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• W2803254201 abstract "Triggering receptor expressed on myeloid cells 2 (TREM2) is an immune receptor expressed on the surface of microglia, macrophages, dendritic cells, and osteoclasts. The R47H TREM2 variant is a significant risk factor for late-onset Alzheimer's disease (AD), and the molecular basis of R47H TREM2 loss of function is an emerging area of TREM2 biology. Here, we report three high-resolution structures of the extracellular ligand-binding domains (ECDs) of R47H TREM2, apo-WT, and phosphatidylserine (PS)-bound WT TREM2 at 1.8, 2.2, and 2.2 Å, respectively. The structures reveal that Arg47 plays a critical role in maintaining the structural features of the complementarity-determining region 2 (CDR2) loop and the putative positive ligand–interacting surface (PLIS), stabilizing conformations capable of ligand interaction. This is exemplified in the PS-bound structure, in which the CDR2 loop and PLIS drive critical interactions with PS via surfaces that are disrupted in the variant. Together with in vitro and in vivo characterization, our structural findings elucidate the molecular mechanism underlying loss of ligand binding, putative oligomerization, and functional activity of R47H TREM2. They also help unravel how decreased in vitro and in vivo stability of TREM2 contribute to loss of function in disease. Triggering receptor expressed on myeloid cells 2 (TREM2) is an immune receptor expressed on the surface of microglia, macrophages, dendritic cells, and osteoclasts. The R47H TREM2 variant is a significant risk factor for late-onset Alzheimer's disease (AD), and the molecular basis of R47H TREM2 loss of function is an emerging area of TREM2 biology. Here, we report three high-resolution structures of the extracellular ligand-binding domains (ECDs) of R47H TREM2, apo-WT, and phosphatidylserine (PS)-bound WT TREM2 at 1.8, 2.2, and 2.2 Å, respectively. The structures reveal that Arg47 plays a critical role in maintaining the structural features of the complementarity-determining region 2 (CDR2) loop and the putative positive ligand–interacting surface (PLIS), stabilizing conformations capable of ligand interaction. This is exemplified in the PS-bound structure, in which the CDR2 loop and PLIS drive critical interactions with PS via surfaces that are disrupted in the variant. Together with in vitro and in vivo characterization, our structural findings elucidate the molecular mechanism underlying loss of ligand binding, putative oligomerization, and functional activity of R47H TREM2. They also help unravel how decreased in vitro and in vivo stability of TREM2 contribute to loss of function in disease. TREM2 (triggering receptor expressed on myeloid cells 2) is an immune receptor expressed on the surface of microglia, macrophages, dendritic cells, and osteoclasts. Mutations in TREM2 have been linked to various neurodegenerative diseases, including Alzheimer's disease (AD), 8The abbreviations used are: ADAlzheimer's diseaseNHDNasu–Hakola diseaseECDextracellular ligand-binding domainsTREM2shed or soluble form of TREM2PSphosphatidylserinePLISpositive ligand–interacting surfaceasuasymmetric unitSASAsolvent-accessible surface areaScshape complementarityCDRcomplementarity-determining regionr.m.s.root mean squareNAGN-acetylglucosaminessODNsingle-stranded oligonucleotideDOPS1,2-dioleoyl-sn-glycero-3-phospho-l-serine sodium salt. frontotemporal dementia, and Nasu–Hakola disease (NHD) (1.Jonsson T. Stefansson H. Steinberg S. Jonsdottir I. Jonsson P.V. Snaedal J. Bjornsson S. Huttenlocher J. Levey A.I. Lah J.J. Rujescu D. Hampel H. Giegling I. Andreassen O.A. Engedal K. et al.Variant of TREM2 associated with the risk of AD.N. Engl. J. Med. 2013; 368 (23150908): 107-11610.1056/NEJMoa1211103Crossref PubMed Scopus (1639) Google Scholar). TREM2 is reported to bind to several putative ligands, including apolipoprotein E (apoE) and apolipoprotein J (apoJ) and is activated by anionic lipids (2.Atagi Y. Liu C.-C. Painter M.M. Chen X.