARC v1 ing with Enabled/Vasodilator-stimulated protein (Ena/VASP) Initial particle count 414,189 proteins (27, 28). Ena/VASP proteins bind their ligands through Final particle count 111,986 conserved proline-rich sequences, but how exactly BChE comes Symmetry C2 to be associated with lamellipodin-derived peptides remains un- Map resolution, Å 5.7 clear. Presumably, HuBChE is synthesized and released into the FSC threshold 0.143 plasma as monomers, which then associate with polyproline-rich Sharpening B factor, Å2 −456.502 peptides to form the functional tetramer (2). Since the HuBChE Refinement and validation polypeptide contains a WAT sequence highly homologous to that Software used Phenix, Coot of AChE near its C terminus (25), it seemed plausible that it Model composition assembles to the native tetramer in a similar way and may thus Chains 5 be formally considered as a heteropentamer rather than as a Atoms 18,174 (hydrogens: 0) homotetramer. Amino acid residues 2,252 Numerous high-resolution structures are available for vari- Water molecules 0 ous forms of the