Article pubs.acs.org/biochemistry
EF5 Is the High-Affinity Mg2+ Site in ALG‑2 John J. Tanner,†,‡ Benjamin B. Frey,† Travis Pemberton,‡ and Michael T. Henzl*,† †
Department of Biochemistry, University of Missouri, Columbia, Missouri 65211, United States Department of Chemistry, University of Missouri, Columbia, Missouri 65211, United States
‡
ABSTRACT: The penta-EF-hand (PEF) protein ALG-2 (apoptosis-linked gene 2) has been implicated in several important physiological processes, including endoplasmic reticulum−Golgi vesicular transport and endosomal biogenesis/transport. ALG-2 was recently shown to harbor a metal ion-binding site with a high affinity for Mg2+ and a low affinity for Ca2+. We herein present the X-ray structure of Mg2+-bound ALG2des23wt. Although the Cα trace is nearly indistinguishable from that of the Ca2+-free protein, the orientation of the C-terminal helix differs in the two structures. Consistent with that observation, replacement of the +x ligand in EF5, D169, with alanine eliminates high-affinity Mg2+ binding. It also eliminates the low-affinity Ca2+ site and lowers the affinity of the remaining Ca2+-binding sites, EF3 and EF1. The coordination environment in EF5 approaches ideal Mg2+ octahedral geometry. The ligand array, consisting of three carboxylates (+x, +y, +z), a backbone carbonyl (−y), and two water molecules (−x, −z), may offer a recipe for a high-affinity, high-selectivity Mg2+-binding site. Sequence data for other PEF proteins indicate that select calpain large subunits, notably CAPN1 and CAPN8, may also possess a high-affinity Mg2+-binding site. In Mg2+-bound ALG-2, the carbonyl of F188 and the C-terminal carboxylate of V191 interact with the ε-ammonium group of K137 in the opposing subunit, suggesting that Mg2+ binding could have an impact on dimerization. Interestingly, EF1 and EF3 are also occupied in the crystal, despite having modest affinity for Mg2+. The results of a calorimetry-based analysis indicate that their Mg2+ binding constants are 2 orders of magnitude lower than that determined for EF5. EF-hand proteins1−6 participate in numerous eukaryotic signal transduction pathways.7−9 The “EF-hand” motif includes a metal ion-binding loop and flanking helical segments, the spatial orientation of which can be suggested by the right hand. Although the motifs typically occur as tandem pairs, the dimeric penta-EF-hand (PEF) proteins10 possess an unpaired Cterminal EF-hand, which serves as a dimerization domain. The PEF protein ALG-2 (short for apoptosis-linked gene 2)11−13 was discovered during a search for gene products that conferred protection from ligand-induced apoptosis in a T-cell hybridoma.14 Highly conserved, ALG-2 displays a broad tissue distribution14,15 and is found in the nucleus and cytoplasm.16,17 Two isoforms are expressed in vivo.13,15 The more abundant one, often denoted ALG-2wt, includes 191 residues. Roughly one-third of the time, an alternative splicing event excises the codons for G121 and F122, yielding ALG-2ΔGF122. The two isoforms can form a heterodimeric complex.15 Ca2+ binding triggers exposure of apolar surface, for interaction with target proteins. ALG-2 is proposed to function as an adaptor molecule, bridging unrelated proteins or stabilizing weak protein−protein complexes. The addition of Ca2+ to cell lysates promotes translocation of ALG-2 to the membranous fraction, implying association with membranelocalized proteins.13,18 A majority of putative biological targets identified to date harbor specific proline-rich regions (PRRs) known as ALG-2-binding motifs (ABMs). The ALG-2 isoforms exhibit distinct target protein specificities. Whereas ALG-2wt associates with either ABM-1 (PPYPXXPGYP) or ABM-2 (PXPGF) motifs, ALG-2ΔGF122 interacts exclusively with ABM2.15,19,20 © 2016 American Chemical Society
Although its name would suggest otherwise, ALG-2 activity can promote either cell death or cell proliferation.11 The influence on cell survival pathways is probably an indirect consequence of its involvement in several important physiological processes. These include (1) endoplasmic reticulum (ER)−Golgi vesicular transport, through interactions with Sec31a and annexin A11,21−27 (2) endosomal biogenesis and transport, via interactions with Alix/AIP128,29 and Tsg101,30 and (3) cell membrane repair,31 likewise via interaction with Alix/AIP1. Curiously, elimination of the ALG-2 gene has no apparent physiological impact: ALG-2−/− mice develop normally and display no obvious immune defect.32 45 Ca2+ flow-dialysis measurements revealed that both ALG-2 isoforms possess two high-affinity Ca2+ sites and one lowaffinity site.15 EF1 and EF3 are the high-affinity sites,33,34 and EF5 is the low-affinity site. A one-residue insertion in the EF5binding loop prevents the glutamyl residue at the C-terminal end of the loop from serving as the canonical bidentate Ca2+ ligand. Inclusion of 2 mM Mg2+ in the flow-dialysis assays had no discernible impact on Ca2+ affinity in either isoform, suggesting that the ALG-2 Ca2+-binding sites are specific for Ca2+. However, a recent ITC study convincingly demonstrated that both ALG-2 isoforms possess a high-affinity Mg2+ site.35 We herein describe the X-ray structure of ALG-2des23wt crystallized in the presence of 1.0 mM Mg2+. The tertiary structure of the Mg2+-bound molecule closely resembles that of Received: June 12, 2016 Revised: August 18, 2016 Published: August 19, 2016 5128
