Structure of the EMMPRIN N‐terminal domain 1: Dimerization via β‐strand swapping (2009)

Extracellular matrix metalloproteinase inducer (EMMPRIN), also known as Hab18G, CD147, Basigin, M6, and neurothelin, is a membrane glycoprotein expressed on the surface of various cell types and many cancer cells. 1 EMMPRIN stimulates adjacent fibroblasts and tumor cells to produce matrix metalloproteinases and plays an important role in tumor invasion and metastasis, angiogenesis, spermatogensis and fertilization, cell–cell adhesion and communication, and other biological processes (reviewed in Ref. 1 and references therein). It was demonstrated that the EMMPRIN extracellular domain (ECD), which structurally belongs to the IgG superfamily, can form homo-oligomers in a cis dependent manner and the N-terminal domain 1 (residues 22–101) was necessary and sufficient to mediate this interaction.2 The crystal structure of the ECD of recombinant human EMMPRIN (Hab18G/CD147) expressed in E. coli was reported at 2.8 Å resolution (Yu et al. 2008). 3 The construct consists of residues 22–205 of the mature protein and has both an N-terminal IgC2 domain (ND1, residues 22–101) and a C-terminal IgC2 domain (ND2, residues 107–205). The two domains are joined by a five amino acid residue linker that constitutes a flexible hinge between the two domains. The crystal form has four copies of the molecule in the asymmetric unit, each of which has a different inter-domain angle that varies from 121° to 144°.3 The two domains each have a conserved disulfide bridge and both are comprised of two β-sheets formed by strands EBA and GFCC′, and DEBA and A′GFCC′ for ND1 and ND2, respectively. Based on the crystal packing in this structure, the authors proposed that lateral packing between the two IgG domains of EMMPRIN ECD represents a potential mechanism for cell adhesion. Here we report the 2.0-Å crystal structure of the N-terminal domain of EMMPRIN ECD (ND1) expressed in mammalian cells. The overall structure of the domain is very similar to that in the full length ECD. Quite unexpectedly, ND1 forms a dimer mediated through the exchange of its last β-strand (strand G). β-strand swapping, which is a subset of 3D domain swapping, has been found to mediate cell–cell adhesion by cadherins. 4 3D domain swapping has been proposed to be a mechanism of protein oligomerization, aggregation, evolution of oligomeric proteins from single domains5 and amyloidogenesis.6, 7 In domain swapped proteins, the same structural elements are involved in the final 3D structure, and so there is little overall energetic difference between the monomer and the swapped oligomers. However, there is often a high energy barrier for the conversion as it often goes through an unfolded state.8 It is also possible that strand-swapping occurs during folding of nascent polypeptide chains. Frequently, the exchange hinges contain proline-rich motifs which are often in high strain conformations.9 Domain swapping appears to be a strategy to resolve such local structural strain. The exchange hinge of ND1 contains a Pro-Glu-Pro tripeptide motif. Both of the proline residues adopt extended trans conformations, when compared with cis in the full-length ECD structure. Proline cis–trans isomerization may be the driving force for this exchange. Strand-exchanged dimerization may be a mechanism for the oligomerization of EMMPRIN ECD and its cis-dependent homophilic interactions in cell–cell adhesion. CMV, cytomegalovirus; HPLC, high performance liquid chromatography; IMAC, immobilized metal ion affinity chromatography; MPD, 2-methyl-2,4-pentanediol; PBS, phosphate buffered saline; Qq/TOF, quadrapole time-of-flight; SLS, static light scattering; UV, ultraviolet. The sequence of the human EMMPRIN (CD147) Ig-like CH2-type domain (residues 1–103) was engineered with a C-terminal His6 tag and subcloned into the pCEP4 (Invitrogen, San Diego, CA) vector, which was modified to contain the CMV intron A immediately following the CMV promoter. This construct (p3995) contains an A19D polymorphic mutation in the signal peptide sequence and an N44D mutation to remove the only N-linked glycosylation site. This N-terminal domain (ND1) was expressed transiently in HEK293 cells. The protein was purified from the supernatant using a Talon/IMAC column and by size exclusion chromatography (HiLoad 26/60 Superdex 200 column, GE Healthcare, Piscataway, NJ). Before crystallization trials, ND1 was further purified by anion exchange chromatography using a Mono Q 5/50 GL column (GE Healthcare, Piscataway, NJ) equilibrated with 20 mM Tris, pH 8.5, 10% glycerol (buffer A) and eluted with 20 mM Tris, pH 8.5, 10% glycerol, 1 M NaCl (buffer B) with a linear gradient of 6–20% buffer B in 60 column volumes. The purified ND1 was concentrated to 11.7 mg/mL in 20 mM Tris, pH 8.5, 10% glycerol, 100 mM NaCl. The molecular mass of ND1 was analyzed using HPLC-Qq/TOF mass spectrometry (Waters Acquity UPLC coupled with a Waters Q-TOF API US MS). The HPLC was run at a flow rate of 150 μL/min (solvent A was 0.1% formic acid in water and solvent B was acetonitrile with 0.1% formic