evolutionaryandstructuralanalysesofmammalianhaloacid ...the first aspartate in this had motif i acts...

Evolutionary and Structural Analyses of Mammalian HaloacidDehalogenase-type Phosphatases AUM and ChronophinProvide Insight into the Basis of Their Different SubstrateSpecificities*

Received for publication, July 18, 2013, and in revised form, December 10, 2013 Published, JBC Papers in Press, December 13, 2013, DOI 10.1074/jbc.M113.503359

Annegrit Seifried‡§, Gunnar Knobloch‡§, Prashant S. Duraphe§1, Gabriela Segerer‡, Julia Manhard‡,Hermann Schindelin§, Jörg Schultz¶, and Antje Gohla‡§2

From the ‡Institute for Pharmacology and Toxicology, §Rudolf Virchow Center for Experimental Biomedicine, University ofWürzburg, 97080 Würzburg and the ¶Biocenter, Department of Bioinformatics, University of Würzburg, 97074 Würzburg, Germany

Background: Substrate specificity determinants of mammalian haloacid dehalogenase (HAD) phosphatases are poorlyunderstood.Results: AUM (aspartate-based, ubiquitous, Mg2�-dependent phosphatase) is a novel tyrosine phosphatase and paralog of theserine/threonine- and pyridoxal 5�-phosphate phosphatase chronophin.Conclusion: Conserved cap residues in AUM or chronophin determine phosphatase substrate specificity.Significance: These findings provide a starting point for structure-based development of HAD phosphatase inhibitors.

Mammalian haloacid dehalogenase (HAD)-type phosphatasesare an emerging family of phosphatases with important functionsin physiology and disease, yet little is known about the basis of theirsubstrate specificity. Here, we characterize a previously unex-plored HAD family member (gene annotation, phosphoglycolatephosphatase), which we termed AUM, for aspartate-based, ubiqui-tous, Mg2�-dependent phosphatase. AUM is a tyrosine-specificparalog of the serine/threonine-specific protein and pyridoxal5�-phosphate-directed HAD phosphatase chronophin. Compara-tive evolutionary and biochemical analyses reveal that a single, dif-ferently conserved residue in the cap domain of either AUM orchronophin is crucial for phosphatase specificity. We have solvedthe x-ray crystal structure of the AUM cap fused to the catalyticcore of chronophin to 2.65 Å resolution and present a detailed viewof the catalytic clefts of AUM and chronophin that explains theirsubstrate preferences. Our findings identify a small number of capdomain residues that encode the different substrate specificities ofAUM and chronophin.

Haloacid dehalogenase (HAD)3-type phosphatases (1– 4)constitute an emerging family of at least 40 human enzymes

with important functions in physiology and disease (5–12).Although these enzymes can specifically dephosphorylate verydiverse substrates, ranging from metabolites, lipids, and DNAto serine/threonine- or tyrosine-phosphorylated proteins,little is currently understood about the structural basis ofthis specificity.

HAD phosphatases are aspartate- and Mg2�-dependentenzymes encompassing a Rossmannoid fold and the active sitesignature sequence hhhDXDX(T/V)(L/I)h (where h is hydro-phobic residue; X is any amino acid; human consensus motif).The first aspartate in this HAD motif I acts as the essentialnucleophile, and additional catalytic residues cluster in a total offour HAD motifs that are positioned within the Rossmannoidcore. The catalytic cleft of HAD phosphatases has to be temporar-ily shielded during the phosphoaspartyltransferase reaction,because the nucleophilic attack requires active site solvent exclu-sion, whereas the subsequent hydrolysis of the phosphoaspartateintermediate is solvent-dependent. Control of active site accessi-bility is achieved by so-called flap structures, which are small,mobile elements bordering the catalytic core, and by highlydiversified cap domains that can provide more extensiveshielding (2, 3).

Cap domains can additionally play a decisive role in the selec-tivity for low or high molecular weight substrates; HAD phos-phatases with small (C0) caps have an open catalytic cavity andtend to process macromolecular substrates, whereas the largercap domains of the C1/C2 subfamily members typically occludethe active site to facilitate the dephosphorylation of low molec-ular weight substrates (12). However, some phosphoproteinswith terminal phosphorylation sites can also be dephosphory-lated by C2-capped HAD phosphatases such as chronophin (8).Importantly, C1/C2 caps can supply structural elementsinvolved in substrate interactions, and thus contribute tophosphatase specificity (13–17).

Although all HAD phosphatases belong to the same fold,their catalytic features have evolved independently multiple

* This work was supported by the Deutsche Forschungsgemeinschaft GrantsSFB688 (to A. G.) and FZ82 (to A. G. and H. S.).

The atomic coordinates and structure factors (code 4BKM) have been depositedin the Protein Data Bank (http://wwpdb.org/).

1 Present address: Dept. of Biotechnology, Abasaheb Garware College,411004 Pune, India.

2 To whom correspondence should be addressed: Dept. of Pharmacology,University of Würzburg, Versbacher Strasse 9, 97078 Würzburg, Ger-many. Tel.: 49-931-3180099; Fax: 49-931-20148539; E-mail: [email protected].

3 The abbreviations used are: HAD, haloacid dehalogenase; AUM, aspartate-based, ubiquitous, Mg2�-dependent phosphatase; PDB, Protein DataBank; Pdxp, pyridoxal 5�-phosphate phosphatase; PGP, phosphoglycolatephosphatase; PLP, pyridoxal 5�-phosphate; pNPP, para-nitrophenylphos-phate; pNP, para-nitrophenol; r.m.s.d., root mean square deviation; TEV,tobacco etch virus; TEA, triethanolamine.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 6, pp. 3416 –3431, February 7, 2014© 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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times, illustrating the functional diversity of the Rossmannoidcore (2, 3). These “large scale” mechanisms for structural andfunctional divergence have been studied and are well under-stood (18). Yet, given one structural type, how different can thesubstrates be? And, given that they are different, how is sub-strate specificity determined? To address these questions, wehave analyzed AUM (aspartate-based, ubiquitous, Mg2�-de-pendent phosphatase; gene annotation,: phosphoglycolatephosphatase, PGP). This gene is the closest paralog of chro-nophin (also known as pyridoxal 5�-phosphate phosphatase) (8,19, 20). Interestingly, AUM acts as a tyrosine phosphatase,whereas chronophin dephosphorylates serine/threonine-phos-phorylated proteins (8, 21–24) and PLP (19, 20, 25). We haveintegrated evolutionary, biochemical, and structural analyses ofAUM and chronophin to unravel the determinants behind thesubstrate specificity of these two closely related proteins.

EXPERIMENTAL PROCEDURES

Phylogenetic Analysis of AUM and Chronophin—Metazoanchronophin and AUM phosphatases were identified usingPFAM 25.0 (26), clan CL0137, as described (12), and humansequences were used as queries for BLAST searches of the indi-cated genomes. Ensembl accession numbers are listed in thelegend of Fig. 1B. The sequences were aligned with Muscle (27)and curated with GBlocks (28). The phylogenetic tree was cal-culated using PhyML with default parameters (29, 30). Con-served sites were identified as 90% consensus in chronophinand AUM, respectively (www.hiv.lanl.gov). Site-specific evolu-tionary rates were identified using MrBayes (31, 32) with a �model for the rate distribution and 10 categories consideringthe AUM and chronophin subfamilies. Differently conservedsites were identified with SDPfox (33). To test for sites underdifferent selective pressure, HMMDiverge was used (34). Allsites with a probability of �0.8, of which the evolutionary rate ishigher in one family, were selected. Branch-specific rate analy-ses were performed with PAML (35). The following modelswere tested: 1) one rate for the whole tree; 2) one additional ratefor the branches of chronophin and AUM, respectively, follow-ing the duplication; and 3) one additional rate for the chro-nophin and the AUM sub-branch, respectively. To test for sig-nificance, the doubled difference of the log likelihood wascompared with the �2 value taking into account the differentdegrees of freedom. Neither model 2 nor model 3 fitted the datasignificantly (p � 0.05) better than the simple model 1.