-F. Verbeeck C. Zheng H. Li X. Rademakers R. Kang S.S. Xu H. Younkin S. Das P. Fryer J.D. Bu G. Apolipoprotein E is a ligand for triggering receptor expressed on myeloid cells 2 (TREM2).J. Biol. Chem. 2015; 290 (26374899): 26043-2605010.1074/jbc.M115.679043Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 3.Bailey C.C. DeVaux L.B. Farzan M. The triggering receptor expressed on myeloid cells 2 binds apolipoprotein E.J. Biol. Chem. 2015; 290 (26374897): 26033-2604210.1074/jbc.M115.677286Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 4.Yeh F.L. Wang Y. Tom I. Gonzalez L.C. Sheng M. TREM2 binds to apolipoproteins, including APOE and CLU/APOJ, and thereby facilitates uptake of amyloid-β by microglia.Neuron. 2016; 91 (27477018): 328-34010.1016/j.neuron.2016.06.015Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, 5.Wang Y. Cella M. Mallinson K. Ulrich J.D. Young K.L. Robinette M.L. Gilfillan S. Krishnan G.M. Sudhakar S. Zinselmeyer B.H. Holtzman D.M. Cirrito J.R. Colonna M. TREM2 lipid sensing sustains the microglial response in an Alzheimer's disease model.Cell. 2015; 160 (25728668): 1061-107110.1016/j.cell.2015.01.049Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar). It also has been shown to play a role in the clearance of β-amyloid plaques (5.Wang Y. Cella M. Mallinson K. Ulrich J.D. Young K.L. Robinette M.L. Gilfillan S. Krishnan G.M. Sudhakar S. Zinselmeyer B.H. Holtzman D.M. Cirrito J.R. Colonna M. TREM2 lipid sensing sustains the microglial response in an Alzheimer's disease model.Cell. 2015; 160 (25728668): 1061-107110.1016/j.cell.2015.01.049Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar), apoptotic cells (6.Takahashi K. Rochford C.D.P. Neumann H. Clearance of apoptotic neurons without inflammation by microglial triggering receptor expressed on myeloid cells-2.J. Exp. Med. 2005; 201 (15728241): 647-65710.1084/jem.20041611Crossref PubMed Scopus (763) Google Scholar), myelin debris (7.Poliani P.L. Wang Y. Fontana E. Robinette M.L. Yamanishi Y. Gilfillan S. Colonna M. TREM2 sustains microglial expansion during aging and response to demyelination.J. Clin. Invest. 2015; 125 (25893602): 2161-217010.1172/JCI77983Crossref PubMed Scopus (289) Google Scholar), and bacterial beads (8.Chen Q. Zhang K. Jin Y. Zhu T. Cheng B. Shu Q. Fang X. Triggering receptor expressed on myeloid cells-2 protects against polymicrobial sepsis by enhancing bacterial clearance.Am. J. Respir. Crit. Care Med. 2013; 188 (23721075): 201-21210.1164/rccm.201211-1967OCCrossref PubMed Scopus (61) Google Scholar). The ability to interact with multiple endogenous and surrogate ligands in various physiological and challenge environments by potentially engaging different molecular interaction surfaces is consistent with the promiscuous nature of this class of receptors (9.Read C.B. Kuijper J.L. Hjorth S.A. Heipel M.D. Tang X. Fleetwood A.J. Dantzler J.L. Grell S.N. Kastrup J. Wang C. Brandt C.S. Hansen A.J. Wagtmann N.R. Xu W. Stennicke V.W. Cutting edge: identification of neutrophil PGLYRP1 as a ligand for TREM-1.J. Immunol. 2015; 194 (25595774): 1417-142110.4049/jimmunol.1402303Crossref PubMed Scopus (100) Google Scholar). Alzheimer's disease Nasu–Hakola disease extracellular ligand-binding domain shed or soluble form of TREM2 phosphatidylserine positive ligand–interacting surface asymmetric unit solvent-accessible surface area shape complementarity complementarity-determining region root mean square N-acetylglucosamine single-stranded oligonucleotide 1,2-dioleoyl-sn-glycero-3-phospho-l-serine sodium salt. Studies on the R47H variant (linked to late-onset AD) have revealed impaired NFAT reporter gene signaling in R47H TREM2/DAP12 cell lines, subtle but consistent differences in the secondary structure of the variant protein, and small differences in glycosylation and potential trafficking compared with WT protein (5.Wang Y. Cella M. Mallinson K. Ulrich J.D. Young K.L. Robinette M.L. Gilfillan S. Krishnan G.M. Sudhakar S. Zinselmeyer B.H. Holtzman D.M. Cirrito J.R. Colonna M. TREM2 lipid sensing sustains the microglial response in an Alzheimer's disease model.Cell. 2015; 160 (25728668): 1061-107110.1016/j.cell.2015.01.049Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar, 10.Kleinberger G. Yamanishi Y. Suarez-Calvet M. Czirr E. Lohmann E. Cuyvers E. Struyfs H. Pettkus N. Wenninger-Weinzierl A. Mazaheri F. Tahirovic S. Lleo A. Alcolea D. Fortea J. Willem M. et al.TREM2 mutations implicated in neurodegeneration impair cell surface transport and phagocytosis.Sci. Transl. Med. 2014; 6 (24990881): 243ra8610.1126/scitranslmed.3009093Crossref PubMed Scopus (473) Google Scholar). Furthermore, the variant has been shown to bind more weakly to apoE isoforms (2.Atagi Y. Liu C.