acid), and the effluent passed directly into the Qq/TOF mass spectrometer post-UV detection. The spray voltage was 3.0 kV and the cone was set at 35 V. Two microliters (200 pmol) of the EMMPRIN ND1 proteins (1.0 mg/mL) were immobilized on a ProSorb cartridge (Applied Biosystems, Foster City, CA) and desalted with 600 μL of 0.1% trifluoroacetic acid (TFA). The protein sequences were obtained for 15 cycles using an ABI Procise 494HT instrument with standard protocols without addition of Biobrene. Crystallization of ND1 was carried out by the vapor-diffusion method in hanging and sitting drops at 20°C. ND1 protein (∼11 mg/mL) with and without the anion exchange purification were mixed in 1:1 volume ratio with crystallization reservoirs and equilibrated over the reservoirs. Two crystal forms were observed. Form I crystals grew from 4.8 M sodium formate, 5% MPD in 0.1 M MES, pH 6.5 and from 0.1 M sodium citrate, pH 3.5, 3.2 M sodium formate and 3% MPD with seeds from the higher pH conditions. Form II crystals of the anion-exchange purified ND1 grew from 4.2 M sodium formate, 3% MPD in 0.1 M sodium citrate buffer, pH 3.5 without seeding. The crystals were harvested and flash frozen in the mother liquor in liquid N2 or the cold stream taking advantage of the high sodium formate concentration as cryo-protectant. 10 The X-ray diffraction data for crystal Form I were collected at Advanced Photon Source beamline 17ID with ADSC CCD TH7899 detector (wavelength 1.0 Å) and processed with HKL2000 (HKL Research, VA).11 The X-ray diffraction data for crystal Form II were collected using the Rigaku MicroMax-007HF microfocus X-ray generator equipped with an Osmic VariMax confocal optics, Saturn 944 CCD detector, and an X-stream 2000 cryocooling system (Rigaku, Woodlands, TX). The X-ray data were processed with the program D*TREK (Rigaku, Woodlands, TX).12 The crystal data statistics are given in Table I. The structure of crystal Form I was determined by molecular replacement (MR) using Phaser 14 with the N-terminal domain of the EMMPRIN ECD structure (PDB ID 3B5H)3 as the search model. The structure of Form II was determined in a similar way with the ND1 dimer from Form I as the search model. Structural models were refined with PHENIX15 without noncrystallographic symmetry restraints to the two ND1 molecules. Water molecules were added at the (Fobs − Fcalc) electron density peaks using the cutoff level of 5σ. Model adjustments were carried out using the program COOT16 with integrated Molprobity clash checking.13 The crystal data and refinement statistics are given in Table I. All molecular graphics figures were generated with PyMOL.17 The atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 3I84 and 3I85. The ND1 samples at ∼1.0 mg/mL at pH 3.5 and ∼1.0mg/mL at pH 7.5 were fractionated by size exclusion exchange chromatography (SEC) on a Superdex 75 (GE Healthcare, Piscataway, NJ) column in phosphate buffered saline (PBS) at pH 7.5 or tri-sodium citrate at pH 3.5, monitored by UV absorbance at 280 nm using an Agilent MWD detector and the refractive index at 685nm using an OptiLab differential refractometer (Wyatt Technology Inc., Santa Barbara, CA). The molecular weights were determined by static light scattering measurements at 690 nm, using a DAWN-EOS MALS detector (Wyatt Technology Inc, Santa Barbara, CA). Mass spectrometry and N-terminal sequencing data show that the expressed ND1 is composed of two main species with N-termini of L13LGTHGDSGAAGT25 (MWT 10,628.0 Da) and D19SGAAGT (9,792.9 Da). These N-termini are longer (9 and 3 residues, respectively) than the mature N-terminus (A22AGT25) of the native EMMPRIN protein. Apparently, the mutation of A19D in the signal peptide affected processing of the pre-protein in the cell. Signal peptides are generally weakly conserved stretches of 13–36 amino acids of the amino terminus of a pre-protein. The fortuitous mutation A19D thus appears to have resulted in a very short signal recognition sequence of 12 amino acids. SignalP 18 predicts the second, but not the first species. There are two ND1 molecules in the asymmetric unit of both the P6122 (Form I) and the P3221 (Form II) crystal forms. The overall structures in both forms are virtually identical. Thus, the following description is based on the 2 Å resolution structure of Form I. Molecule A, comprised of residues 23–102, is well defined in the electron density with the exception of residue 47. For molecule B, additional density for L13LGTHG18 was clearly visible in Form I and L13LGT16 was also visible in Form II. The two monomers are nearly identical with a backbone rmsd of 1.21 and 0.68 Å for Forms I and II, respectively. The overall structure of the ND1 is very similar to an IgG C2 domain and to that in the full-length ECD of EMMPRIN (see Fig. 1), as reported 3 with the exception of a short segment G58GVVLKED65, which appears to be mistraced. This region in the structure of the full ECD is out of register by one residue. Pair-wise root mean square differences for the backbones range from 0.82 to 1.56 Å. The two independent monomers in the crystal form