Bacterial Expression Constructs—The full-length cloneencoding for murine AUM (gene annotation, phosphoglycolatephosphatase) was obtained from the German Resource Centrefor Genome Research. For bacterial expression, full-lengthAUM cDNA was cloned into the bacterial expression vectorpETM11 (EMBL), producing AUM with an N-terminal His6 tagthat is followed by a tobacco etch virus (TEV) protease cleavagesite. Murine chronophin was reverse-transcribed from adultmouse brain tissue, and the PCR product was subcloned intopETM11 to create N-terminally His6-tagged chronophin. Site-directed mutagenesis was performed using the QuikChangemutagenesis kit (Stratagene). The AUM(1–113)-chro-nophin(101–207)-AUM(234 –321) (ACA) hybrid in pETM11was obtained by gene synthesis after optimizing the sequence

for maximal protein production without changing the murineAUM or chronophin amino acid sequences (Geneart). NestedPCR was used to create the chronophin(1–100)-AUM(114 –233)-chronophin(208 –292) (CAC) hybrid in pETM11. All con-structs were verified by sequencing.

Recombinant Protein Expression and Purification—pETM11constructs encoding for His6-tagged AUMWT and AUM vari-ants were transformed into Escherichia coli BL21 (DE3) andexpressed for 20 h at 28 °C after induction with 0.5 mM isopro-pyl �-D-1-thiogalactopyranoside supplemented with 20 �g/mlchloramphenicol, 50 �g/ml kanamycin, and 1 ng/ml tetracy-cline. To increase solubility, AUM was co-expressed with thechaperones groES-groEL-tig from the pG-Tf2 plasmid (Takara)according to the manufacturer’s instructions. Cells were har-vested at 8000 � g for 10 min and resuspended in TNM (50 mM

triethanolamine (TEA), 200 mM NaCl, 5 mM MgCl2; pH 7.5)supplemented with 10 mM imidazole and protease inhibitors(EDTA-free protease inhibitor tablets; Roche Applied Science).All purification steps were carried out at 4 °C. Cells were lysedin the presence of 150 units/ml DNase I (Applichem) using acell disruptor (Constant Systems), and cell debris was removedby centrifugation (10,000 � g, 30 min, 4 °C). For His6-AUMpurification, cleared supernatants were loaded on a nickel-ni-trilotriacetic acid-agarose column (HisTrap HP, GE Health-care) operated on an ÄKTA liquid chromatography system (GEHealthcare) in TNM, and His6-tagged proteins were elutedusing a linear 10 – 400 mM imidazole gradient in TNM. Peakfractions were tested for phosphatase activity using pNPP as asubstrate (see below). Active fractions were pooled, and theHis6 tag was cleaved with TEV protease for 4 days at 4 °C. Sub-sequently, cleaved AUM was separated from uncleaved AUMand from the His-tagged TEV protease on a HisTrap HP col-umn. Active fractions containing untagged AUM were pooled,concentrated (10-kDa MWCO; Amicon Ultra-15, Millipore),further purified on a HiLoad 16/60 Superdex 200 pg size exclu-sion chromatography column (GE Healthcare), and eluted inTNM.

Chronophin and the CAC hybrid were expressed and puri-fied as described above for AUM with the following modifica-tions: His6-tagged enzymes were expressed for 18 h at 20 °Cafter induction with isopropyl �-D-1-thiogalactopyranoside.After centrifugation, cells were resuspended in 100 mM TEA,500 mM NaCl, 20 mM imidazole, 5 mM MgCl2; pH 7.4, and lysedusing a cell disruptor in the presence of protease inhibitors andDNase I. Cleared supernatants were loaded on a HisTrap HPcolumn in binding buffer (50 mM TEA, 500 mM NaCl, 20 mM

imidazole, 5 mM MgCl2; pH 7.4), and His6-tagged proteins wereeluted using a linear gradient of up to 50% elution buffer (50 mM

TEA; 250 mM NaCl; 500 mM imidazole; 5 mM MgCl2; pH 7.4).The fractions containing His6-chronophin were pooled; theHis6 tag was cleaved with TEV protease, and untagged chro-nophin was isolated using a HisTrap HP column and furtherpurified on a HiLoad 16/60 Superdex 200 pg size exclusionchromatography column in 50 mM TEA; 250 mM NaCl; 5 mM

MgCl2; pH 7.4.Analytical Size Exclusion Chromatography—To determine

the oligomeric state of AUM, analytical size exclusion chroma-tography was performed using a HiLoad 16/60 Superdex 200 pg

Characterization of the Novel Mammalian Tyrosine Phosphatase AUM

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column, calibrated with globular proteins of known molecularweight (gel filtration LMW calibration kit, GE Healthcare). Bluedextran was used to determine the void volume of the column.Protein elution volumes were determined by monitoring theabsorption at 280 nm. The partition coefficient (Kav) was calcu-lated with the formula Kav � (Ve � Vo)/( Vt � Vo), where Ve isthe elution volume; Vois the void volume, and Vt is the totalcolumn volume. The apparent molecular weight was thenderived from the inverse logarithm of the partition coefficient.

Analytical Ultracentrifugation—Sedimentation velocity ana-lytical ultracentrifugation was carried out using a BeckmanOptima XL I analytical ultracentrifuge (Beckman Coulter) withan eight hole An-50 Ti rotor at 40,000 rpm and 20 °C. Fourhundred �l of highly purified recombinant murine AUM,murine chronophin, or reference buffer solution were loaded instandard double-sector charcoal-filled Epon centerpiecesequipped with sapphire windows. Protein concentrations cor-responded to an A280 of 0.25– 0.8. Data were collected in con-tinuous mode at a step-size of 0.003 cm, using absorption opti-cal detection at a wavelength of 280 nm. Data were analyzedusing the software SEDFIT (National Institutes of Health) todetermine continuous distributions for solutions to the Lammequation c(s), as described previously (36). Analysis was per-formed with regularization at confidence levels of 0.68 andfloating frictional ratio (f/f0 �1.22 0.04 for AUM and f/f0�1.32 0.02 for chronophin, suggesting a globular conforma-tion for both enzymes), time-independent noise, base line, andmeniscus position to root mean square deviation (r.m.s.d.) val-ues of �0.0064 for AUM and �0.012 for chronophin. Consis-tent results were obtained in three independent experiments.

Protein Crystallization and Data Collection—The chronophin-(1–100)-AUM(114 –233)-chronophin(208 –292) (CAC) hybridprotein was concentrated to 10 mg/ml (as determined byabsorption at 280 nm using a calculated molar extinction coef-ficient of 21,430 M�1 cm�1) in crystallization buffer (10 mM

TEA; 0.1 M NaCl; 1 mM MgCl2; pH 7.4) using 10-kDa molecularmass cut off centrifugal filter devices (Amicon Ultra-15, Milli-pore). Crystals were grown at 20 °C in 15% (w/v) PEG 3350 and0.2 M Mg(NO3)2 using the sitting-drop vapor diffusion method,by mixing 0.6 �l of protein solution with 0.4 �l of reservoirsolution. CAC crystals appeared as thin plates with dimensionsof �0.25 � 0.5 � 0.05 mm after 2–3 days, and the majority ofcrystals displayed very high mosaicity. Crystals were cryopro-tected for flash-cooling in liquid nitrogen by soaking in motherliquor containing 30% (v/v) glycerol. Diffraction data were col-lected on an R-axis HTC image plate detector mounted on aMicromax HF-007 rotating anode x-ray generator. Data wereprocessed using iMosflm (37) and scaled with Scala from theCCP4 program suite (38). The structure was solved by molec-ular replacement with the program Phaser (39) with humanpyridoxal-5�-phosphatase (PDB entry 2OYC) as a searchmodel. The structure was refined at 2.65 Å resolution with Phe-nix (40), incorporating torsion angle noncrystallographic sym-metry (ncs) restraints. Data collection and refinement statisticsare summarized in Table 3. The figures of the chronophin andCAC structures were generated with PyMOL (The PyMOLMolecular Graphics System, Version 1.5.0.4, Schrödinger,LLC.). Topology diagrams were generated with the pro-origami

system (41) using PDB files 2P69 and 4BKM. Dimer interfacecalculations were performed with the PISA on-line tool (42).