-C. Painter M.M. Chen X.-F. Verbeeck C. Zheng H. Li X. Rademakers R. Kang S.S. Xu H. Younkin S. Das P. Fryer J.D. Bu G. Apolipoprotein E is a ligand for triggering receptor expressed on myeloid cells 2 (TREM2).J. Biol. Chem. 2015; 290 (26374899): 26043-2605010.1074/jbc.M115.679043Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 3.Bailey C.C. DeVaux L.B. Farzan M. The triggering receptor expressed on myeloid cells 2 binds apolipoprotein E.J. Biol. Chem. 2015; 290 (26374897): 26033-2604210.1074/jbc.M115.677286Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar), which was confirmed in an unbiased library screening in which several proteins, including apoE and apoJ, showed reduced binding to R47H TREM2 compared with WT (4.Yeh F.L. Wang Y. Tom I. Gonzalez L.C. Sheng M. TREM2 binds to apolipoproteins, including APOE and CLU/APOJ, and thereby facilitates uptake of amyloid-β by microglia.Neuron. 2016; 91 (27477018): 328-34010.1016/j.neuron.2016.06.015Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). Finally, immunofluorescence studies on brain sections from R47H carriers with AD also suggested loss of a novel microglial barrier function (11.Yuan P. Condello C. Keene C.D. Wang Y. Bird T.D. Paul S.M. Luo W. Colonna M. Baddeley D. Grutzendler J. TREM2 haplodeficiency in mice and humans impairs the microglia barrier function leading to decreased amyloid compaction and severe axonal dystrophy.Neuron. 2016; 90 (27196974): 724-73910.1016/j.neuron.2016.05.003Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). At the molecular level, the complete loss of function associated with some mutations linked to NHD and frontotemporal dementia, including Q33X and Y38C, can be explained by truncation (Q33X) or misfolding of the protein due to aberrant disulfide bond formation (Y38C). The effects of other mutations like R47H, R62H, T66M, and D87N, however, appear to be more subtle. The analysis of a 3.1 Å structure of WT TREM2 ECD revealed that mutations in NHD are buried, whereas AD risk variants are found on the surface, providing valuable preliminary insight into structural features that impact TREM2 function (12.Kober D.L. Alexander-Brett J.M. Karch C.M. Cruchaga C. Colonna M. Holtzman M.J. Brett T.J. Neurodegenerative disease mutations in TREM2 reveal a functional surface and distinct loss-of-function mechanisms.eLife. 2016; 5 (27995897): e20391Crossref PubMed Scopus (110) Google Scholar, 13.Kober D.L. Wanhainen K.M. Johnson B.M. Randolph D.T. Holtzman M.J. Brett T.J. Preparation, crystallization, and preliminary crystallographic analysis of wild-type and mutant human TREM-2 ectodomains linked to neurodegenerative and inflammatory diseases.Protein Expr. Purif. 2014; 96 (24508568): 32-3810.1016/j.pep.2014.01.015Crossref PubMed Scopus (10) Google Scholar). An understanding of the structural and biophysical properties of WT and variant ECD is also important for better understanding the properties of the shed or soluble form of TREM2 (sTREM2). sTREM2 is composed of the extracellular putative ligand binding domain of TREM2, where the Ig domain is followed by a short stalk terminating at His157 (14.Schlepckow K. Kleinberger G. Fukumori A. Feederle R. Lichtenthaler S.F. Steiner H. Haass C. An Alzheimer-associated TREM2 variant occurs at the ADAM cleavage site and affects shedding and phagocytic function.EMBO Mol. Med. 2017; 9 (28855300): 1356-136510.15252/emmm.201707672Crossref PubMed Scopus (113) Google Scholar, 15.Thornton P. Sevalle J. Deery M.J. Fraser G. Zhou Y. Ståhl S. Franssen E.H. Dodd R.B. Qamar S. Gomez Perez-Nievas B. Nicol L.S. Eketjäll S. Revell J. Jones C. Billinton A. et al.TREM2 shedding by cleavage at the H157–S158 bond is accelerated for the Alzheimer's disease-associated H157Y variant.EMBO Mol. Med. 2017; 9 (28855301): 1366-137810.15252/emmm.201707673Crossref PubMed Scopus (78) Google Scholar). Several of the disease-associated variants (most notably T66M) present with reduced surface TREM2 expression and a concomitant reduction in sTREM2 levels (16.Kleinberger G. Brendel