Crystal structure of elegans HER-1 and characterization of the interaction between HER-1 and TRA-2A

Brent Y. Hamaoka, Charles E. Dann III, Brian V. Geisbrecht, and Daniel J. Leahy*

Department of Biophysics and Biophysical Chemistry and Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205

Edited by Barbara J. Meyer, University of California, Berkeley, CA, and approved June 28, 2004 (received for review April 9, 2004) HER-1 is a secreted protein that promotes male development in the questions about the relationships between specific pathway . HER-1 inhibits the function of components. TRA-2A, a multipass integral membrane protein thought to serve To provide a molecular basis for interpreting HER-1 function as its receptor. We report here the 1.5-Å crystal structure of HER-1. and to investigate the relationship of HER-1 to other signaling The structure was solved by the multiwavelength anomalous molecules, we have determined the crystal structure of C. elegans diffraction method by using selenomethionyl-substituted HER-1 HER-1. We have also developed an immunofluorescence assay produced in Chinese hamster ovary cells. The HER-1 structure and used it to demonstrate an interaction between HER-1 and consists of two all-helical domains and is not closely homologous TRA-2A-expressing cells and examine regions on both proteins to any known structure. Sites of amino acid substitutions known to important for mediating this interaction. impair HER-1 function were mapped on the HER-1 structure and classified according to the likely mechanism by which they affect Materials and Methods HER-1 activity. A subset of these and other amino acid substitutions Expression and Purification. A cDNA encoding the C. elegans her-1 on the HER-1 surface were assayed for their ability to disrupt gene (GI:297387) was kindly provided by M. Perry (University interactions between HER-1 and TRA-2A-expressing cells, and a of Toronto, Toronto). DNA encoding the mature protein, amino localized region on the HER-1 surface important for mediating this acids 19–175, was amplified by PCR by using 5Ј and 3Ј primers interaction was identified. that encoded NheI and EcoRI restriction sites, respectively. The PCR product was digested with these enzymes, subcloned into exual differentiation in Caenorhabditis elegans is determined XbaI and EcoRI sites in the expression vector pSGHV0 (17), and sequenced. The resulting vector directs expression of a fusion by the ratio of sex chromosomes (X) to autosomes, with XO S protein with human growth hormone (hGH) at the N terminus normally developing as males and XX animals as followed by an octahistidine tag, a tobacco etch virus protease hermaphrodites (1–3). Activity of the her-1 gene is required for recognition sequence (18), and HER-1. It is not known whether male development, and its expression is repressed in XX her- endogenous HER-1 is glycosylated or whether glycosylation maphrodites (4, 5). Loss of her-1 function in XO animals results plays a role in HER-1-mediated signaling, but attached carbo- in conversion to self-fertile hermaphrodites but has no effect in hydrate frequently inhibits crystallization. Point mutations XX animals (4). Gain of her-1 function in XX animals results in changing both Asn-98 and Asn-163 to glutamate were thus masculinized intersex phenotypes but has no effect in XO introduced by site-directed mutagenesis to remove consensus animals (6). N-linked glycosylation sites. The HER-1 product produced after her-1 encodes a Cys-rich secreted protein and acts just up- tobacco etch virus proteolysis begins with GSSS followed by the stream and inhibitory to tra-2a, which is itself an inhibitor of HER-1 sequence starting at Thr-19 and containing the altered downstream components of the sex determination pathway (4, glycosylation sites. This protein will be referred to as HER-1GM. 7–9). tra-2a encodes a multipass integral membrane protein that To remain consistent with existing literature (9, 19), numbering is believed to serve as a receptor for HER-1 (10). A single amino for amino acids will start with the first amino acid of the signal acid substitution in the TRA-2A extracellular region, Arg-177 to sequence. Lys, results in constitutively active TRA-2A and has been A Chinese hamster ovary cell line that stably expressed 9 proposed to function by disrupting an inhibitory interaction ␮g͞ml hGH͞HER-1GM was generated after two methotrexate between HER-1 and TRA-2A (11). A direct interaction between selections (17). Four liters of HER-1GM-conditioned media HER-1 and TRA-2A has yet to be observed, however. were collected from roller bottles, concentrated to Ϸ250 ml, and HER-1 homologs have only been found in the dialyzed against 20 mM Tris, pH 8.0͞500 mM NaCl by tangential Caenorhabditis briggsae, , and Teladorsagia circum- flow filtration with 30-kDa cutoff filters (Millipore). Glycerol cincta (12), and insight into the evolution and mechanism of and imidazole were added to final concentrations of 10% HER-1 from comparison with homologous proteins has been (vol͞vol) and 10 mM, respectively, and the concentrated medium limited. In contrast, TRA-2A shares topological similarity and was applied to a Nickel Hi-Trap chelating column (Amersham an extended region of sequence homology with the Patched Pharmacia). Bound fusion protein was eluted with 200 mM family of proteins (10, 13). Patched proteins serve as receptors imidazole and cleaved at room temperature with Ϸ5,000 units of for Hedgehog proteins and mediate cell and tissue differentia- tobacco etch virus protease per 40 ml of Ϸ0.8 mg͞ml hGH͞ tion in species ranging from flies to humans (14). Reminiscent of TRA-2A, Patched is both inhibited by its ligand and inhibitory to downstream pathway components (15). The structural and This paper was submitted directly (Track II) to the PNAS office. functional similarities between Patched and TRA-2A led to Abbreviation: hGH, human growth hormone. speculation that Hedgehog and C. elegans sex determination Data deposition: The coordinates and structure factors reported in this paper have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 1SZH). pathways may be related (10). Hedgehog proteins are absent in BIOLOGY *To whom correspondence should be addressed. E-mail: [email protected].