In Vitro Phosphatase Activity Assays—Phosphatase activityassays were conducted in 96-well microtiter plates. For assaysusing pNPP as a substrate, 0.8 �g of the purified proteins werepreincubated for 30 min at 37 °C in TNM, in the presence orabsence of phosphatase inhibitors or their respective solventcontrols. To study the effect of NaF or BeF3

� on phosphataseactivity, AUM or chronophin was preincubated with 1 mM NaFor with 1 mM NaF � 0.1 mM BeCl2 (BeF3

�). The reaction wasstarted by the addition of pNPP (final concentration rangingfrom 0.5 to 9 mM in a total assay volume of 100 �l and 3.5 mM forsingle point assays). The kinetics of pNP generation were fol-lowed spectrophotometrically by measuring the absorbance at405 nm every 30 s on a microplate reader (Envision 2104;PerkinElmer Life Sciences). pNP generation was quantitatedusing pNP standard curves. To derive Km and kcat values, thedata were fit by nonlinear regression to the Michaelis-Mentenequation using GraphPad Prism, version 4.01. For nucleotide orPLP dephosphorylation assays, 0.16 �g of protein were prein-cubated for 10 min at 22 °C in TNM. The reactions were startedby the addition of nucleotides (0 –3 mM) or PLP (final concen-tration ranging from 0 to 1 mM in a total volume of 50 �l, and 0.5mM for single point assays) and stopped after 5.5 min by theaddition of 100 �l of malachite green solution (Biomol Green;Enzo Life Sciences). Released phosphate was determined bymeasuring A620 and extrapolating the values to a phosphatestandard curve, and Km and kcat values were calculated usingGraphPad Prism, version 4.01. All pNPP and PLP phosphataseassays were performed with three independently purified pro-tein batches. For phosphopeptide array assays, phosphoty-rosine and mixed phosphotyrosine/-serine/-threonine phos-phatase substrate sets (JPT Peptide Technologies) were probedwith purified AUM according to the manufacturer’s protocol.Briefly, peptides were incubated with 83 ng of AUM in 25 �l ofphosphatase assay buffer (40 mM TEA, pH 7.5; 30 mM NaCl;0.1% (v/v) Triton X-100), yielding a final substrate concentra-tion of 10 �M. The plate was incubated at 37 °C under agitation,and the reaction was quenched after 45 min with 25 �l of mal-achite green solution. Released phosphate was determined bymeasuring A620.

Phosphatase Overlay Assays—To test the phosphatase activ-ity of AUM against cellular phosphoproteins, HeLa cells weretreated with 100 �M freshly prepared pervanadate solution for15 min at 37 °C to block the activity of cellular tyrosine phos-phatases. Cells were rinsed in phosphate-buffered saline (PBS),scraped in ice-cold lysis buffer (150 mM NaCl, 1% (v/v) TritonX-100, 1 mM �-glycerophosphate, 2.5 mM sodium pyrophos-phate, 1 mM sodium orthovanadate, 10 �g/ml aprotinin, 10�g/ml leupeptin, 1 mM pepstatin, 1 mM phenylmethylsulfonylfluoride (PMSF)) supplemented with phosphatase inhibitorcocktails I and II (Sigma), and lysed by repeatedly passingthrough a 20-gauge needle. Insoluble material was removed bycentrifugation at 21,000 � g for 10 min at 4 °C, and the clearedsupernatants were mixed with 2� Laemmli’s sample buffer.Proteins were separated by SDS-PAGE using a single-wellcomb and transferred onto nitrocellulose membranes (HybondC; Amersham Biosciences). The nitrocellulose membrane was




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then cut into vertical strips and incubated for 1 h at 37 °C underrotation with the indicated concentrations of AUMWT orAUMD34N in phosphatase assay buffer supplemented with 0.5%(w/v) nonfat milk powder and 2 mM MgCl2. Tyrosine phosphor-ylation patterns were visualized by probing with �-phospho-tyrosine antibodies (clone 4G10, Millipore).

RNA Interference and Analysis of Cellular Tyrosine Phos-phorylation—For RNA interference-mediated knockdown ofAUM in the murine spermatogonial cell line GC1-spg, lentiviralparticles containing AUM-directed or nontargeting control shR-NAs (MISSION shRNA panel SHCLND-NM_025954 or SHC002,respectively; Sigma) were generated in human embryonic kidney293F cells by co-transfection with the packaging vector psPAX2and the envelope vector pCR-VSV-G. Cells stably expressing theshRNA constructs were selected in medium containing 1 �g/mlpuromycin for 2–3 days. GC1-spg cells expressing AUM shRNA(TRCN0000081477) or control shRNA (SHC002) were seeded on3-cm diameter dishes, precoated with 0.1 mg/ml poly-L-lysine(Sigma), and starved in DMEM without FCS for 20 h. Cells werestimulated with human epidermal growth factor (EGF; 100 ng/ml,Sigma) for the indicated time points and lysed on ice in 250 �l of2� Laemmli’s sample buffer. Proteins were separated by SDS-PAGE and blotted onto nitrocellulose, and membranes wereprobed with 4G10 �-phosphotyrosine antibodies. Blots werestripped and reprobed with antibodies against AUM to assessAUM depletion and with �-tubulin antibodies (clone DM1A,Sigma) to control for comparable protein loading.

Generation of AUM Antibodies—AUM-specific rabbit poly-clonal antibodies were generated by Charles River using puri-fied full-length murine untagged AUMD34N as an immunogenin New Zealand White rabbits, and antibodies were purified byaffinity chromatography.

Preparation of Tissue and Cell Lysates—An adult C57BL/6male mouse was sacrificed by cervical dislocation and dissected,and the indicated tissues/organs were immediately snap-frozenand pulverized in liquid nitrogen. One hundred mg of therespective tissue powder were solubilized in 1 ml of tissue lysisbuffer (50 mM Tris-HCl, pH 7.2; 150 mM NaCl; 0.1% (w/v) SDS;1% (v/v) Triton X-100; 1 mM EDTA; 0.5 mM sodium orthovana-date; 1 mM sodium fluoride; 1% (w/v) sodium deoxycholate; 10�g/ml aprotinin; 10 �g/ml leupeptin; 1 mM pepstatin; 1 mM

PMSF) supplemented with phosphatase inhibitor cocktails Iand II. Insoluble material was removed by centrifugation at21,000 � g for 10 min at 4 °C.

All cell lines were purchased from ATCC/LGC Promochemand cultured in DMEM containing 4.5 g/liter glucose supple-mented with 10% (v/v) fetal calf serum, 2 mM L-glutamine, 100units/ml penicillin, and 100 �g/ml streptomycin. Cell lysateswere prepared in ice-cold RIPA buffer (50 mM Tris-HCl, pH 8.0;150 mM NaCl; 1% (v/v) Nonidet P-40; 0.1% (w/v) SDS, 0.5%(w/v) sodium deoxycholate) containing freshly added proteaseinhibitors (10 �g/ml aprotinin, 10 �g/ml leupeptin, 1 mM pep-statin, and 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride(Pefabloc)), and insoluble material was removed by centrifuga-tion. Blots were incubated in stripping buffer (62.5 mM Tris-HCl, pH 6.7; 2% (w/v) SDS; 100 mM �-mercaptoethanol) for 30min at 60 °C under agitation, washed in PBS, and reprobed withantibodies against chronophin (clone C85E3, Cell Signaling

Technology) or AUM, respectively (see above), and with �-ac-tin (clone C4, Millipore) and �-GAPDH antibodies (clone14C10, Cell Signaling Technology) to control for comparableprotein loading.

Accession Code—The x-ray crystal structure of the murinechronophin(1–100)-AUM(114 –233)-chronophin(208 –292)(CAC) hybrid protein has been deposited in the Protein DataBank under accession code 4BKM.

RESULTS

AUM Is a Chronophin Paralog—The overall amino acidsequence identity between HAD phosphatases is typically�15%. By database mining, we identified AUM as the closestchronophin homolog in metazoa with an overall amino acidsequence identity of 45% between murine AUM and chro-nophin and 47% between the two human enzymes. Fig. 1Ashows that AUM contains the four HAD-type phosphatase sig-nature motifs that are strictly conserved across evolution. Thecorresponding amino acid residues are largely identical inAUM and chronophin, yet markedly different from the moredistantly related C2-capped phosphomannomutases PMM1and -2. Based on the overall sequence identity with chronophinand the insertion of the capping domain between HAD motifsII and III, AUM likely belongs to the structural subfamily ofNagD-like, C2-capped HAD phosphatases (2, 12). To deter-mine the phylogenetic relationships of AUM and chronophin,we analyzed their transcripts from different vertebrate proteinstogether with urochordate sequences by calculating a phyloge-netic tree (43). As shown in Fig. 1B, AUM and chronophinevolved via duplication of an ancestral gene at the origin of thevertebrates. Retained duplicated genes usually undergo eitherneofunctionalization (i.e. one of the genes evolves a new func-tion) or subfunctionalization (i.e. each gene retains a subset ofthe original function) (44). A typical indication of neofunction-alization is an increased evolutionary rate in one of the twoduplicated genes. To test for such a rate difference in the case ofAUM and chronophin, models allowing for different rates atthe base of each subgroup as well as in the whole subgroupswere compared with models allowing only a single rate overthe entire phylogenetic tree. None of the complex modelsimproved the fit to the data significantly (p � 0.05). Thus, theevolutionary rate of both genes cannot be distinguished, dis-agreeing with a typical model of neofunctionalization.