DOI: 10.1021/acs.biochem.6b00596 Biochemistry 2016, 55, 5128−5141
Article
Biochemistry
were harvested with loops and flash-cooled by rapid immersion in liquid nitrogen. X-ray Diffraction Data Collection and Processing. Xray diffraction data were collected at beamline 4.2.2 of the Advanced Light Source, using a CMOS-based Taurus-1 detector in shutterless mode. The data set used for refinement, collected at a detector distance of 210 mm, consisted of 1800 frames spanning 180°. The total exposure time was 360 s. Data were integrated and scaled with XDS.37 Intensities were merged and converted to amplitudes with Aimless.38 ALG-2des23wt crystallized in space group P21212, with the following unit cell dimensions: a = 76.5 Å, b = 48.5 Å, and c = 54.3 Å. Because the data were initially processed assuming a < b < c, the standard convention for primitive orthorhombic space groups, the REINDEX module of ccp4i39 was used to reindex the reflections so that the 54.3 Å axis corresponds to the c-axis, i.e., transformation of (h,k,l) to (l,h,k). Refinement. Refinement with PHENIX40 was initiated with the coordinates from a structure of N-terminally truncated human ALG-2 [Protein Data Bank (PDB) entry 2ZND] that likewise crystallized in P21212, the same cell used here for mouse ALG-2des23wt. The first round of refinement used rigidbody refinement and simulated annealing. The B-factor model consisted of one TLS group encompassing the entire protein chain and an isotropic B-factor for each non-hydrogen atom. All atoms have an occupancy of 1.0. Estimation of the Uncertainties of Bond Lengths and Bond Angles. The uncertainties in the bond lengths and angles for the Mg2+ sites were estimated from structures refined against six diffraction data sets collected from four crystals (Table 5). The six data sets included the one used to generate the deposited structure (Table 1) and five others having highresolution limits between 1.75 and 1.80 Å (Table 5). For each data set, the refined 1.72 Å resolution structure (see Table 1) was used to initiate 10 independent simulated annealing refinement calculations. Prior to each refinement calculation, the coordinates of each atom were perturbed by applying a random shift, using the “shake” option of phenix.pdbtools (mean displacement parameter set to 0.4 Å). The 60 resulting structures were used to estimate standard deviations for the bond lengths and angles. Sedimentation Velocity Analyses. ALG-2des23wt and the D169A variant were analyzed by sedimentation velocity at 20 °C in a Beckman XL-I Optima analytical ultracentrifuge. Aliquots of protein (400 μL) and buffer (430 μL), in 0.15 M KCl, 0.025 M Hepes, and 1.0 mM EDTA (pH 7.4), were loaded into the sample and solvent chambers, respectively, of a sedimentation velocity cell, equipped with a charcoal-Epon dual-sector centerpiece. The absorbance of the sample was 0.80 at 280 nm, in a 1.0 cm cuvette. Assuming a molar absorptivity of 36100 M−1 cm−1, the nominal protein (monomer) concentration was 22 μM. Following temperature equilibration, the sample was centrifuged at 30000 rpm, with data being acquired continuously until 300 radial scans had been collected. The resulting data set was analyzed globally with Sedfit to obtain the sedimentation coefficient, c(s), and molecular weight, c(M), distributions. Urea Denaturation Studies. The impact of Mg2+ on the apparent conformational stability was evaluated by titrating samples of ALG-2des23wt and D169A with urea at 25 °C, in the absence or presence of 1.0 mM Mg2+. Unfolding was monitored by circular dichroism in a 1.0 cm cuvette, employing an AVIV 202 spectrometer, equipped with a Hamilton Microlab 500
the apoprotein structure reported previously.36 The most prominent structural difference appears in the C-terminal helix, suggesting that EF5 is the high-affinity Mg2+ site. Consistent with that hypothesis, replacement of D169 (full-length ALG-2wt numbering system) with alanine abolishes high-affinity Mg2+ binding, eliminates the low-affinity Ca2+ site, and attenuates the Ca2+ affinity at the two remaining sites. The structural changes that accompany the binding of Mg2+ in EF5 could potentially influence the kinetics and/or energetics of dimerization. Because the dimeric structure of ALG-2 is integral to its proposed adaptor function, Mg2+ binding could modulate ALG2 activity. EF1 and EF3 are also occupied by Mg2+ in the crystal. The unanticipated presence of Mg2+ in these low-affinity sites prompted an effort to obtain estimates for their Mg2+ binding parameters, employing an ITC-based analysis of competitive Ca2+ and Mg2+ binding.
