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Structure, Vol. 12, 2257–2263, December, 2004, ©2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.str.2004.10.007 Crystal Structure of a FYVE-Type Domain from the Caspase Regulator CARP2

Michael D. Tibbetts,1 Eric N. Shiozaki,1 tion and lack the three signature sequences (Ostermeier Lichuan Gu,1 E. Robert McDonald III,2 and Brunger, 1999; Stenmark and Aasland, 1999). FYVE Wafik S. El-Deiry,2 and Yigong Shi1,* and FYVE-related domains have been identified in sev- 1Department of Molecular Biology eral , such as EEA1 and Rabphilin-3A, respec- Lewis Thomas Laboratory tively, both of which are involved in membrane traffick- Princeton University ing and bind to members of the Rab subfamily of GTP Princeton, New Jersey 08544 hydrolases (Simonsen et al., 1998; Stahl et al., 1996). 2 Laboratory of Molecular Oncology The crystal structure of the EEA1 FYVE domain in and Cell Cycle Regulation complex with the soluble phosphoinositide analog inosi-

Howard Hughes Medical Institute tol-(1,3)-bisphosphate (Ins(1,3)P2) revealed the molecu- Departments of Medicine, Pharmacology, lar mechanism for specific binding to PtdIns(3)P (Dumas and Genetics et al., 2001). As predicted by the structure of an unli- University of Pennsylvania School of Medicine ganded FYVE domain and mutagenesis studies, the li- Philadelphia, Pennsylvania 19104 gand is coordinated exclusively by residues from the conserved motifs, primarily the basic pocket R(R/K) HHCR and the WxxD motif (Gaullier et al., 2000; Misra Summary and Hurley, 1999). Specific hydrogen bonds are made with the 1 and 3 phosphate and 4, 5, and 6 hydroxyl The caspase-associated ring proteins (CARP1 and groups of Ins(1,3)P2; however, this interaction is not suf- CARP2) are distinguished from other caspase regula- ficient for membrane recruitment. Dimerization, medi- tors by the presence of a FYVE-type zinc finger do- ated by a coiled-coil region adjacent to the FYVE do- main. FYVE-type domains are divided into two known main, is necessary for the functional targeting of EEA1 to classes: FYVE domains that specifically bind to phos- membranes. In addition, EEA1 and other FYVE domains phatidylinositol 3-phosphate in lipid bilayers and FYVE- such as Vps27p contain a tip of hydrophobic residues related domains of undetermined function. Here, we re- that are predicted to extend into lipid bilayers (Misra port the crystal structure of the N-terminal region of and Hurley, 1999). These nonspecific interactions are CARP2 (44–139) including the FYVE-type domain and responsible for a 6-fold increase for the -mem- its associated helical bundle at 1.7 A˚ resolution. The brane binding affinity (Kutateladze et al., 2004). Addi- structure reveals a cramped phosphoinositide binding tional interactions between acidic phospholipids and a pocket and a blunted membrane insertion loop. These conserved basic site in the domains further facilitate structural features indicate that the domain is not opti- membrane targeting (Kutateladze et al., 2004). mized to bind to phosphoinositides or insert into lipid The homologous caspase-associated ring proteins, bilayers. The CARP2 FYVE-like domain thus defines a CARP1 and CARP2, are a recently identified class of third subfamily of FYVE-type domains that are func- proteins with FYVE-type zinc finger domains (McDonald tionally and structurally distinct. Structural analyses and El-Deiry, 2004). The CARPs are distinguished from provide insights into the possible function of this other FYVE-type proteins by their important role in the unique subfamily of FYVE-type domains. negative regulation of apoptosis, specifically targeting two initiator caspases, caspase 8 and caspase 10. The CARPs contain a C-terminal RING domain with signifi- Introduction cant homology to the inhibitor of apoptosis proteins (IAPs) (Deveraux and Reed, 1999; Shi, 2002). The CARP FYVE-type zinc finger domains have been best char- RING domain has ubiquitin-ligase activity in vitro, and acterized in proteins targeted to lipid membranes. thus the CARPs have been proposed to target caspase Most FYVE domains target proteins to endosomes by 8 and caspase 10 for ubiquitin-mediated proteolysis binding specifically to phosphatidylinositol-3-phos- (McDonald and El-Deiry, 2004). phate (PtdIns(3)P) at the membrane surface (Burd and The activation of death receptors such as Fas and Emr, 1998; Gaullier et al., 1998; Patki et al., 1998; Sten- TNF-␣ initiates the extrinsic cell death pathway and re- mark et al., 1996). However, some FYVE-domain-con- sults in the activation of caspases 8 and 10 (Boldin et taining proteins, such as the Fgd1 family, localize to al., 1996; Fernandes-Alnemri et al., 1996). The CARPs the plasma membrane and do not bind exclusively to inhibit these initiator caspases, thereby impeding the PtdIns(3)P (Sankaran et al., 2001). FYVE domains are cell death pathway. In addition, the regulation of these distinguished from other zinc fingers by three signature initiator caspases affects cellular proliferation signals, sequences: an N-terminal WxxD motif, a basic R(R/ thereby regulating a wide range of processes in the cell K)HHCR patch, and a C-terminal RVC motif (Burd and (Tibbetts et al., 2003). Emr, 1998; Gaullier et al., 1998; Patki et al., 1998; Sten- The CARPs do not localize to membranes in the cell, mark et al., 1996). The FYVE-related domains are struc- and thus the role of the FYVE-type domain is unclear turally similar to FYVE domains but have no known func- (McDonald and El-Deiry, 2004). Furthermore, the CARPs have an altered sequence in the basic ligand binding *Correspondence: [email protected] patch and lack the WxxD motif. To decipher the function Structure 2258