We raised polyclonal antibodies against murine AUM tocompare the distribution of AUM and chronophin. Fig. 2Ashows that AUM-directed antibodies recognize recombinantAUM and do not cross-react with recombinant chronophin.AUM is expressed in all investigated mouse tissues, with highlevels present in testis, whereas chronophin appears to be rela-tively enriched in brain (Fig. 2B). Fig. 2C demonstrates thatAUM is also expressed in commonly used cell lines, includingthe mouse spermatogonial cell line GC1-spg, which we used forfurther studies (see Fig. 3F).

AUM Is a HAD-type Protein-tyrosine Phosphatase—We ver-ified that AUM has intrinsic phosphatase activity by first ana-lyzing the enzyme kinetics of recombinant AUM against pNPP,a generic phosphatase substrate. Fig. 3A shows that AUMdephosphorylates pNPP, whereas the activity of chronophin




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measured in parallel was very low, as reported earlier (8, 19, 20).The replacement of the putative nucleophilic Asp in AUM withAsn (AUMD34N) abolishes pNPP dephosphorylation, demon-

strating that AUM is indeed an aspartate-dependent phospha-tase. The catalytic efficiency of AUM is about 1000-fold higherthan the pNPP phosphatase activity of the HAD-type protein-

FIGURE 1. Comparison of AUM and chronophin. A, HAD motifs of AUM and chronophin are closely related. Alignment of the HAD motifs of vertebrate AUMorthologs in comparison with chronophin and phosphomannomutase (PMM) 1 and -2. M. m., Mus musculus. The consensus motifs reflect an alignment of 40human phosphatases. B, phylogenetic tree of vertebrate AUM and chronophin proteins together with urochordate sequences predating the duplication of acommon ancestor. Branch support is indicated by the result of the approximate likelihood ratio test (72). Accession numbers are given on the right.




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tyrosine phosphatase Eya3, as determined for the recombinantmurine enzyme (45), yet it is about 1000-fold lower in compar-ison with classical tyrosine phosphatases such as PTP1B, TC-PTP, or SHP1 (46).

AUM also dephosphorylates adenine and guanine nucleotidedi- and triphosphates (ADP ATP � GDP � GTP; Table 1),whereas chronophin exhibits no measurable activity againstthese substrates (19). In contrast to chronophin, AUM dephos-phorylates the low molecular weight chronophin substrate PLPonly poorly (Fig. 3B).

HAD phosphatases form a stable complex with BeF3� that

structurally mimics their tetragonal phosphoaspartate inter-

mediate (16, 47, 48). Fig. 3C shows that the hydrolytic activity ofboth AUM and chronophin is completely blocked by BeF3

� butis insensitive to NaF. AUM was insensitive to inhibitors of type1 and 2A Ser/Thr protein phosphatases, such as okadaic acidand calyculin A (both tested up to a concentration of 1 �M) butwas concentration-dependently inactivated by orthovanadate,a phosphate mimic that can act as a product inhibitor (IC50 �41.4 �M). Consistent with the critical role of Mg2� for HADphosphatases, the Mg2�-chelator EDTA (5 mM) completelyabolished AUM activity, and AUM was also inhibited by Ca2�,which can displace Mg2� from the active site (49) (IC50 �0.5mM, measured in the presence of 5 mM Mg2�). Taken together,

A B

Ponceau

kDa

75 -

50 -

37 -

100 -

25 -

kDa

α-actin

α-GAPDH

α-chronophinkD

a

75 -

50 -

37 -

100 -

25 -

α-AUM

kDa

75 -

50 -

37 -

100 -

25 -

2.5 1 0.5 0.1 2.5 1 0.5 0.1 [μg]

α-chronophin

rec. AUM rec. chronophin

50 -

150 -100 -75 -

37 -kDa

25 -

50 -

150 -100 -75 -

37 -kDa

25 -

rec. AUM rec. chronophin

α-AUM

2.5 1 0.5 0.1 2.5 1 0.5 0.1 [μg]

C

α-AUM

α-actin

25 -

37 -

50 -

FIGURE 2. Antibody validation, comparison of AUM and chronophin expression levels in murine tissues, and analysis of AUM expression in culturedcell lines. A, purified, recombinant (rec.) AUM and chronophin (0.1–2.5 �g/lane) were separated by SDS-PAGE, blotted onto nitrocellulose membranes, andprobed with rabbit polyclonal �-AUM (upper panel) or with rabbit monoclonal �-chronophin antibodies (lower panel). B, comparison of endogenous AUM andchronophin expression levels in mouse tissues. Mouse tissue lysates (50 �g of protein/lane) were separated by SDS-PAGE, blotted onto nitrocellulose mem-branes, and analyzed by immunoblotting with �-AUM antibodies. The blot was stripped and reprobed with �-chronophin antibodies. The identity of the fastermigrating bands is unknown. Blots were reprobed with �-actin and �-GAPDH antibodies to test for comparable protein loading. Additionally, the Ponceau-stained membrane is shown. C, whole cell lysates (25 �g of protein/lane) of human cervical carcinoma (HeLa), Chinese hamster ovary (CHO), mouse spermato-gonial (GC1-spg), and monkey kidney (COS7) cells were separated by SDS-PAGE, blotted onto nitrocellulose membranes, and analyzed by immunoblottingwith �-AUM antibodies. A–C, the bands corresponding to the expected AUM or chronophin molecular weights are indicated by arrows.




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these results support the classification of AUM as a Mg2�-de-pendent HAD phosphatase.

Some HAD phosphatases display in vitro activity againstphosphopeptides (7). We therefore explored the activity andpotential substrate preferences of AUM in array assays contain-ing a total of 720 different human phosphopeptides. AUMdephosphorylated a small fraction (�3.5%) of the 488 testedTyr(P) peptides (Fig. 3D), whereas no activity was detectedagainst any of the 174 Ser(P) or the 58 Thr(P) peptides. We next

examined the potential protein phosphatase activity of AUMin phosphatase overlay assays. Fig. 3E shows that AUMdirectly hydrolyzes tyrosyl-phosphorylated proteins isolatedfrom HeLa cell extracts, whereas the activity of AUMD34N ismarkedly compromised. Like Eya3, AUM thus displays invitro activity against denatured tyrosyl-phosphorylated pro-teins at concentrations that are equivalent to those reportedfor SHP1 and PTP1B in comparable in vitro phosphataseoverlay assays (50).

control NaF BeF3-0

100

200

300

v [n

mol

/min

/mg]

(pN

PP)

control NaF BeF3-0

1000

2000

3000

v [n

mol

/min

/mg]

(PLP

)

AUM chronophinC

0 5 10 15 20 25 300.00

0.02

0.04

0.06

0.08

0.10

0.12 AUMWT

chronophinAUMD34N

t [min]

OD

405n

m

A

0 100 200 300 400 500 600 700 800 900 10000

1000

2000

3000

4000

5000

6000 chronophin

AUM

PLP [µmol/L]

v [n

mol

/min

/mg]

B

D

E

α-pTyr (5 sec. exposure)

170 -130 -70 -55 -

40 -35 -

25 -

kDaunstim.

AUMWT AUMD34N

pervanadate

− 10 30 50 100 100 μg

α-pTyr (15 sec. exposure)

170 -130 -70 -55 -

40 -35 -

25 -

kDaunstim.

AUMWT AUMD34N

pervanadate

− 10 30 50 100 100 μg

37 -

50 -

75 -100 -150 -250 -

α-tubulinα-AUM

α-pTyr

kDactrl ctrl ctrlAUMAUM AUM shRNA

0` 3` 5` EGFF




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When we depleted endogenous AUM by RNA interferencein the mouse spermatogonial cell line GC1-spg and stimulatedthe cells with epidermal growth factor (EGF), the loss of AUMresulted in a transient increase in EGF-induced protein tyrosinephosphorylation. After 3 min of EGF treatment, we detectedhyperphosphorylated bands of �150 and �250 kDa in AUM-depleted cells compared with control shRNA cells, and an �75-kDa band was hyperphosphorylated after 5 min of EGF treat-ment in AUM-depleted cells (Fig. 3F). These data demonstratethat in contrast to the Ser/Thr phosphatase chronophin, whichis inactive against standard Tyr(P)-containing peptides andtyrosyl-phosphorylated proteins (8), AUM acts as a tyrosinephosphatase with narrow protein substrate specificity in cells.