M. Mracsko E. Wefers B. Groeneweg L. Xiang X. Focke C. Deussing M. Suárez-Calvet M. Mazaheri F. Parhizkar S. Pettkus N. Wurst W. Feederle R. Bartenstein P. et al.The FTD-like syndrome causing TREM2 T66M mutation impairs microglia function, brain perfusion, and glucose metabolism.EMBO J. 2017; 36 (28559417): 1837-185310.15252/embj.201796516Crossref PubMed Scopus (114) Google Scholar). Recently, Song et al. (17.Song W.M. Joshita S. Zhou Y. Ulland T.K. Gilfillan S. Colonna M. Humanized TREM2 mice reveal microglia-intrinsic and -extrinsic effects of R47H polymorphism.J. Exp. Med. 2018; 215 (29321225): 745-76010.1084/jem.20171529Crossref PubMed Scopus (132) Google Scholar) noted that R47H TREM2 5x FAD mice (AD genetic mouse models carrying the R47H variant) appeared to have lower levels of sTREM2 in the brain compared with CV (common variant) TREM2 5x FAD mice. However, similar transcript and surface TREM2 levels have been reported (17.Song W.M. Joshita S. Zhou Y. Ulland T.K. Gilfillan S. Colonna M. Humanized TREM2 mice reveal microglia-intrinsic and -extrinsic effects of R47H polymorphism.J. Exp. Med. 2018; 215 (29321225): 745-76010.1084/jem.20171529Crossref PubMed Scopus (132) Google Scholar), and the differences could not be explained by differences in cleavage propensity by known TREM2 proteases like ADAM17. Key outstanding questions include how the R47H variant affects levels of sTREM2 in vivo; what link exists between sTREM2 levels, TREM2 receptor activity, and microglia activity; and how sTREM2 interferes with therapeutics designed to target the receptor. To elucidate the molecular mechanism of the loss of function conferred by the R47H variant and to inform effective therapeutic strategies targeting TREM2 in neurodegenerative diseases, we have carried out structural, biochemical, functional, and biophysical studies on the ECDs of both R47H and WT TREM2. Here we report high-resolution crystal structures of R47H TREM2 and WT TREM2 at 1.8 and 2.2 Å, respectively. We also describe the co-crystal structure of TREM2 complexed with a putative ligand, phosphatidylserine (PS), at 2.2 Å. Furthermore, we describe the effect of the variant on both in vitro and in vivo stability of sTREM2. Together, these data advance our understanding of the molecular interactions underpinning the effects of the R47H TREM2 variant on the fold, stability, functional activity, and putative positive ligand–interacting surface (PLIS) impacting ligand binding. Human TREM2 is a polypeptide chain of 230 amino acids that consists of a single 156-amino acid N-terminal mature ECD (residues 19–174, with residues 1–18 constituting signal peptide), a membrane-spanning region (residues 175–195), and a C-terminal cytosolic tail (residues 196–230). Here, we focused our structural and biophysical efforts on the ECD of WT and R47H TREM2 proteins for the reasons discussed above. To generate soluble and homogenous proteins, we engineered various forms of WT and R47H TREM2 ECDs, including truncations and glycosylation-site mutations (Asn20 and Asn79) and also screened different expression systems (see “Experimental procedures” and Table S4). We were ultimately successful in obtaining diffracting crystals for R47H TREM2 produced by Escherichia coli expression and WT TREM2 with an N20D mutation produced by mammalian expression. The crystal structure of R47H TREM2 (residues 19–131) was solved to 1.8 Å resolution in the P21212 space group with two molecules per asymmetric unit (asu) arranged in a dimeric “tail-to-tail” configuration (see “Experimental procedures,” Table S1, and Fig. S1A). The total buried solvent-accessible surface area (SASA) between the two molecules in the asu is 605 Å2 with a shape complementarity (Sc) score of 0.49, suggesting that the protein is unlikely to exist as a dimer in solution, consistent with size-exclusion chromatography data. Analysis of the three antibody-equivalent complementarity-determining region (CDR) loops reveals a distinct and nonstandard 2-turn helix CDR2 loop conformation between β-strands C′ and C″ (Fig. 1A). The structure of WT TREM2 ECD (residues 19–141) was determined to 2.2 Å resolution (see “Experimental procedures” and Table S1) in the P41212 space group with six molecules per asu (Fig. 1B and