C. elegans (16), however, and the absence of complete HER-1- DEVELOPMENTAL and Hedgehog-signaling pathways in any single organism raises © 2004 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0402559101 PNAS ͉ August 10, 2004 ͉ vol. 101 ͉ no. 32 ͉ 11673–11678 Downloaded by guest on September 23, 2021 Table 1. Immunofluorescence assay statistics No. of TRA-2A TRA-2A-expressing Cells labeled transfection HER-1GM media Amino acid substitutions in HER-1GM cells counted with HER-1, %

WT HER-1GM None 254 96.8 R177K HER-1GM None 218 2.3 WT Mutant set 1 K60A, D85A, K152A, E154A 224 2.4 WT Mutant set 2 V135A, I151A, V155A, E160A 230 0.9 WT Mutant set 3 E39A, K46A, F47A, T49A, R52A 212 92.9 WT Mutant set 4 D96A, D99A, R102A, R103A, E105A 209 92.3 WT Mutant set 5 E77A, K80A, T121A, K127A, N134A 212 83.0 WT Mutant set 6 E23A, K26A, K31A, L61A, K76A 200 84.0 WT Mutant set 7 Q70A, K81A, N83A, R147A, K148A 205 62.9 WT HER-1GM E77K 224 71.0 WT HER-1GM E154K 209 1.4