Determinants of Substrate Specificity—Which features deter-mine the difference in AUM and chronophin substrate prefer-ences? To address this question, we swapped the cap domainsbetween the two enzymes. Interestingly, the exchange of theAUM and chronophin cap modules resulted in a clear change ofsubstrate specificity (Fig. 4). Although the phosphatase hybridconsisting of the chronophin cap and the AUM core domain(AUM(1–113)-chronophin(101–207)-AUM(234–321), referredto as “ACA”) is inactive against pNPP, the presence of the chro-nophin cap is sufficient for substantial PLP dephosphorylation. Incontrast, the presence of the AUM cap in the chronophin core/AUM cap phosphatase hybrid (chronophin(1–100)-AUM(114-233)-chronophin(2(08 –292), termed “CAC”) is incompatiblewith phosphatase activity against PLP. Consistent with a role ofresidues in the AUM core domain in efficient pNPP dephos-phorylation (Table 2), the pNPP phosphatase activity of the

CAC hybrid is substantially reduced compared with AUMWT

but comparable with the low basal pNPP activity of chro-nophinWT. The fact that pNPP dephosphorylation by CAC wasnot enhanced compared with chronophin supports the conceptthat caps provide features geared toward the specific recogni-tion of physiological substrates.

We performed an evolutionary analysis of residues that arestrictly retained in the respective orthologs to predict sites thatcould be of importance for substrate specificity (Table 3). Leu-204 in AUM and His-182 in the corresponding position in chro-nophin were identified as the most significant candidate resi-dues with a Z-score of 6.88. When we introduced a His residuein the AUM position 204 (AUML204H), the pNPP activity ofAUML204H was reduced to �64.3% of the AUMWT activity (Fig.4A), yet AUML204H dephosphorylated PLP almost as well as theACA mutant (Fig. 4B). This result confirms that residues in thecatalytic core of AUM are important for efficient pNPP dephos-phorylation (see Table 2). Furthermore, it clearly demonstratesthat the introduction of a His residue at position 204 in theAUM cap module can transfer chronophin-like specificity ontoAUM, while simultaneously attenuating pNPP dephosphory-lation. We conclude that this position is critical for the substratespecificity of AUM and chronophin.

Structural Basis of Substrate Specificity—Further insightsinto the structural basis of the different substrate specificities ofAUM and chronophin were obtained from x-ray crystallo-graphic studies. Although we were unable to crystallize full-length AUM, we succeeded in growing and analyzing crystals ofthe CAC hybrid, containing the AUM cap domain inserted intothe chronophin catalytic core (PDB entry 4BKM; Table 4). Theatomic model of the CAC hybrid was refined at 2.65 Å resolu-tion to an R-factor of 19.8% and an Rfree of 25.6%. The proteincrystallized in the space group P21 with four molecules (twohomodimers) per asymmetric unit. The overall structure of themurine chronophin core/AUM cap hybrid is highly similar tohuman chronophin (Fig. 5A). When only the amino acids cor-responding to the catalytic core of human chronophin (PDB2P69) and the catalytic core of murine chronophin in the CAChybrid are structurally aligned (residues 1–100 and 207–292 inchronophin or 1–100 and 200 –306 in CAC, respectively), the

FIGURE 3. AUM substrate specificity in vitro and in cells. A, in vitro pNPP phosphatase assays were performed in 96-well microtiter plates in a total assayvolume of 100 �l, using recombinantly expressed and purified AUM, AUMD34N, or chronophin (0.8 �g of protein/well) and 3.5 mM pNPP as a substrate. Thekinetics of pNP generation were followed spectrophotometrically by measuring the absorbance at 405 nm. B, in vitro PLP phosphatase assays with purifiedAUM and chronophin were performed in 96-well microtiter plates in a total assay volume of 50 �l, using recombinantly expressed, purified AUM or chronophin(0.16 �g of protein/well), and 0 –1 mmol/liter PLP. The reaction was stopped with malachite green, and released phosphate was determined by measuring A620.The enzyme velocity toward increasing PLP concentrations is shown. C, effect of BeF3

� on AUM or chronophin activity toward pNPP or PLP. The recombinantpurified enzymes (0.8 �g of AUM or 0.16 �g of chronophin/well) were preincubated for 30 min at 37 °C (AUM) or for 10 min at 22 °C (chronophin) in the absence(control) or presence of 1 mM NaF (NaF) or 1 mM NaF � 0.1 mM BeCl2 (BeF3

�) before phosphatase activity against pNPP (3.5 mM) or PLP (0.5 mM) was measuredas described above. Left panel, velocity of AUM-dependent pNPP dephosphorylation inhibitors; right panel, velocity of chronophin-dependent PLP dephos-phorylation inhibitors. A–C, results are mean values S.E. of n � 3 independent experiments. D, activity of AUM in phosphopeptide array assays. A set of 720different phosphopeptides phosphorylated on tyrosine, serine, or threonine residues (final substrate concentration, 10 �M) was incubated with 100 nM purifiedAUM in a 384-well microtiter plate (final assay volume, 25 �l) for 45 min at 37 °C. The reaction was quenched with malachite green, and released phosphate wasdetermined by measuring A620. Absorbance values were normalized to the peptide substrate yielding the highest reading (100% hydrolysis), and all peptideswith absorbance values of �66% over the background are listed. In these sequences, acidic residues are highlighted in red, basic residues in blue, and prolineresidues in gray. Swiss-Prot accession numbers of the corresponding proteins are given on the right. E, determination of AUM and AUMD34N activity towardtyrosine-phosphorylated proteins in overlay assays. HeLa cells were left unstimulated (unstim.) or treated with pervanadate. After SDS-PAGE, cell lysates wereblotted onto nitrocellulose, and the membrane was cut and incubated in the absence (�) or presence of the indicated concentrations of AUMWT or AUMD34N.Protein tyrosine phosphorylation was analyzed with 4G10 �-phosphotyrosine antibodies. A short (left panel) and longer exposure (right panel) is shown (n �3). F, comparison of cellular phosphotyrosine levels in control (ctrl) or AUM-depleted GC1-spg cells. GC1-spg cells expressing control (ctrl) shRNA or AUM shRNAwere stimulated with 100 ng/ml EGF for the indicated time points. Cells were lysed, and proteins were separated by SDS-PAGE and transferred onto nitrocel-lulose membranes for immunoblotting. Cellular tyrosine phosphorylation levels were analyzed with 4G10 �-phosphotyrosine (pTyr) antibodies; AUM deple-tion was assessed with �-AUM antibodies, and tubulin served as a loading control (n � 3).

TABLE 1Catalytic constants of AUM toward nucleotidesNucleotide dephosphorylation was measured in 96-well microtiter plates in a totalassay volume of 50 �l, using recombinantly expressed, purified AUM (0.16 �g ofprotein/well) and 0 –3 mM nucleotides. The reactions were stopped with malachitegreen, and released phosphate was determined by measuring A620. Results aremeans S.E.; n � 3.

Km kcat

mM s�1

ADP 0.42 0.04 0.73 0.02ATP 1.23 0.19 0.48 0.03GDP 1.48 0.31 0.47 0.05GTP 1.47 0.21 0.46 0.03




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low root mean square deviation (r.m.s.d.) of 0.58 Å indicatesthat the introduction of the AUM capping domain has no majorimpact on the folding of the catalytic core of chronophin.

The inclusion of the cap domain in the superpositionincreases the r.m.s.d. to 1.1 Å, still indicating a significant struc-tural homology between the two proteins. However, the super-imposition of only the catalytic cores of the chronophin andCAC structures reveals that the orientation of the cappingdomain in the CAC hybrid differs from chronophin by a 6.5°rotation as determined by LSQKAB of the CCP4 program suite,resulting in a slightly more open conformation of the cap rela-tive to the core domain.