Figure 1. Overall Structure of the CARP2 N-Terminal Core Domain (A) Schematic representation of CARP2 N-terminal structural core domain (residues 44–139) comprising the FYVE domain (cyan) and the adjoining helical bundle (pink). The secondary structural elements are indicated. The two Zn2ϩ atoms are shown as white spheres. (B) The electrostatic potential of the molecu- lar surface of the CARP2 N-terminal structural core domain. Basic and acidic surfaces are colored blue and red, respectively. The two panels are related by a 180Њ rotation along a vertical axis in the plane of the paper. The structure in the left panel is in the same orien- tation as in (A). These two panels were gener- ated using GRASP (Nicholls et al., 1991). (A) was prepared using MOLSCRIPT (Kraulis, 1991); all other structural figures were made using PyMol (DeLano, 2002). (C) Sequence alignment of the N-terminal structural core domain of human CARP2 with the human paralog CARP1 and the orthologs of mouse CARP1 and CARP2. Key basic resi- dues in the cleft between the FYVE domain and helical bundle are indicated by blue squares, and the core hydrophobic residues of the helical bundle are indicated by a white circle.

of the CARPs, we have determined the crystal structure ces, ␣2, ␣3, and ␣4 (Figure 1). The helical bundle con- of the CARP2 FYVE-type domain and its associated tains a well-packed hydrophobic core composed of helical bundle at 1.7 A˚ resolution. Structural analysis Phe91, Phe96, Tyr112, Leu113, and Val133. These resi- reveals that the CARPs constitute a subfamily of unique dues are highly conserved in mouse CARP2 and mouse FYVE-type domains. and human CARP1, suggesting that the helical bundle is conserved among the CARPs (Figure 1C). Results and Discussion The molecular surface of the CARP2 N-terminal struc- tural core domain contains several highly basic patches Crystallization and Structure Determination (Figure 1B). Most notably, close packing between the We first identified the structural core domain of the FYVE-type domain and the adjoining helical bundle CARP2 N-terminal region (residues 1–289) by limited gives rise to a deep basic cleft. This cleft is lined by proteolysis (data not shown). Results from N-terminal Arg90 and Lys107 on opposing sides of the cleft and sequencing and mass spectroscopic analyses revealed could be an ideal binding pocket for an acidic ligand. The a trypsin-resistant fragment (residues 26–145), which only known interacting proteins of CARP2 are caspase 8 includes the FYVE-type domain (amino acids 39–95) and and caspase 10, which bind to another region of the an additional 50 amino acids at its C terminus. We sub- protein (McDonald and El-Deiry, 2004). Future work will cloned this fragment, purified it to homogeneity, and be needed to determine if the positively charged cleft crystallized it. The structure was determined by multi- binds to a ligand and if this binding is critical to CARP2 wavelength anomalous dispersion (MAD) using anoma- function. lous signals from two native Zn2ϩ ions. The final atomic model contains residues 44–139 and was refined at 1.7 A˚ Comparison with Other FYVE Domains resolution (Figure 1 and Table 1). The CARP2 N-terminal structural core domain, like the Vps27p FYVE domain and Rabphilin-3A FYVE-related Overall Structure domain, was crystallized as a monomer (Misra and Hur- The N-terminal structural core domain of CARP2 forms ley, 1999; Ostermeier and Brunger, 1999). In contrast, a highly organized tertiary structure consisting of a zinc both the Hrs VHS-FYVE tandem domains and the EEA1 binding FYVE-type domain and an adjoining helical bun- FYVE domain with associated coiled-coil region form dle (Figure 1). The CARP2 FYVE-type domain comprises dimers (Dumas et al., 2001; Mao et al., 2000). The CARP2 two pairs of antiparallel ␤ strands and a C-terminal ␣ FYVE-type domain shares a similar overall fold with both helix (␣1). The first zinc atom is coordinated by cysteines the FYVE and FYVE-related classes. The core structural 47, 50, 71, and 74, and the second zinc is coordinated elements can be superimposed with a root-mean- by cysteines 63, 65, 85, and 88. The CARP2 FYVE-type square deviation (rmsd) of 0.95 A˚ for 156 atoms between domain forms a single folding unit with a closely associ- CARP2 and EEA1 and 164 atoms between CARP2 and ated helical bundle, which is composed of three ␣ heli- Rabphilin-3A (Figure 2). All three domains contain two Structure of a FYVE-Type Domain from CARP2 2259