HER-1GM. After Ϸ16 h, the reaction mixture was dialyzed to modified to result in addition of a c-myc epitope and a stop decrease the imidazole concentration to 10 mM and reapplied to codon after the tra-2a sequence. A form of this vector encoding the nickel column. Flow-through from this column was collected, the tra-2a(eg) mutant (in which Arg-177 is substituted with Lys) concentrated, and applied to a Superdex 75 gel filtration column was produced by using the QuikChange kit (Stratagene). Both (Amersham Pharmacia). Purified HER-1GM was dialyzed into tra-2a alleles were sequenced. 2 mM Tris, pH 8.0͞20 mM NaCl and concentrated to 2–5mg͞ml. Transient transfections of tra-2a into HEK 293 cells attached To prepare selenomethionyl-substituted HER-1GM, cells to glutaraldehyde cross-linked gelatin-coated coverslips were were washed once with Hanks’ balanced salt solution and performed by using SuperFect reagent (Qiagen, Valencia, CA) incubated in ExCell 301 medium (JRH Biosciences, Lenexa, KS) according to manufacturer’s instructions. tra-2a-transfected cells lacking Met but supplemented with 50 ␮g͞ml L-selenomethi- were allowed to recover for 24 h and then incubated in condi- onine (Sigma). To ensure full incorporation of selenomethionine tioned media containing Ϸ150 nM hGH͞HER-1GM fusion into HER-1GM, the selenomethionine-containing media was protein for4hatroom temperature. Cells were then washed with exchanged after the first day (16–24 h) and the initial media was a wash buffer (WB) of PBS containing 1 mM MgCl2 and discarded. Selenomethionyl-substituted HER-1GM-conditioned saturated CaCl2, fixed with WB plus 4% paraformaldehyde, and medium containing Ϸ2 ␮g͞ml of hGH͞HER-1GM fusion pro- finally permeabilized with WB plus 1% Triton X-100. WB tein was subsequently collected, and HER-1GM was prepared as containing 0.1% Tween 20 and 5% FBS was next added to block described above. nonspecific antibody binding. Cells were then incubated with 1 ␮g͞ml mouse anti-myc monoclonal antibody 9E10 and a 1:1,000 Crystallization. HER-1 crystals were grown by the hanging drop dilution of polyclonal rabbit anti-hGH sera (Research Diagnos- vapor diffusion method. One microliter of protein was mixed tics, Flanders, NJ). Finally, 625 ng͞ml 4Ј,6-diamidino-2- with 1 ␮l of a 1:1 mixture of water and 50 mM sodium acetate, phenylindole plus 5 ␮g͞ml each of Alexa Fluor 488-coupled goat pH 4.3͞10% polyethelene glycol 3350͞80 mM ammonium sul- anti-mouse and Alexa Fluor 568-coupled goat anti-rabbit sec- fate. Hexagonal plate-like crystals with typical dimensions of ondary antibodies (Molecular Probes) were added. The cover- 350 ϫ 250 ϫ 30 ␮m3 grew after 7–14 days. slips were mounted on glass slides with Aqua-Poly͞Mount (Polysciences) and viewed with a ZEISS Axiovert 135 TV Data Collection and Structure Determination. Native and selenome- fluorescence microscope with a ϫ40 objective. Images were thionyl-substituted HER-1GM crystals were exchanged into recorded with a CoolSnap HQ camera (Photometrics, Tucson, crystallization buffer supplemented with 22.5% ethylene glycol AZ). Cells not expressing TRA-2A failed to bind significant and flash frozen in liquid nitrogen. Diffraction data were levels of either HER-1GM or the antibodies used to detect collected at beamline X4A of the National Synchrotron Light HER-1GM or TRA-2A and provide negative controls for non- Source at Brookhaven National Laboratory. Multiwavelength specific binding of each of these reagents. anomalous diffraction data at selenium inflection, peak, and Genes encoding seven HER-1GM variants containing multi- remote wavelengths were collected by using an inverse beam ple Ala substitutions (Table 1) were assembled by the mega- strategy (Table 2, which is published as supporting information primer method (24) and subcloned into the pSGHV0 expression on the PNAS web site). vector. Two additional HER-1GM variants containing single- Positions of all four unique selenium atoms in the asymmetric site substitutions known to impair HER-1 activity, E77K and unit were identified by SOLVE (20). Density modification and E154K (19), were created by site-directed mutagenesis. Nine initial model building were performed with RESOLVE (21), which stable cell lines, each secreting an hGH͞HER-1GM variant, built backbone atoms for 234 of 322 residues in the asymmetric were created as described above with the exception that expres- unit. Side chains and the remainder of the backbone were built sion levels were not amplified by methotrexate selection. All manually by using O (22). Refinement was carried out with native HER-1GM variants were expressed at levels comparable to or data by using CNS (23). slightly below WT, and expression of full-length protein was confirmed in each case by anti-hGH Western blot. Immunoflu- Immunofluorescence Assays. A cDNA encoding tra-2a was kindly orescence assays were carried out for the variant HER-1GM provided by P. Kuwabara (University of Bristol, Bristol, U.K.). proteins as described above. Independent assays were conducted PCR was used to add a Kozak consensus sequence, GGAGCC, at least five times for native HER-1GM, three times for each just upstream of the tra-2a initiator Met codon and eliminate the HER-1GM mutant cluster, and twice for the E77K and E154K native stop codon. By using the NheI and NotI sites, this tra-2a variants. Similar results were obtained in each instance; statistics clone was ligated into a pCDNA 3.1(ϩ) vector (Invitrogen) presented in Table 1 represent those from a single experiment.