Both caps consist of a central parallel �-sheet in 8 –7-9 –10�13 (CAC) or 7– 8-9 –12 (chronophin) orientation, with�-helices (CAC: F, E, G, H, I; chronophin: E, F, G, H) connectingthe strands of the sheet (Fig. 5B). Preceding strand 10 (CAC) or9 (chronophin), a �-hairpin followed by a helix (I in CAC and Hin chronophin) is inserted into the capping domain (�-hairpinresidues, 201–211 in AUM, 190 –200 in CAC, and 181–191 inchronophin). This �-hairpin covers the entrance of the activesite and harbors the substrate specificity determining residue(Leu-204 in AUM/Leu-191 in CAC, His-182 in chronophin)and is therefore referred to as “substrate specificity loop.” Com-pared with the orientation of the substrate specificity loop inthe chronophin cap, the corresponding �-hairpin is notablyskewed in AUM (Fig. 5A, open arrow). The AUM cap also fea-

tures an additional extensive loop not present in chronophin,indicated by the closed arrow in Fig. 5A (residues 140 –164 inAUM/127–151 in CAC; referred to as “transverse loop,” seebelow), which is located between strands 8 and 9 and comprisesa short helical stretch (helix G in CAC, see Fig. 5B). Thus, con-sistent with the structural conservation of the cap domains inHAD subfamilies, the overall structures of the AUM and chro-nophin capping domains are closely related in terms of struc-tural elements and folding with an r.m.s.d. of 1.28 Å, althoughremarkable differences exist (see below and Fig. 6A).

The CAC hybrid crystallizes as a dimer (Fig. 5C), and weconfirmed that AUM also predominantly exists as a dimer insolution, by employing size exclusion chromatography andanalytical ultracentrifugation sedimentation velocity measure-ments (Fig. 5, D and E). The AUM phosphatase activity in thesize exclusion chromatography fractions containing AUMdimers or AUM tetramers was comparable. CAC dimerizationis mediated via the AUM capping domain, mainly by helices Hand I located between strands 9 and 13 of the �-sheet and fol-lowing the �-hairpin substrate specificity loop. Interestingly,the transverse loop in the AUM cap of molecule A in the dimeris positioned in close proximity to the substrate specificity loopin the cap of molecule B. Arg-197 in the �-hairpin and Leu-144in the CAC transverse loop (corresponding to Arg-201 andLeu-157 in AUM) may be important residues for this interac-tion between two CAC protomers. This characteristic loop in

FIGURE 4. Effects of the AUM-L204H substitution and of AUM and chronophin cap domain exchanges on phosphatase specificity. The indicatedrecombinant proteins were tested for their in vitro phosphatase activity toward pNPP (using 0.8 �g of the respective purified proteins in an assay volume of 100�l) and PLP (using 0.16 �g of the respective purified proteins in an assay volume of 50 �l). Enzyme velocities against pNPP (A) and PLP (B) are plotted againstincreasing substrate concentrations. Results are mean values S.E. of n � 3 independent experiments. ACA, hybrid protein consisting of AUM core domainfused to the chronophin cap domain; CAC, chronophin core domain fused to the AUM cap domain. C, purity of the employed recombinant proteins. Shown areCoomassie Blue-stained gels (see also Table 2).




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the AUM cap also partially occludes the active site entrance ofthe adjacent protomer. When the four CAC protomers (twohomodimers) of one asymmetric unit are superimposed, nomarked differences are seen in the orientation of this loop, indi-cating that this is the predominant conformation rather than anartifact of crystal packing. Thus, both the substrate specificityloop and the large and presumably flexible transverse loop arelikely to determine AUM phosphatase substrate accessibilityand selectivity.

A comparison of the active sites of chronophin and the CAChybrid provides a detailed view of residues important for sub-strate specificity (Fig. 6A). Although all core domain residues

that are directly involved in the dephosphorylation reaction areidentical between AUM and chronophin (with the exception ofThr-67 in AUM, which contributes to the orientation of thesubstrate for nucleophilic attack by forming a hydrogen bond

TABLE 2Catalytic constants of AUM, AUM mutants, and chronophin towardpNPPExchange of the cap domains and of amino acid residues in AUM with the corre-sponding chronophin residues in regions of AUM/chronophin sequence diver-gence. pNPP dephosphorylation assays were performed as described in the legend toFig. 3. The substitution of three divergent amino acids adjacent to HAD motif I ofAUM for the corresponding chronophin residues (AUMR41N; T44R; A45I) slightly ele-vates the activity toward pNPP. In contrast, the swap of residues in the seven-aminoacid stretch of AUM that immediately succeeds the conserved Ser/Thr residue inHAD motif II (AUMT67S; S71R; K72R; T73A) abolishes AUM phosphatase activitytoward pNPP, which is partially restored in the presence of the HAD motif II residueThr-67 (AUMS71R; K72R; T73A; �58.7% of AUMWT activity). Ser/Thr of motif II formsa hydrogen bond with the substrate’s phosphoryl group and thus contributes to theorientation of the substrate for nucleophilic attack. The catalytic constantsof AUMT67S; K72R; T73A, AUMT73A, the CAC hybrid, and chronophin could not bedetermined (ND) because the pNPP activity of these proteins was too low. None ofthe investigated AUM variants dephosphorylated the chronophin substrate PLP.The protein concentrations of those AUM mutants that could not be purifiedto apparent homogeneity (AUMT67S; S71R; K72R; T73A, AUMT67S; K72R; T73A,AUMT67S; S71R; T73A, see Fig. 4C) were determined densitometrically. To this end,proteins were subjected to SDS-PAGE and stained with Coomassie Blue, and thebands corresponding to the respective AUM mutant were analyzed using the ImageJsoftware (National Institutes of Health). Various concentrations of BSA were run onthe same SDS-polyacrylamide gels and were analyzed as described above to con-struct protein concentration standard curves. Results are mean values S.E. ofthree independent experiments performed in triplicate with three independentlypurified protein batches.

Km kcat

mM s�1

AUMWT 3.13 0.36 0.47 0.023AUMR41N; T44R; A45I 4.58 1.55 0.57 0.096AUMT67S; S71R; K72R; T73A 0.09 0.60 0.006 0.002AUMT67S; K72R; T73A ND NDAUMT67S; S71R; T73A 0.28 0.84 0.008 0.003AUMT73A ND NDAUMS71R; K72R; T73A 4.31 0.74 0.28 0.023AUML204H 7.97 1.58 0.30 0.04ACA 3.75 0.36 0.001 0.001CAC ND NDChronophin ND ND

TABLE 3Differently conserved sites between AUM and chronophin

Position in AUMPosition inchronophin

Amino acidin AUM

Amino acid inchronophin

Amino acidin Ciona Z-score

204 182 Leu His M 6.88203 181 Arg Trp Arg 6.58199 177 Asn Asp Asn 6.56128 117 Ala Gly Ala 6.55244 222 Val Ile Ile 5.87263 241 Asp Glu Asn 5.58178 156 Tyr Phe Tyr 5.56217 195 Cys Ser Cys 5.47101 88 Thr Ser Thr 5.3572 63 Lys Arg Lys 5.34268 246 Leu Phe Phe 5.31136 125 Val Ala Leu 5.00197 175 Gly Ala Ala 4.96169 147 Val Leu Leu 4.94226 204 Ala Ser Ala 4.80100 87 Gly Ser Ser 4.78

TABLE 4Data collection and refinement statistics

Data collectionWavelength 1.5418 ÅSpace group P 21Unit cell parameters

a, b, c 67.50, 91.96, 105.92 Å�, �, � 90, 90.2, 90°

Resolution rangea 41.74 to 2.65 Å (2.79 to 2.65 Å)Rsym

b 0.125 (0.816)Rp.i.m.

c 0.070 (0.456)�I/�I d 6.6 (1.4)Completeness 96.2% (94.9%)Multiplicity 4.1 (4.1)Total reflections 150,275Unique reflections 36,203 (3531)

RefinementWilson B-factor 50.0 ÅAverage B-factor 86.9 Å

Macromolecules 87.4Solvent 64.0

Rcryste 0.1976 (0.2878)

Rfreee 0.2564 (0.3290)

No. of non H-atoms 9450Macromolecules 9236Ligands 12Water 202

r.m.s.d. inBond lengths 0.004 ÅBonds angles 0.83°Planar groups 0.004 ÅDihedral angles 13.54°

Coordinate errorf 0.36 ÅRamachandran statisticsg

Favored 97.68%Allowed 2.07%Outliers 0.25%

MolProbity clash scoreh 12.17a Numbers in parentheses refer to the respective highest resolution data shell in

the data set.b Rsym � �hkl�i�Ii � �I �/�hkl�iIi, where Ii is the ith measurement, and �I is the

weighted mean of all measurements of I.c Rp.i.m. � �hkl (1/(n � 1))1/2 �i�Ii � �I /�hkl�iIi, where n is the multiplicity of the

observed reflection.d �I/�I indicates the average of the intensity divided by its S.D. value.e Rcryst � ��Fo � Fc�/��Fo�, where Fo and Fc are the observed and calculated struc-

ture factor amplitudes. Rfree is same as Rcryst for 5% of the data randomly omit-ted from the refinement.

f Estimated coordinate error is based on Rfree.g Ramachandran statistics indicate the fraction of residues in the favored, allowed,

and disallowed regions of the Ramachandran diagram, as defined by MolProbity(73).

h Number of serious clashes per 1000 atoms is shown in Ref. 73.