Table 1. Statistics of Crystallographic Analysis Data Collection (at NSLS-X25) Zn (Peak) Zn (Inflection) Zn (Remote)

Space group P31 P31 P31 Wavelength (A˚ ) 1.2831 1.2835 1.2575 Resolution (A˚ ) 99.0–1.7 99.0–1.7 99.0–1.7 Total observations 75,959 56,842 62,116 Unique observations 12,241 12,029 12,106 Data coverage (outer shell) 97.3% (85.2%) 95.4% (76.7%) 97.0% (93.6%)

Rsym (outer shell) 0.071 (0.446) 0.051 (0.475) 0.057 (0.414) Refinement Resolution range (A˚ ) 20.0–1.7 No. of reflections (|F|Ͼ0) 12,426 Data coverage (outer shell) 97.3% (85.2%)

Rworking/Rfree 0.2163/0.2304 No. of atoms 698 No. of waters 138 Rmsd bond length (A˚ ) 0.007 Rmsd bond angles (Њ) 1.37 Ramachandran Plot Most favored (%) 91.5 Additionally allowed (%) 8.5 Generously allowed (%) 0.0 Disallowed (%) 0.0

Rsym ϭ⌺h⌺i|Ih,i Ϫ Ih|/⌺h⌺i Ih,i, where Ih is the mean intensity of the i observations of symmetry-related reflections of h.Rϭ⌺|Fobs Ϫ Fcalc|/⌺Fobs,

where Fobs ϭ FP and Fcalc is the calculated factor from the atomic model (Rfree was calculated with 5% of the reflections). Rmsd in bond lengths and angles are the deviations from ideal values, and the rmsd deviation in B factors is calculated between bonded atoms.

pairs of antiparallel ␤ strands, ␤1/␤2 and ␤3/␤4, and a could not form many of the important interactions re- C-terminal ␣ helix, ␣1. However, compared to EEA1 and quired for binding to PtdIns(1,3)P (Figure 2C). Most nota- Rabphilin-3A, CARP2 has a truncation of eight and three bly, CARP2 has a leucine in the same position as residues, respectively, in the region between the ␤3 and Arg1375 of EEA1, which hydrogen bonds to the 3-phos- ␤4 strands. Consequently, these three proteins assume phate group of PtdIns(1,3)P (Figure 3A). Mutation of quite different conformations in the ␤3-␤4 loop, with Arg1375 to Ala in EEA1 resulted in a more than 100-fold the Rabphilin-3A loop extending out farthest from the decrease in binding affinity for PtdIns(1,3)P (Gaullier et structural core (Figure 2A). As will be discussed in detail, al., 2000). In addition, a pair of highly conserved histidine another important difference between the CARP2 FYVE- residues in other FYVE domains is replaced by Gln61- type domain and other FYVE and FYVE-related domains Thr62 in CARP2. is the membrane insertion loop (Figures 2A and 2B). In the crystal structures of the EEA1 and Vps27p FYVE domains, the N-terminal sequence contains the WxxD Comparison of the Ligand Binding Pocket motif, which forms part of the ligand binding pocket by The CARP2 FYVE-type domain has several unique struc- packing against the ␤2 strand (Figure 3A). The bulky tural features, all of which appear to have a negative tryptophan side chain stabilizes the pocket, and the impact on its ability to bind to phosphoinositides or to aspartate hydrogen bonds to the 5- and 6- hydroxyl