11674 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0402559101 Hamaoka et al. Downloaded by guest on September 23, 2021 Fig. 1. Alignment of HER-1 primary and secondary structures. The site of signal sequence cleavage (9) is indicated by a black arrow. Blue and pink coils above the sequences indicate the positions of ␣-helices, and navy blue and purple indicate positions of 310-helices in domains 1 and 2 of the HER-1GM structure, respectively. The two consensus sites of N-linked glycosylation altered in HER-1GM are indicated by stars. Colored triangles indicate the HER-1 mutant sets used in the immunofluorescence assays (red, set 1; green, set 2; magenta, set 3; purple, set 4; blue, set 5; yellow, set 6; orange, set 7). Orange squares and triangles indicate amino acids that are located in the basic pocket on the HER-1 surface. Amino acids conserved among all HER-1 sequences are highlighted in red (12). The last amino acid visible in the HER-1 crystal structure is indicated with a black circle.

More details about this assay are provided in Supporting Text, of C␣ atoms Ͼ 4 Å) and structure-based sequence alignments do which is published as supporting information on the PNAS web not provide conclusive evidence for a divergent evolutionary site. relationship between HER-1 and these proteins, however. No- Results and Discussion HER-1GM Crystal Structure. The crystal structure of C. elegans HER-1GM was determined by the multiwavelength anomalous diffraction method by using selenomethionyl-substituted protein produced in Chinese hamster ovary cells and refined to a final ͞ ͞ Rcryst Rfree of 0.209 0.227 and rms deviation from ideality of 0.018 Å͞1.50° for bonds͞angles. Data collection and refinement statistics are listed in Table 2. The final model includes all of the HER-1GM sequence except 11 C-terminal residues (165–175) for which electron density was not interpretable. Two essentially identical HER-1GM molecules are present in the crystallo- graphic asymmetric unit (1.2-Å rms deviation for all C␣ atoms), but this crystallographic dimer is unlikely to be physiologically relevant because HER-1GM is monomeric in solution at Ϸ40 ␮M as judged by gel filtration chromatography. HER-1GM adopts an all-helical structure with two subdo- mains (Figs. 1 and 2A). Residues 19–80 comprise a left-handed three-helix bundle (domain 1) with an overhand connection between the second and third helices; residues 81–164 comprise a left-handed anti-parallel four-helix bundle (domain 2) in which the first helix consists of four consecutive turns of 310-helix. Fourteen Cys are conserved in all known HER-1 sequences and form seven disulfide bonds in C. elegans HER-1GM. Domains 1 and 2 share a homologous pattern of three disulfide bonds with an additional disulfide bond formed between Cys at the C terminus of domain 2. The similar topology and shared disulfide pattern found in the HER-1 subdomains suggest that they may have arisen by duplication of an original helical bundle. Super- Fig. 2. Structure of C. elegans HER-1GM. (A) A stereo ribbon diagram of the position of the structures and alignment of amino acid sequences HER-1GM structure. Domains 1 and 2 are colored blue and pink, respectively, of these domains provides no additional indication of such a with 310-helices colored navy blue and purple. The N and C termini are labeled. relationship, however. Disulfide bonds are shown in yellow and green, with yellow indicating Cys that A search of structures deposited in the Protein Data Bank by result in a her-1 hypomorph when mutated (19). (B) An alternate view of the using DALI (25) fails to identify unequivocal homologs of HER-1. HER-1 structure is shown with the six sites of non-Cys hypomorphic her-1 Several weak HER-1 homologs were identified, including the mutations indicated (2). (C) Two views of the electrostatic surface potential of acetyl-Lys-recognizing domains (bromodomains) of RNA poly- HER-1, rotated 180° about a vertical axis relative to one another. The color scale is calibrated to Ϫ10 kT per electron and ϩ10 kT per electron for red and merase II initiation factor (26) and histone acetyltransferase blue, respectively. The orientation of the molecule in the right image is