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MATERIALS AND METHODS Reagents. These items were purchased from Fisher Scientific: ampicillin, CaCl2·H2O, MgCl2·2H2O, Na2EDTA· 2H2O, glycerol, Hepes, KCl, lysozyme, and 2-propanol. The following were obtained from Sigma-Aldrich: chloramphenicol, EGTA, NTA, 1.0 M Tris buffer solutions, Tween 20, and highpurity (>99.5%) urea. IPTG was obtained from Gold Biotechnology. LB broth capsules were purchased from Research Products International. Mutagenesis. The coding sequence for ALG-2des23wt, an ALG-2wt construct lacking residues 1−23, had previously been cloned into pET11. Asp 169 (full-length ALG-2wt sequence numbering) was replaced with alanine using the QuikChange mutagenesis kit (Agilent), employing oligonucleotides purchased from Integrated DNA Technologies (Coralville, IA). The fidelity of the mutated sequence was confirmed by automated DNA sequencing at the University of Missouri DNA Core Facility. Protein Expression and Isolation. Rosetta 2(DE3) cells (Novagen) harboring the construct of interest (ALG-2des23wt or ALG-2des23wt/D169A) were incubated at 37 °C in LB broth containing ampicillin (100 μg/mL) and chloramphenicol (30 μg/mL). When the absorbance at 600 nm reached 0.6, the 1 L cultures were chilled to approximately 20 °C in an ice−water bath. IPTG was added to a final concentration of 0.25 mM, and the cultures were maintained at 23 °C while being shaken for an additional 16 h. The bacteria were collected by centrifugation, resuspended in 20 mM Hepes (pH 7.4), and lysed by being treated with lysozyme and extrusion from a French pressure cell. The proteins were purified to apparent homogeneity, as assessed by SDS−PAGE, employing the procedure described by Lo et al.33 Crystallization. Crystals of ALG-2des23wt were grown at 4 °C in hanging drops, using 24-well VDX plates (Hampton Research). The precipitant contained 2-propanol [29−32% (v/ v)], 0.10 M Tris-HCl (pH 7.4, 25 °C), 1.0 mM EGTA, and 1.0 mM Mg2+. The inclusion of EGTA prevented binding of (contaminating) Ca2+ to the EF-hand motifs. The drops were prepared by combining 2.0 μL of ALG-2des23wt [5.0 mg/mL in 10 mM Hepes-KOH (pH 7.4)] with 2.0 μL of the precipitant solution. The crystals, which appeared in 2−4 days, were pulverized and used to seed additional wells, resulting in larger crystals. The latter were prepared for low-temperature data collection by being transferred to a cryobuffer (precipitant containing 25% glycerol) at 4 °C. The cryoprotected crystals 5129
DOI: 10.1021/acs.biochem.6b00596 Biochemistry 2016, 55, 5128−5141
Article
Biochemistry Table 1. Data Collection and Refinement Statisticsa beamline space group unit cell parameters (Å) wavelength resolution (Å) no. of observations no. of unique reflections Rmerge(I) Rmeas(I) Rpim(I) mean CC1/2 mean I/σ completeness (%) multiplicity no. of protein residues no. of atoms/ions protein Mg2+ water Rcryst Rfreeb rmsd for bond lengths (Å) rmsd for bond angles (deg) Ramachandran plotc (%) favored outliers Clashscore (percentile)c MolProbity score (percentile)c average B (Å2) protein Mg2+ water coordinate error (Å)e PDB entry