Fig. S1, B–D). Three of the six molecules in the asu assumed a core trimeric arrangement (molecules A, B, and C), with the other three molecules packed in the interfaces between each pair of neighboring molecules in the trimer core (molecules D, E, and F) (Fig. S1, B–F). The total buried SASAs between the molecule pairs within the core trimer (A-B, A-C, and B-C) are between 672 and 687 Å2, with the Sc score ranging from 0.68 to 0.73 (Tables S2 and S3). In contrast, the total buried SASAs of the exterior TREM2 molecules with the neighboring trimer exhibit a broader range from 472 to 672 Å2. Correspondingly, the shape complementarities of these surfaces also exhibit a broader range from 0.60 to 0.81. Here the Sc scores are borderline to typical Sc scores of 0.70–0.76 for protein oligomeric interfaces and the buried SASAs are approaching the cutoff of 850 Å2 for monomeric proteins (18.Lawrence M.C. Colman P.M. Shape complementarity at protein/protein interfaces.J. Mol. Biol. 1993; 234 (8263940): 946-95010.1006/jmbi.1993.1648Crossref PubMed Scopus (1102) Google Scholar, 19.Chothia C. Janin J. Principles of protein–protein recognition.Nature. 1975; 256 (1153006): 705-70810.1038/256705a0Crossref PubMed Scopus (853) Google Scholar). These scores, along with solution state characterization by size exclusion and dynamic light scattering, indicate that the 6-mer observed in the crystal packing of WT TREM2 is less likely to be the biologically relevant form. However, the interface analysis also indicates that WT TREM2 has a higher propensity to oligomerize than R47H TREM2, and the trimeric interfaces provide one example of putative interaction interfaces. In addition, at 2.2 Å resolution, most residues in our WT TREM2 structure are well defined in the electron density maps, enabling us to clearly elucidate atomic interactions. Whereas the R47H variant and WT structures are highly similar in the core fold, there is a remarkable structural change in the CDR2 loop region, with CDR2 loop in the R47H structure adopting a completely different conformation compared with the WT structure (Fig. 1D). The downstream β-strand C″ and C″-D loop are disordered in the R47H variant as well (Fig. 1D). Superposition of the R47H and WT structures results in an r.m.s. deviation of 1.1 Å over all of the backbone Cα atoms (residues 19–130). When residues 67–81 from the CDR2 loop, β-strand C″, and loop C″-D are excluded, however, the r.m.s. deviation drops to 0.52 Å, indicating that the variant structure differs from WT mainly in the region of the CDR2 loop to loop C″-D. At the molecular level, the CDR2 loop remodels as a short helix in the R47H variant. This occurrence can be described by an entirely different set of interactions in the region surrounding the R47H mutation, which resides in the C-terminal region of the CDR1 loop, compared with the WT structure. In the WT structure, the Arg47 side chain projects into the N-terminal region of the CDR2 loop, participating in extensive hydrogen bond networks with residues in the CDR2 loop, including the side chain of Ser65, the backbone carbonyls of Thr66 and His67, and the side chain of Asn68 (Fig. 2A). As a result, Arg47 plays a critical structural role in maintaining the fold of the N-terminal area of the CDR2 loop. Other hydrogen bond interactions in the region extend from the carbonyl of Lys48 to the amide of Thr66 and from the amide of Lys48 to the side-chain hydroxyl of Thr66 (Fig. 2A). Whereas the R47H variant maintains the two hydrogen bond interactions between Lys48 in the C terminus of the CDR1 loop and Thr66 in the N terminus of CDR2 loop, the hydrogen bond network interactions from Arg47 to Ser65, Thr66, His67, and Asn68 of the CDR2 loop noted in the WT structure are all absent in the R47H variant structure. The imidazole group of the His47 side chain in the R47H variant of CDR1 makes no direct hydrogen-bond interactions with the backbone of the CDR2 loop. Instead, the His47 imidazole moiety directly hydrogen-bonds with the side-chain hydroxyl of Thr66 and also forms π–π stacking interactions with the His67 imidazole ring from the CDR2 loop (Fig. 2B). His67 undergoes a dramatic conformational change to swing ∼180° toward the area of the C terminus of CDR1, facilitating interactions with