(27). The closest structural match to an extracellular protein is equivalent to the orientation of the molecule in A rotated Ϸ60° about a BIOLOGY

to the lipid transporter apolipophorin-III (28). The quality of the vertical axis. A and B were made with RIBBONS (35) and POV-RAY, and C was made DEVELOPMENTAL structural superpositions (DALI scores Յ 3.3 and rms deviations with GRASP (36).

Hamaoka et al. PNAS ͉ August 10, 2004 ͉ vol. 101 ͉ no. 32 ͉ 11675 Downloaded by guest on September 23, 2021 tably, the HER-1 structure bears no resemblance to Hedgehog for HER-1GM but not TRA-2A. Either HER-1GM does not proteins (29). interact directly with TRA-2A or our ability to detect TRA-2A at the cell surface is masked. For example, a factor coupling Mutation Analysis. Genetic screens have uncovered several mis- TRA-2A to the endocytic pathway may block antibody access to sense mutations that diminish her-1 activity (19). These muta- the C-terminal myc tag. Although we do not consistently detect tions may be placed into three general classes based on the likely TRA-2A at the cell surface, Sokol and Kuwabara (31) readily effects of the resulting amino acid substitutions on the HER-1 observed TRA-2A at the surface of transfected insect cells by structure. Substitution of individual Cys residues is the most using TRA-2A specific antisera. common cause of hypomorphic her-1 phenotypes; changes have HER-1GM binding to TRA-2A expressing cells is not an been found in all disulfides except those between helices 1 and artifact of our transfection procedure. A mutation that results in 2 and helix 2 and a coil in domain 1 (Fig. 2A). These substitutions a single amino acid substitution in the extracellular region of are distributed throughout the HER-1 structure and occur at TRA-2A, R177K, releases TRA-2A from HER-1 inhibition and positions that are substantially buried. A likely explanation for has been postulated to disrupt interactions with HER-1 and map their effects on HER-1 function is that they result in global the interaction site for HER-1 on TRA-2A (11). Consistent with destabilization of the HER-1 structure. this interpretation, the ability of HER-1GM to bind to cells A second class of her-1 mutations results in amino acid expressing TRA-2A bearing the R177K substitution (TRA-2A͞ substitutions that either bury a charged residue (V65D) or occur R177K) is impaired (Fig. 3B). TRA-2A͞R177K is expressed at at the interface between domains 1 and 2 (P50L, P115L, and levels comparable to native TRA-2A, and the inability of S116F) (Fig. 2B, green residues). These mutations are also likely HER-1GM to bind to TRA-2A͞R177K-expressing cells impli- to disrupt the global structure of HER-1. In particular, the cates TRA-2A as the critical factor conferring HER-1GM substitutions that occur at the interdomain interface are likely to binding to cells transfected with native tra-2a. affect the relative orientation of the two subdomains, and the existence of several such substitutions suggests that the relative Regions on HER-1 Important for Mediating Interactions with TRA-2A- orientation of the HER-1 subdomains is important for optimal Expressing Cells. Development of an assay to detect an interaction HER-1 activity. between HER-1 and TRA-2A-expressing cells allowed identifi- Two final substitutions, E77K and E154K, affect residues at cation of regions on the HER-1 surface important for mediating distant sites (Ϸ35 Å apart) on the HER-1 surface (Fig. 2B, this interaction. Seven nonoverlapping clusters of four to five yellow residues). Changes in surface residues are unlikely to alter solvent-exposed residues were selected for Ala substitution the global structure or solubility of HER-1, and the simplest based on inspection of the HER-1GM structure. The clusters explanation for their effects is that they disrupt interactions were selected such that they each form a localized patch on the between HER-1 and a binding partner. HER-1 surface but collectively sample all areas of the HER-1 surface. Clusters rather than individual residues were chosen for Interaction Between HER-1GM and TRA-2A. The integral membrane substitution to allow all surface regions to be targeted with a