insert into lipid bilayers. Most notably, the CARP2 FYVE- groups of Ins(1,3)P2. In CARP2, the N-terminal linker type domain contains a distinct kink in the loop preced- region lacks the WxxD motif and is occluded from pack- ing the ␤1 strand from Ala55 to Arg59 (Figure 2A). This ing against the ␤2 strand by the substitution of a glycine, kinked loop folds back into the ligand binding pocket, conserved in more than 40 FYVE domains, with lysine likely interfering with ligand binding; in other FYVE and (Figure 2C). This glycine is the only invariant residue in FYVE-related domains, this region extends away from all FYVE domains outside of the ligand binding pocket the pocket. When superimposed onto the EEA1 struc- and is positioned at the beginning of the ␤2 strand (Du-

ture with bound Ins(1,3)P2, Ala58 in CARP2 is only 1.0 A˚ mas et al., 2001; Stenmark and Aasland, 1999). In EEA1,

away from the 1-phosphate group of Ins(1,3)P2, which Gly1378 is within van der Waals contact distance of the clearly represents steric hindrance (Figure 3A). Thus, backbone groups of Lys1348-Trp1349, and thus any

Ins(1,3)P2 is unlikely to bind to the CARP2 FYVE-type substitution would disrupt the packing between the domain in the same orientation as that observed in EEA1. N-terminal linker region and the core of the FYVE domain The mutation of the conserved tryptophan or basic (Dumas et al., 2001). In CARP2, this glycine is replaced residues in the EEA1 FYVE domain was found to result by Lys67. in a reduction of the binding affinity for PtdIns(1,3)P by The analyses above indicate that the FYVE-type do- 6- to 100-fold (Gaullier et al., 2000). An analysis of the main in CARP2 may not bind to phosphoinositides. Inter- residues lining the binding pocket indicates that CARP2 estingly, the FYVE-related zinc-finger-containing pro- Structure 2260

Figure 2. The CARP2 FYVE-Type Domain Has Several Unique Structural Features (A) Comparison of the CARP2 FYVE-type domain with the EEA1 FYVE domain and the Rabphilin-3A FYVE-related domain. Several distinguishing structural features are highlighted, including the phosphoinositide binding pocket for EEA1, the ␤3-␤4 loop, the membrane insertion tip, and the distinct kink in the loop close to the binding pocket in CARP2. Details of the membrane insertion region of all three proteins are shown in the inset. The CARP2 tip region lacks the hydrophobic property that is conserved in EEA1 and Rabphilin-3A. (B) A stereo view of the membrane insertion loop of CARP2. The 2Fo-Fc electron density map, colored magenta and contoured at 1.2 ␴,is shown at the tip of membrane insert loop. The three amino acid residues at the tip, Ala55, Asn56, and Thr57, are labeled. (C) Sequence alignment of the FYVE-type domains from CARP2, CARP1, Fgd1, Vps27p, Hrs, EEA1, SARA, Rabphilin-3A, and NOC2. The alignment highlights the conservation of the zinc-coordinating cysteines and key sequence differences between the different subfamilies of FYVE-type domains. Note that the WxxD motif is conserved only in phosphoinositide binding FYVE domains. The solid line identifies the R(R/ K)HHCR signature motif, and the double line indicates the C-terminal RVC motif. The membrane insertion tip residues are indicated by a wavy line. The eight conserved cysteines that coordinate two zinc atoms are indicated with asterisks. The conserved glycine in FYVE domains is indicated by a yellow box, and the conserved glycine in FYVE-related domains is indicated by a blue triangle. Structure of a FYVE-Type Domain from CARP2 2261