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with its transferring phosphoryl group and thus is required forAUM activity rather than specificity; see Table 2), remarkabledifferences exist in the AUM and chronophin cap domains.

Importantly, the imidazole ring of His-182 in the chronophincap directly coordinates and orients the PLP pyridine ring,which explains its critical function for substrate recognition




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(see Fig. 4). In contrast, the aliphatic Leu-204 of AUM (Leu-191in CAC) is unable to coordinate PLP. Two other notable aminoacid exchanges in the cap are the replacement of the acidicAsp-177 in chronophin for the charge-neutral Asn-199 inAUM (Asn-186 in CAC) and the substitution of Tyr-150 inchronophin for Phe-173 in AUM (Phe-159 in CAC).

Finally, we have conducted a topological comparison of the cat-alytic clefts in chronophin and the CAC hybrid. Fig. 6B illustratesthat the cap portion of the active site pocket in CAC that is dedi-cated to substrate recognition is deeper than in the chronophinpocket. This result is consistent with the observed AUM phospha-tase activity toward the Tyr(P)-mimetic pNPP substrate andtoward Tyr-phosphorylated peptides and proteins, and it indicatesthat in contrast to the shallower binding groove of chronophin, theconformation of the substrate binding pocket of AUM allows forthe accommodation of bulkier Tyr residues.

Evolution of Substrate Specificity—To identify further sitesresponsible for the different substrate specificities of AUM andchronophin, we adopted a comparative evolutionary approach.If, following gene duplication, the functions of two paralogschange, two types of sites can typically be distinguished. Pro-vided that a corresponding position in the two proteins is essen-tial for their respective functions, this site is highly conserved inboth proteins but harbors different residues (class II sites (51)).The substrate switch resulting from the His-182/Leu-204 swaphas clearly revealed the importance of such differentially con-served residues for AUM and chronophin functions. We haveidentified 15 additional class II sites that may be involved infunctional differentiation (Table 3).

When mapping these sites on the AUM cap structure (Fig.7A), some differentially conserved residues stand out as candi-dates involved in the determination of substrate specificity:

FIGURE 5. Crystal structure of the murine chronophin core/AUM cap hybrid (CAC). A, overall CAC structure (PDB 4BKM) compared with human chronophinin the PLP-bound state (PDB 2P69). Chronophin is in gray; the CAC murine chronophin core is in lime, and the AUM cap is in pink. The catalytic cores of humanand murine chronophin are organized in a superimposable manner, whereas the AUM and chronophin caps differ. Open arrows, AUM substrate specificity loop;closed arrows, AUM transverse loop (see C). B, topology diagrams of chronophin and CAC. Left panel, organization of chronophin. Right panel, CAC organizationwith the core domain in lime and the cap in pink. The �-strands are numbered consecutively, and �-helices are in alphabetical order from the N to the Cterminus. Blue star, nucleophilic Asp-25; flap, �-strands 3 and 4; substrate specificity loop, �-strands 10 and 11 (chronophin) or 11 and 12 (CAC). C, overallstructure of the CAC homodimer, shown in ribbon representation with transparent surfaces. One protomer (mol A) is represented in gray with the substratespecificity loop shown in magenta. Mol B is in rainbow colors (N terminus in blue). One inset shows the magnified spatial arrangement of the small helix in thetransverse loop of the AUM cap (mol B, green) and the specificity loop of the AUM cap (mol A, pink). The 2nd inset shows helix I of mol A (gray) and of mol B(orange) forming the dimer interface. Red star, Asp-25. D, elution profile of purified, untagged murine AUM on a size exclusion chromatography column. Thepeak elution volume of AUM (theoretical molecular mass, 34.5 kDa) corresponds to a calculated molecular mass of 79.6 kDa. The elution volume of a fraction(19.3%) of AUM corresponds to a calculated molecular mass of 163.6 kDa. E, analytical ultracentrifugation sedimentation velocity experiments of purified AUMand chronophin. AUM exists in equilibrium between dimers (88.3%) and tetramers (11.7%) in solution and has a greater propensity for tetramer formation thanchronophin (only 2.2% of chronophin particles sediment as tetramers). D and E, n � 3. Arrowheads indicate the expected/observed position of AUM monomers(M), dimers (D), and tetramers (T).

FIGURE 6. View of the active sites of chronophin and the CAC chronophin/AUM cap hybrid. A, left panel, view of the chronophin/PLP binding interface (PDB2P69). PLP is shown in yellow, and the catalytic core residues that build up the binding site for the substrate’s phosphate moiety are in gray and consist of HADmotif I (Asp-25 and Asp-27), II (Ser-58 and Ser-61), III (Lys-213), and IV (Asp-238 and Asp243) residues. Middle panel, view into the active site of the CAC hybrid(PDB 4BKM). The catalytic residues of the murine chronophin core domain are shown in lime, and the residues of the AUM cap domain are in pink. Amino acidresidues are labeled according to their position in CAC. The PLP molecule is modeled according to the human chronophin structure. Right panel, superpositionof the active site residues of human chronophin in the PLP-bound state (gray) and the corresponding residues of the AUM cap domain in the CAC hybrid (pink).Amino acid residues are labeled according to their position in murine chronophin and murine AUM. B, superposition of the CAC (pink) and chronophin (gray)active sites in surface representation, revealing the different spatial arrangements of the substrate binding pockets. Shown is a zoom into the active siteunderneath the nucleophilic Asp-25 (left panel). A lateral view is presented in the right panel. Black dotted lines outline the borders of the active site sections inCAC or chronophin, respectively.




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Asn-199 in AUM (Asp-177 in chronophin) is located in thesubstrate recognition part inside the active cleft (see also Fig.6A), whereas Arg-203 in AUM (Trp-181 in chronophin) mapsto the substrate specificity loop but is oriented toward the outerprotein surface and may thus function in substrate recognitionat the entrance to the active cleft. Other differentially conservedresidues, including Ala-128, Tyr-178, Cys-217, and Ala-226, inAUM are exposed on the cap surface and may play a role inregulatory protein-protein interactions. Sites involved in func-tional differentiation can also be highly conserved in one para-log but under poor selection constraint in the other (class I sites;see Table 5). Of interest, Glu-207 in AUM is positioned on topof the specificity loop and may be involved in initial substratecontact of AUM but not of chronophin (Fig. 7B), and Arg-41 in

AUM is located at the entrance to the active cleft (Fig. 7B andTable 2).

To visualize catalytic core residues that are differently con-served, we have mapped them on the chronophin structure. Fig.7C shows that these residues cluster around the active center ofchronophin. Chronophin-Arg-63 is located at the active siteentrance and corresponds to AUM-Lys-72, a core domain res-idue important for AUM activity (see Table 2 for biochemicalindications on the relevance of this residue). The differentlyconserved chronophin residues Ser-195, Phe-156, and Gly-117and residues Arg-74 and Ala-198 that are only conserved inchronophin are located on the chronophin surface and arepotential candidates for protein-protein interaction sites. Therelevance of these conserved residues for substrate specificitycan be systematically explored once a physiological AUM sub-strate has been identified.