His47 (Fig. 2, A and B). By contrast, in the WT structure, residue His67 from the CDR2 loop adopts an extended conformation toward the exterior of the protein (Fig. 2A). As a result of numerous intramolecular interactions from Arg47 to CDR2, the CDR2 loop in the WT structure is very stable, as indicated by its low B-factors (Fig. S2A). In the R47H variant structure, however, whereas the N-terminal part of the CDR2 loop that interacts with the CDR1 loop remains stable, the rest of the CDR2 loop (residues Leu69–Leu75 in the short helix) presents high B-factors due to lack of intramolecular interactions, followed by a disordered β-strand C″ compared with the rest of the protein (Fig. S2B). Consequently, Arg47 plays a critical structural role in maintaining the tertiary architecture in the region around the C terminus of the CDR1 loop and the N terminus of the CDR2 loop. The arginine-to-histidine mutation in the R47H variant abolishes the extensive interactions specific for the arginine residue, reducing the stability of the CDR2 loop and its downstream β-strand C″. In this manner, CDR2 loop remodeling in the R47H variant can be traced back readily to the single amino acid mutation of R47H in the CDR1 loop, probably causing the rest of the CDR2 loop to be moved out of register with its partner strands. The drastic conformational change in the CDR2 loop in the R47H variant also results in substantial modifications in the putative PLIS on the A-G-F-C-C′ sheet of WT TREM2 composed mainly of a few basic residues, including Arg47 (CDR1 loop), Arg62 (β-strand C′), His67 (CDR2 loop), and Arg77 (β-strand C″) (Fig. 2, C and D). Kober et al. (12.Kober D.L. Alexander-Brett J.M. Karch C.M. Cruchaga C. Colonna M. Holtzman M.J. Brett T.J. Neurodegenerative disease mutations in TREM2 reveal a functional surface and distinct loss-of-function mechanisms.eLife. 2016; 5 (27995897): e20391Crossref PubMed Scopus (110) Google Scholar) also reported a similar PLIS. The altered interactions in the R47H variant discussed above, especially the swing of His67 to make His47–His67 stacking, result in a disruption in the close interactions of the CDR2 loop with CDR1 and β-strand C″, subsequently resulting in a net reduction in the PLIS of the variant and the appearance of a significantly altered surface (Fig. 2D). With regard to the influence of crystal contacts on the altered conformation of the CDR loops, His47 in the R47H variant completely remodels the fold encompassing CDR2, which is not involved in crystal packing in the asymmetric unit or between symmetry-related molecules. As well, examination of CDR1 and CDR3 reveals that these loops are not involved in crystal contacts involving CDR2, again within the asymmetric unit or between symmetry-related molecules. The WT TREM2 CDR2 loop does have crystal contacts with both the CDR1 and D-E loops of neighboring molecules. However, the CDR2 loop has a fold highly similar to the fold observed in the recently reported WT TREM2 crystal structure (12.Kober D.L. Alexander-Brett J.M. Karch C.M. Cruchaga C. Colonna M. Holtzman M.J. Brett T.J. Neurodegenerative disease mutations in TREM2 reveal a functional surface and distinct loss-of-function mechanisms.eLife. 2016; 5 (27995897): e20391Crossref PubMed Scopus (110) Google Scholar), although the two structures are determined in two different space groups with different crystal packing(s) (Fig. 1C). Due to the numerous interactions of Arg47 with the CDR2 loop, it is more likely that the extensiveness of this interaction makes intramolecular contact more dominant and thus less likely to be impacted by crystal packing. Comparison of the R47H and WT structures clearly reveals the impact of an arginine-to-histidine mutation at residue 47 on the TREM2 ECD structural fold. The protein sources for each structure differ, however, and this difference must be addressed. The WT structure was derived from mammalian protein, whereas the R47H variant structure was derived from E. coli expressed protein and lacks the glycosylation observed at Asn79 in the WT structure. To address the possible contribution of Asn79 glycosylation to the observed differences between the WT and R47H TREM2 structures, we examined the interactions of the NAG glycan with other residues in the WT structure in greater detail. In the WT structures presented here and the previously reported structure (Protein Data Bank code 5ELI) (12.Kober D.L. Alexander-Brett J.M. Karch C.M. Cruchaga C. Colonna M. Holtzman M.J. Brett T.J. Neurodegenerative disease mutations in TREM2 reveal a functional surface and distinct loss-of-function mechanisms.eLife. 