a dimer with the last β strand (residues 93–102) of each ND1 domain exchanged, i.e. ND1 is a strand exchanged swap dimer (see Fig. 1). The exchange point is the P91EP93 which forms a β turn in the full-length ECD with P91 and P93 both in the cis conformation.3 However, in the ND1 structures, this region is extended with both P91 and P93 adopting the trans conformation. A, Molecular structure of ND1 strand swapped dimer. Molecules A and B of ND1 are colored green and cyan, respectively. The signal sequence segment of Mol B is colored red. B, The N-terminal domain of full length EMMPRIN (PDB ID 3B5H) is overlaid on Mol A and shown in gold. The residues of 58–65 are colored blue in panels A and B. For the un-swapped ND1 of 3B5H, this segment was replaced with the same segment ofthe current ND1 structure. The domain swap hinge P91EP93 is colored magenta on the dimer and unswapped monomer. C, A schematic of β-strand swapping of ND1. One interesting feature of the crystal lattices is that the signal peptide fragment (L13LGTHG18) mediates crystal packing (see Fig. 2). The L14GT16 segment of this short peptide adopts a β-strand bridging the parallel C′ strands of the GFCC′ sheets of symmetry related ND1 dimers. Residues 19–23 are disordered in both protomers in the two crystal forms. This suggests that the signal peptide fragment would most likely be disordered in solution and thus would have no impact upon the dimer formation of the ND1. It is interesting that we have not obtained crystals of ND1 species lacking the LLGTHG sequence, consistent with its role in promoting crystal lattice formation. Signal peptide mediated lattice packing. Two symmetry-related dimers are shown (magenta/purple, Mol A and B, and cyan/green, Mol A′ and B′). The ordered parts of the signal peptide, strand A′ (L13LGTHG18) are colored red. This short β-strand mediates the GFCC′ sheets of symmetry related dimers to form a larger β sheet. A noncrystallographic 2-fold is perpendicular to the paper in the center. The crystals of ND1 were obtained from conditions of low pH (3.5–4.5). To investigate whether strand-exchange was induced by low pH, we performed SEC followed by molecular weight determination by static light scattering (SLS) at pH 7.5 and 3.5 and at several different concentrations. The SEC-SLS data are shown in Figure 3. ND1 elutes as slightly polydispersed molecular species of 13–16 kDa at pH 7.5 and ∼11 kDa at pH 3.5 with mass recovery of 80–99%. The size of the molecular species observed is fairly constant and therefore independent of ND1 sample load on SEC between 0.1 and 1.0 mg/mL. The decrease in apparent molecular size of ND1 at pH 3.5 suggests that lowering the pH does not cause dimerization of the starting ND1 protein, which would correspond to an increase in the apparent molecular weight. Thus, there is no evidence of dimerization under the conditions of the static light scattering experiments. SEC-SLS of elution profiles of ND1 at pH 7.5 and 3.5. The ND1 protein was injected at 1.0 mg/mL onto Superdex-75 in PBS pH 7.5 or in 20 mM sodium citrate pH 3.5, 100 mM NaCl. Results show an average MW 13–16 kDa at pH 7.5 and 11 kDa at pH 3.5 based on static light scattering (SLS). The estimated molecular mass (g/mol) and protein concentration (RI, refractive index in volts) are labeled on the left and right axes, respectively. The exchange hinge in ND1 is composed of two proline residues with an intervening glutamate (P91EP93). Both of the Pro residues adopt the cis conformation in the full-length ECD structure, 3 whereas they are both in trans conformation in ND1. The cis–trans proline isomerization, a very low energy barrier, is a relatively slow process that can affect the protein folding pathway. For example, the cell-cycle regulatory protein suc1 exists in both the monomer and the strand exchanged dimer forms.19, 20 The hinge of suc1 contains the P90EP92 sequence, and it was found to be critical for the strand-exchanged dimer formation by mutational analysis.8 Both Pro residues are in the trans conformations in the exchanged dimer, whereas the coordinates for the monomer form were not deposited. Thus, similar to suc1, the P91 and P93 in ND1 are likely to be critical for the strand exchange and cis–trans isomerization of the hinge proline residues may indeed be the driving force. The EMMPRIN ECD domain has been shown to form oligomers in a cis-dependent fashion on the same cell and the ND1 is sufficient for this interaction. 2 Yu et al. suggested that the crystal packing interactions between ND1 are the likely mechanisms of EMMPRIN oligomer formation.3 Here we suggest that ND1–ND1 might form strand swapped dimers on the plasma membrane, which can then make use of the lateral β-strand packing to form higher order oligomers, both on the same cell or across cells, consistent with its role in oligomer formation on the cell surface2 and also serving as its own receptor in homophilic interactions.21 The authors thank Mark Cunningham, Juliane Mills, and Yi Tang for their technical assistance and discussion, and Marta Abad for assistance in synchrotron data collection.