absence and presence of chelators (EDTA, EGTA, and NTA). The resulting data were analyzed globally, using the binding parameters for the small-molecule chelators reported previously.35 As described elsewhere,42,43 the strategy used to calculate the heat associated with the ith titrant injection involves estimation of the free divalent ion concentration(s), calculation of the cumulative binding enthalpy after the ith injection, and subtraction of the cumulative binding enthalpy associated with the previous injection. The D169A mutation abolishes high-affinity Mg2+ binding. Because it also eliminates the low-affinity Ca2+ site, the Ca2+ binding behavior of the resulting protein can be described with a two-site Adair model. Estimation of the free Ca 2+ concentration was achieved by minimizing the following FC function, employing a bisection strategy:
ALS 4.2.2 P21212 a = 76.5, b = 48.5, c = 54.3 1.0000 54.35−1.72 (1.75−1.72) 152354 22193 0.045 (1.014) 0.049 (1.103) 0.018 (0.429) 1.000 (0.770) 25.8 (1.6) 100.0 (100.0) 6.9 (6.5) 168
FC = [Ca 2 +] + n[P]t ⎛ K [Ca 2 +] + 2K K [Ca 2 +]2 ⎞ Ca,1 Ca,2 Ca,1 ⎜⎜ ⎟ − [Ca 2 +]t 2+ 2+ 2 ⎟ ⎝ 1 + K Ca,1[Ca ] + K Ca,2K Ca,1[Ca ] ⎠
1398 3 106 0.1797 (0.3082) 0.2075 (0.3279) 0.006 0.694
where KCa,1 and KCa,2 are stepwise macroscopic binding constants for the first and second Ca2+ binding events, respectively, [Ca2+]t and [P]t represent the total Ca2+ and protein concentrations, respectively, and n is a stoichiometric factor that allows for uncertainty in the protein concentration. In the presence of a low-molecular weight chelator (e.g., EGTA), FC assumes the following form:
100.00 0.00 0.72 (99th) 1.05 (100th)
2+ ⎛ K ⎞ EGTA [Ca ] ⎟ FC = [Ca 2 +] + [EGTA]t ⎜ 2+ ⎝ 1 + KEGTA[Ca ] ⎠
29.6 26.9/26.5/27.2d 37.7 0.20 5JJG
⎛ K [Ca 2 +] + 2K K [Ca 2 +]2 ⎞ Ca,1 Ca,2 Ca,1 ⎟ + n[P]t ⎜⎜ 2+ 2+ 2 ⎟ ⎝ 1 + K Ca,1[Ca ] + K Ca,2K Ca,1[Ca ] ⎠
a
− [Ca 2 +]t
Values for the outer resolution shell of data are given in parentheses. Random 5% test set. cGenerated with MolProbity.51 dValues are listed as EF1, EF3, EF5. eMaximum likelihood-based coordinate error estimate from PHENIX refine. b
where [EGTA]t is the total EGTA concentration and KEGTA is the Ca2+ binding constant for EGTA. Given an estimate for [Ca2+], the cumulative binding heat after the ith injection (Qi) is calculated with the following equation, where ΔHCa,1 and ΔHCa,2 represent the molar enthalpy changes associated with the binding events:
automated titrator. The protein samples (3.0 μM) and the 10.0 M urea titrant were prepared in 0.15 M KCl, 0.010 M potassium phosphate, and 0.5 mM EGTA (pH 7.4) in the presence or absence of 1.0 mM Mg2+. The titrant concentration was confirmed by refractometry. Titrations were conducted in a constant volume of 2.0 mL. Following each addition of titrant, the sample was stirred for 60 s, and the ellipticity at 222 nm was then measured for 30 s. Isothermal Titration Calorimetry. All ITC data were collected at 25 °C with a VP-ITC instrument (Malvern Instruments). The experiments were conducted in 0.15 M KCl, 0.025 M Hepes (pH 7.4), and 0.5% (v/v) Tween 20. As described previously,35 because Tween 20 is susceptible to hydrolysis, the detergent was added to the samples just prior to the titration. Divalent metal ions were removed from the protein preparations before ITC analysis by passage over EDTA-agarose.41,42 The residual Ca2+ content, measured by flame atomic absorption at 422.7 nm, was