Figure 3. CARP2 Lacks Conserved Features of the Phosphoinositide Binding Pocket (A) Comparison of the EEA1 (left) and CARP2 (right) ligand binding pockets. In the left panel, the residues in the EEA1 ligand binding pocket are colored in orange, and the Ins(1,3)P2 ligand is colored in red and pink, with the positions of the 1 and 3 phosphate groups indicated (Dumas et al., 2001). In the right panel, the CARP2 residues that correspond to those of the EEA1 binding pocket are shown with the superimposed ligand Ins(1,3)P2. The characteristic positioning of key residues important for phosphoinositide binding in EEA1 is missing in CARP2. Most notably, CARP2 lacks the WxxD motif, and the kink in the loop containing Ala58 cramps the binding pocket.

(B) EEA1 but not CARP2 binds to Ins(1,3)P2. Binding of Ins(1,3)P2 to EEA1 and CARP2 was examined by isothermal titration calorimetry (ITC). Representative results for both ITC experiments are shown.

teins, such as Rabphilin-3A, also contain a basic residue ever, there was no detectable binding between Ins(1,3)P2 in place of the conserved glycine (Figure 2C) (Stenmark and CARP2 (26–145) or CARP1 (33–158), which contain and Aasland, 1999). As a result, the N-terminal linker the minimal FYVE-type domain and the associated heli- region does not pack closely against the ␤2 strand. This cal bundle. key substitution distinguishes the CARP2 FYVE-type do- main and the FYVE-related domains from the phospho- Membrane Insertion inositide binding FYVE domains. The specific interaction with a single head group of a In contrast to CARP2/CARP1 and the FYVE domains, phospholipid is insufficient for targeting to the cell mem- the FYVE-related domains have a universally conserved brane by the FYVE domains (Sankaran et al., 2001). Addi- glycine after the sixth coordinating cysteine for the zinc tional nonspecific interactions are required for inserting atoms (Figure 2C). CARP2 and most FYVE domains con- the FYVE domain into the lipid bilayer (Kutateladze and tain a serine residue in the position. This analysis indi- Overduin, 2001; Misra and Hurley, 1999). These interac- cates that the CARP proteins differ from the FYVE- tions are conserved in several FYVE domains, involving related domains by one conserved structural feature. tip sequences Val1367-Thr1368-Val1369 and Leu185- Leu186 in EEA1 and Vps27p, respectively. FYVE do- Binding to Phosphoinositides by CARP2 mains with mutations in the membrane insertion tip fail To further examine the FYVE-type domain of CARP2 to localize to membranes and have decreased affinities and to corroborate our structural analysis, we attempted for PtdIns(3)P-containing vesicles. For example, muta- to measure the binding affinity between CARP2 and tion of Leu185-Leu186 in Vps27p decreased binding to the soluble analog Ins(1,3)P2 using isothermal titration vesicles by 7-fold (Stahelin et al., 2002). A similar inser- calorimetry (Figure 3B). As a positive control, the EEA1 tion sequence is found in the of protein ki- FYVE domain (residues 1287–1411) was found to bind nase C, which also penetrates into lipid bilayers (Xu et to Ins(1,3)P2 with a dissociation constant of approxi- al., 1997; Zhang et al., 1995). Although it is not entirely mately 33 ␮M. This value agrees well with the previously clear if the FYVE-related domains also insert into lipid determined binding affinity of 24 ␮M measured by intrin- bilayers, Rabphilin-3A contains an extended, hydropho- sic tryptophan fluorescence (Dumas et al., 2001). How- bic tip with Met103-Leu104. In contrast, CARP2 has Structure 2262

a blunted tip with the sequences Ala55-Asn56-Thr57 BL-21(DE3) at room temperature for 14 hr. The His-tagged proteins (Figure 2A). These residues are considerably less hy- were purified as described (Shiozaki et al., 2003). CARP2 (26–145) Њ drophobic than those in the membrane insertion loops, crystals were grown at 23 C using the hanging drop diffusion method. The well buffer contained 0.50 M Na, K tartrate, and 0.1 suggesting that the blunted tip of CARP2 may not be M HEPES (pH 7.5). Crystals grew over a period of 2–3 days with able to effectively insert into lipid bilayers. Further sup- approximate dimensions of 400 ␮m by 400 ␮m by 200 ␮m. The

porting this conclusion, the CARP2 membrane insertion crystals belong to the space group P31 and contain one molecule loop is not involved in crystal packing yet is well ordered, per asymmetric unit. The unit cell has dimensions of a ϭ b ϭ as judged by the electron density map (Figure 2B). 51.196 A˚ ,cϭ 38.564 A˚ , and ␥ϭ120Њ.