protein TRA-2A has been suggested as a likely receptor for manageable number of variant proteins. This approach also HER-1 (10, 11), but a direct biochemical interaction between minimizes false negatives that may occur because single amino HER-1 and TRA-2A has not been demonstrated. To investigate acid substitutions at a binding interface often do not significantly this potential interaction, labeled HER-1GM was assayed by perturb binding (31). Variant HER-1GM proteins, each con- immunofluorescence for its ability to bind to TRA-2A- taining a cluster of Ala substitutions, were expressed in Chinese expressing cells. As shown in Fig. 3B, HER-1GM bound effi- hamster ovary cells and assayed for their ability to bind TRA- ciently to HEK 293 cells expressing TRA-2A. The simplest 2A-expressing cells. All mutant sets retained some capacity to explanation for this result is a direct interaction between HER- bind TRA-2A-expressing cells except sets 1 and 2 (Fig. 3D). 1GM and TRA-2A, but the possibility of an indirect interaction Percentages of transfected cells that bound each HER-1GM mediated by either a coreceptor or modification of a different variant are listed Table 1. factor by TRA-2A cannot be ruled out. For example, TRA-2A Mutant sets 1 and 2 map out adjacent regions of the HER- is distantly related to the Hedgehog receptor Patched, which is 1GM surface and coincide with the largest contiguous area of related to proton-driven bacterial transporters and has been surface residues conserved between C. elegans and C. briggsae shown to signal in a catalytic fashion, likely through a transport HER-1 (Fig. 3E). C. elegans and C. briggsae HER-1 share 57% activity (29). amino acid sequence identity and are functionally interchange- To investigate in more detail the nature of the interaction able (12) (Fig. 3E). Residues important for HER-1 activity are between HER-1GM and TRA-2A, the subcellular localization of likely to be conserved between these two species. In contrast, the HER-1GM and TRA-2A was examined by confocal microscopy surface residues encompassed by mutant sets 1 and 2 are not (Fig. 4, which is published as supporting information on the conserved in B. malayi HER-1, which is not active in C. elegans PNAS web site). These images show HER-1GM localized to the or C. briggsae (12). Mutant set 1 also includes Glu-154, which cell surface and intracellular ‘‘spots.’’ HER-1GM almost cer- results in a her-1 hypomorphic phenotype when substituted with tainly interacts initially with a cell-surface component because Lys (19). The E154K substitution is sufficient by itself to cause TRA-2A-expressing cells were exposed to HER-1GM and loss of detectable binding of HER-1GM to TRA-2A-transfected washed several times before permeabilization and labeling of the cells (Fig. 3D). The most likely explanation for these results is cytosolic TRA-2A myc tag. The intracellular HER-1GM seems that mutant sets 1 and 2 map out a region on the HER-1 surface likely to have arisen through endocytosis, but whether endocy- that mediates direct interactions with TRA-2A-expressing cells tosis occurs in nematode cells or plays a role in HER-1 signaling and that substitutions in this region disrupt this interaction. is not certain at this point. TRA-2A staining is strong in HER-1GM with the E77K substitution results in a hypomor- perinuclear regions as well as intracellular spots. Curiously, phic phenotype (19), yet retains most of the ability to recognize strong HER-1GM and TRA-2A staining do not always coincide, TRA-2A-expressing cells (Fig. 3D). Substitution of E77 and four although several intracellular spots stain strongly for both HER- surrounding residues with Ala (mutant set 5) also does not 1GM and TRA-2A. The addition of HER-1GM before perme- severely impair HER-1GM binding to TRA-2A-expressing cells. abilization of cells explains the presence of intracellular regions Because E77 is distant from mutant sets 1 and 2 (Fig. 3C), these that stain only for TRA-2A, but it is more difficult to explain results suggest that E77 may mediate a functionally important regions, primarily at the cell surface, that appear to stain strongly interaction with a component other than TRA-2A.