2016; 5 (27995897): e20391Crossref PubMed Scopus (110) Google Scholar), the Asn79-NAG shares the same hydrogen bond interaction through the amide of Asn79 to the backbone carbonyl of Val63 of β-strand C′, and the GlcNAc moieties have only weak van der Waals contacts with Arg62, Val63, and Val64 of β-strand C′ (Fig. 3A). In addition, the NAG moieties in the two WT structures have different orientations (Fig. 1C) and much higher B-factors in both instances compared with TREM2 residues in the vicinity of the NAGs (Fig. 3A), suggesting dynamic, nonspecific NAG conformations. As a result, the presence or absence of glycan is unlikely to play a significant role in the structural fold of the TREM2 protein. Whereas the lack of a glycan on Asn79 in the R47H variant might contribute to some extent to the disordering of the more distal β-strand C″, it is unlikely to result in the dramatic N-terminal remodeling of the CDR2 loop. Instead, the dramatic conformational change of His67 is well supported by the altered atomic interactions resulting from the single arginine-to-histidine mutation at residue 47. To further probe the effect of glycosylation on R47H TREM2, we conducted structural and biophysical studies on mammalian R47H TREM2. Despite intensive effort, we were unable to obtain crystals of mammalian expressed R47H TREM2 or E. coli expressed WT TREM2 (Table S4). We were, however, able to obtain pure, homogeneous protein for mammalian expressed WT and R47H TREM2 ECD (residues 19–174) for biophysical characterization. An analysis of glycan content for these two protein samples of WT and R47H variant revealed nearly equivalent glycan type and content at both the Asn20 and Asn79 positions (Fig. 3, B and C). Thermal stability measurements (melting temperature (Tm)), however, revealed a 10 °C difference between constructs with a Tm = 55.7 °C for the R47H variant compared with Tm = 67.5 °C for WT TREM2 (Fig. 3D). These data clearly support the profound effect of the R47H mutation in driving conformational changes that result in stability loss. Both WT and R47H variant proteins utilized in these experiments have the same glycan type and content, and hence the observed differences in stability can be explained by the variant alone. Finally, our structural observations, including the remodeled short α-helix and disordered β-strand (C″) in the R47H variant compared with WT TREM2, are consistent with and provide detailed and unprecedented molecular insight into the differences in WT and R47H TREM2 observed by lower-resolution CD spectroscopy (12.Kober D.L. Alexander-Brett J.M. Karch C.M. Cruchaga C. Colonna M. Holtzman M.J. Brett T.J. Neurodegenerative disease mutations in TREM2 reveal a functional surface and distinct loss-of-function mechanisms.eLife. 2016; 5 (27995897): e20391Crossref PubMed Scopus (110) Google Scholar). To determine whether the in vitro differences in stability and structure of WT and R47H ECDs translate to differences in in vivo levels, we developed a homogeneous ELISA to determine steady-state, homeostatic levels of soluble and total TREM2 in plasma and brains of WT and R47H TREM2 mice (for a brief description of mice, see “Experimental procedures”; for a detailed description, see Cheng et al. (33.Cheng Q. Danao J. Talreja S. Wen P. Yin J. Sun N Li C.-M. Chui D. Tran D. Koirala S. Chen H. Foltz I.N. Wang S. Sambashivan S. J. Biol. Chem. 2018; 293 (29599291): 12620-1263310.1074/jbc.RA118.001848Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar)). The assay developed specifically recognizes TREM2, as evidenced by no detectable levels in knockout animal plasma and brains (data not shown). The standard curves using recombinant TREM2 protein confirm that the assay recognizes WT and R47H TREM2 equivalently. R47H variant mice revealed significantly reduced sTREM2 (plasma, Fig. 3E) and tot" @default.
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