The C-Terminal Helical Bundle of CARP2 Data Collection and Structure Determination The crystals were equilibrated in a cryoprotectant buffer containing The helical bundle C-terminal to the FYVE-type domain 0.50 M Na, K tartrate, 0.1 M HEPES (pH 7.5), and 25% glycerol by of CARP2 is unique compared to other adjacent domains slowly increasing the concentration prior to placement under the in FYVE domain structures. The EEA1 FYVE domain was cryostream. These crystals diffracted to 2.2 A˚ on an R-AXIS-IV im- crystallized with a coiled-coil region at the N terminus, aging plate detector mounted on a Rigaku 200HB generator. We and the Hrs FYVE domain was crystallized with an adja- then collected multiwavelength anomalous dispersion (MAD) data cent, N-terminal VHS domain (Dumas et al., 2001; Mao at the zinc edge on the NSLS-X25 beamline at Brookhaven National et al., 2000). In contrast, the helical bundle C-terminal Laboratories. The data were processed using HKL (Otwinowski and Minor, 1997). The positions of the zinc atoms were determined, to the FYVE-type domain of CARP2 comprises three heavy atom positions were refined, and MAD phases were calcu- ␣ tightly packed helices. To identify its potential func- lated using SOLVE (Terwilliger, 1994a; Terwilliger, 1994b; Terwilliger tion, we performed a Dali search for this helical bundle and Berendzen, 1996) and refined using CNS (Brunger et al., 1998). (Holm and Sander, 1995). Interestingly, the first and sec- The final model contains residues 44–139. The chain contains a ond helices of the helical bundle are structurally homolo- single break in the flexible loop from Val78 to Gly81. gous to domains in several nucleic acid binding proteins. Notably, the helical bundle is structurally similar to a Isothermal Titration Calorimetry region in Ku70, a protein involved in DNA double- His-tagged CARP2 (26-145) was purified as described for protein ˚ crystallization. CARP1 residues 33–158 were subcloned into pET15b stranded break repair, with an rmsd of 1.49 A for 42 based on a sequence alignment with CARP2. An EEA1 construct aligned C␣ residues; to rho, a transcription termination (1287–1411) in pET15b was generously provided by David Lam- factor, with an rmsd of 1.37 A˚ for 41 C␣ residues; and bright. CARP1 (33–158) and EEA1 (1287–1411) were purified in the to reverse gyrase, a topoisomerase that positively su- same manner as CARP (26–145). The final buffer for all three proteins percoils DNA, with an rmsd of 1.75 A˚ for 38 aligned C␣ contained 10 mM HEPES (pH 7.5) and 100 mM NaCl. The proteins ␮ residues (Allison et al., 1998; Rodriguez and Stock, 2002; were diluted to a concentration of 20 M, and Ins(1,3)P2 was resus- pended to a concentration of 2.25 mM in the same buffer. The Micro Walker et al., 2001). The homologous residues in Ku70 Calorimetry System (Microcal, Amherst, MA) was used to perform constitute part of the SAP motif, a putative DNA binding the ITC measurements. The titration data, collected at 25ЊC, were motif involved in chromosomal organization (Aravind analyzed using the ORIGIN data analysis software (Microcal Soft- and Koonin, 2000). This analysis suggests a possible link ware, Northampton, MA). between the function of the CARP2 N-terminal structural core domain and binding to nucleic acids, although di- Acknowledgments rect evidence is lacking. Nonetheless, supporting this hypothesis, the N-terminal core domain of CARP2 con- We thank M. Becker and S. Vaday at the NSLS X25 beamline for help with data collection, N. Yan for assistance with ITC, and other tains prominent, positively charged surface patches members of the Shi lab for advice and discussion. This work was (Figure 1B). supported by grants from the National Institutes of Health.

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