11676 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0402559101 Hamaoka et al. Downloaded by guest on September 23, 2021 Fig. 3. HER-1GM interacts with TRA-2A-expressing cells. (A) A schematic diagram of the experimental design is shown with details described in Materials and Methods. Briefly, a green-fluorescing antibody locates TRA-2A, and a red-fluorescing antibody locates HER-1GM. (B) Cells transfected with either the WT tra-2a sequence (Upper)ortra-2a R177K (Lower) were incubated with HER-1GM-conditioned media. TRA-2A-expressing cells were labeled with a green fluorophore- conjugated antibody, HER-1 was labeled with a red fluorophore-conjugated antibody, and nuclei were stained with 4Ј,6-diamidino-2-phenylindole (DAPI), which fluoresces blue. (C) The HER-1GM surface is shown. Sites of residues substituted in variant HER-1GM proteins are indicated in color (red, set 1; green, set 2; magenta, set 3; purple, set 4; blue, set 5; yellow, set 6; orange, set 7). Mutant sets 1 and 5 include E154 and E77, respectively, which have previously been shown to reduce HER-1 activity when substituted with Lys (19). (D) Binding of each of the variant HER-1 proteins to TRA-2A-expressing cells as assayed by

immunofluorescence. The mutant set being assayed is indicated in the bottom left corner of images in the second column; the residues substituted in each set BIOLOGY

are listed in Table 1. (E) Dark-purple regions indicate sites of surface residues that are conserved among C. elegans, C. briggsae, and B. malayi HER-1. Pink regions DEVELOPMENTAL indicate sites of residues that are conserved only between C. elegans and C. briggsae.

Hamaoka et al. PNAS ͉ August 10, 2004 ͉ vol. 101 ͉ no. 32 ͉ 11677 Downloaded by guest on September 23, 2021 Analysis of the electrostatic surface potential of HER-1GM for HER-1 but do not rule out a role for heparin in either HER-1 reveals a region of concentrated positive charge adjacent to the binding to cells or HER-1-mediated signaling. surface mapped out by mutant sets 1 and 2 (Figs. 1 and 2C). The HER-1GM structure reported here shows that HER-1 is Lys-67, Lys-81, Arg-147, and Lys-148 make up this region of not structurally homologous to any known signaling molecule positive charge, which is conserved in C. briggsae but not B. and provides a basis for interpreting the effects of her-1 missense malayi HER-1 (19). Many signaling molecules involved in mutations. An immunofluorescence assay for HER-1 binding to development, including Wnt, Hedgehog, and fibroblast growth TRA-2A-expressing cells was developed and, coupled with the factors, exhibit physiologically important interactions with hep- HER-1GM structure, used to identify a conserved region on the arin (33), and the region of positive charge on HER-1GM raises HER-1 surface important for mediating this interaction. These the possibility that such an interaction might also be important results are consistent with a direct interaction between HER-1 for HER-1 function. Attempts to demonstrate a role for heparin and TRA-2A but do not rule out an indirect mechanism in mediating interactions between HER-1 and TRA-2A proved involving, for example, a coreceptor or modification of another inconclusive, however. HER-1GM binds to a heparin Sepharose factor by TRA-2A. column in physiological salt concentrations but elutes in 450 mM NaCl, a salt concentration somewhat below what is needed to We thank M. Perry and P. Kuwabara for providing cDNAs encoding HER-1 and TRA-2A, respectively; K. Ramyar and J. Beneken for help elute proteins known to bind heparin with high affinity (34). in early stages of this project; R. Abramowitz, C. Ogata, and X. Yang for Moreover, binding of HER-1GM to TRA-2A-expressing HEK assistance at Beamline X4A of the National Synchrotron Light Source 293 cells is not inhibited by 500 ␮g͞ml heparin (data not shown). at Brookhaven National Laboratories, and S. Bouyain, G. Seydoux, and These results suggest that cellular heparin is not a primary ligand W. Yang for comments on the manuscript.

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