DISCUSSION

We have identified and characterized the previously unex-plored chronophin paralog AUM as a mammalian HAD-typetyrosine phosphatase and have characterized it by biochemical,structural, and computational techniques. Although physiolog-ical AUM substrates are currently unknown, the elevated levelsof tyrosine-phosphorylated proteins observed upon stimula-tion of AUM-depleted cells with EGF implicate AUM as a tyro-sine phosphatase involved in growth factor-induced signalingpathways. The gene encoding for human AUM was previouslyannotated as phosphoglycolate phosphatase (PGP) based on

Y178/F156

G144

C217/S195

R203/W181

L204/H182

N199/D177

A128/G117

V169/L147

G197/A175

Y122/F111

A226/S204

D27

D25

V136/A125

E207

A198

AUM cap

E207N32(R41)

E207

N32(R41)

CAC

E207

specificity loop

R203N32(R41)

E207

B C

G144Y178/F156

H182

R63

A198S195

A92

H182

R63 G117

F156

R74

S195

F156

chrono-phin

A

FIGURE 7. Localization of differently conserved residues in AUM and chronophin. A, loop diagram representing the overall structure of the AUM capdomain (in pink; Asp-25/27 of the chronophin core in gray, PLP in yellow). Differently conserved AUM or chronophin residues (class II sites) are labeled in pinkor cyan, respectively (residue numbering according to murine AUM or chronophin). Chronophin residues are represented according to their spatial orientationin the chronophin cap (which is not shown for clarity) after structural alignment with and superimposition onto the CAC structure. Sites conserved in onesubfamily, but not in the other (class I sites), are labeled in orange (AUM) or green (chronophin). B, loop diagram (upper panel) and surface representation (lowerpanel, two different orientations are shown) of a zoom into the active site of the CAC hybrid (chronophin core domain in lime, AUM cap domain in pink, and PLPin yellow). The position of Glu-207 in the AUM cap and the putative position of AUMArg-41 (corresponding to chronophinAsn-32) in the chronophin core areshown in orange. Both residues are conserved in AUM but not in chronophin. C, loop diagram (upper panel) and surface representation (lower panel) of amagnification of the active center of human chronophin (PDB code 2P69). The differently conserved residue Arg-63 in chronophin (shown in cyan; stickrepresentation in the upper panel) is located at the active site entrance and corresponds to Lys-72 of AUM (see also Table 2). The differently conservedchronophin residues Ser-195, Phe-156, and Gly-117 (cyan) and residues Arg-74 and Ala-198 that are only conserved in chronophin (green) are located on thechronophin surface. Ala-92 is only conserved in chronophin; its function is currently unknown. (Note: Ala-198 is not visible in the lower panel.)

TABLE 5Sites conserved in one family but not in the other

Position inAUM

Position inchronophin

Aminoacid

Samerate

High ratein AUM

High rate inchronophin

In chronophin105 92 Ala 0.04 0.96 0.0083 74 Arg 0.04 0.96 0.0096 83 Glu 0.05 0.95 0.0022 13 Arg 0.14 0.86 0.00220 198 Ala 0.20 0.80 0.00

In AUM207 185 Glu 0.03 0.00 0.97317 292 Pro 0.06 0.00 0.9441 32 Arg 0.16 0.00 0.84144 132 Gly 0.20 0.00 0.80




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genetic linkage analyses (52). Although we were unable to testthe potential PGP activity of AUM (2-phosphoglycolate is nolonger commercially available), we note that there are clear dis-crepancies between the reported properties of PGP enzymesand AUM. Although multiple isozymes of human PGP havebeen described (53–57), the human gene annotated as PGP onlyhas one alternative splice variant (Ensembl transcript IDENST00000562001), producing a protein fragment of 57-a-mino acid residues, that is likely to be subject to nonsense-mediated decay. Except for the conserved Rossmannoid fold,the eight available x-ray crystal structures of bona fide PGPs(from bacteria and archaea) show no structural relationshipwith the CAC hybrid. In fact, the substrate binding pocket ofthe high specificity T. acidophilum PGP (PDB 1L6R) is sur-rounded by a network of acidic residues that interact with theglycolate leaving group (58), which is clearly distinct from theproperties of the substrate binding groove in AUM as shown inthis study. Still, should AUM indeed have PGP activity in addi-tion to its tyrosine phosphatase activity, this would add yetanother dimension to the functional potential of this HADphosphatase subfamily.

Contrasting AUM, its paralog chronophin is a PLP- and Ser/Thr-directed phosphatase. Classical Ser/Thr- or Tyr-directedphosphatases constitute separate, structurally unrelatedenzyme families (59, 60). In contrast, all HAD phosphatasesshare a structurally conserved Rossmannoid catalytic core. Pre-vious work has identified HAD family members that possessprotein Ser/Thr or Tyr phosphatase activity (2, 3). Althoughclassical Ser/Thr-directed phosphatases recruit an array of reg-ulatory subunits to precisely target their substrates (61, 62), andclassical Tyr phosphatases have acquired their specificity bydomain fusion events (63, 64), HAD phosphatases have special-ized their functions by the fusion of a “generic” catalytic corewith structurally diversified cap domains (2, 63, 65). Crystallo-graphic studies in prokaryotes (14, 66), mammalian nucleoti-dases (16, 67), human phosphomannomutases (68), and humanphosphatases (69) have led to the realization that HAD caps canprovide specificity domains, yet little experimental evidencehas been available so far to support this presumed role. We havetherefore created functional HAD phosphatase cap hybridsbetween two HAD paralogs and show that these domain swapscan indeed toggle between chronophin- and AUM-like phos-phatase specificities.

Employing a combination of biochemical, evolutionary, andstructural data, we have identified the mechanisms behind thisstriking functional flexibility. We show that the functional spe-cialization of HAD phosphatases cannot only be attributed tothe presence of different cap structures but also to seeminglysmall changes within cap domains. Despite their highly con-served fold, the caps of chronophin and AUM contain residuesinvolved in substrate recognition. Using an evolutionary-basedprediction of residues determining specificity, we identified acandidate site responsible for the change in function. Indeed, asingle, differently conserved residue in AUM and chronophin issufficient to switch between the characteristics of a Tyr- orSer/Thr-directed phosphatase, respectively. This cap residue(Leu-204 in AUM and His-182 in chronophin) is positioned aspart of a characteristic �-hairpin structure referred to as sub-

strate specificity loop (13, 14). Although the presence of thisloop is common to both AUM and chronophin, AUM addition-ally contains a transverse loop that may serve to orient thesubstrate specificity loop of the adjacent monomer in thehomodimer.

In addition to their preference for Tyr- or Ser/Thr-phosphor-ylated residues, phosphatases also have to evolve specificitytoward their particular target proteins. To discover such sites,we combined structural and evolutionary data. We identifiedfour AUM-specific residues around the substrate entry site tothe catalytic core (Arg-41, Lys-72, Arg-203, and Glu-207) thatmay be involved in initial AUM/substrate contact, and two dif-ferentially conserved residues inside the AUM substrate bind-ing groove (Asn-199 and Leu-204) that may be involved indefining the electrostatic environment of the active site and insubstrate recognition. These sites are therefore excellent can-didates for further functional characterization of AUM. Thus,structural information allowed us to map the functional diver-gence of AUM and chronophin to defined substitutions in thesubstrate binding pocket.

Yet the following question remains. How did these differ-ences evolve? AUM and chronophin arose from a duplication ofan ancestral gene at the base of the vertebrates. Unexpectedly,we did not find a significant difference between the evolution-ary rates of the two paralogs, indicating that they both differ infunction from the ancestral gene. The orthologs of AUM andchronophin in the urochordates Ciona savigny and Ciona intes-tinalis harbor neither a histidine nor a leucine residue in thespecificity determining site but instead a methionine. It will beinteresting to test the function of this gene or even to recon-struct the ancestral precursor gene.

When looking at the large and ancient family of HAD pro-teins, the evolution of their functional divergence can be tracedback to the addition of different cap domains to a Rossmannoidcore (2). We thus propose a two-step process in the evolution ofHAD phosphatase specificity. In the first evolutionarily oldstep, different cap domains were added to the Rossmannoidcore. Subsequently, phosphatase functions within structuralHAD subclasses were further diversified by amino acid substi-tutions within the caps. It seems that this evolutionary mecha-nism has equipped HAD phosphatases with a high degree offunctional flexibility. Indeed, orthologs of AUM and chro-nophin were expanded independently in nematodes andarthropods (12). Differences in the residues positioned in thespecificity determining sites hint at supplementary functions inthese model organisms. Thus, the AUM/chronophin subfamilymight provide an excellent model system to study the evolutionof functional divergence of HAD phosphatases.

Despite their important roles in human diseases, which makesome HAD phosphatases conceptually attractive drug targets(5, 12, 25, 70, 71), knowledge of mammalian HAD phosphatasespecificity determinants is currently very limited. We demon-strate that HAD substrate specificity can be encoded by a verysmall number of predictable amino acid residues. This conceptmay be instrumental in the search for specific HAD phospha-tase inhibitors that target the unique substrate recognitionpockets of individual enzymes. Finally, it will be important toelucidate the in vivo functions of the Ser/Thr- and PLP-directed




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phosphatase chronophin and the related phosphotyrosinephosphatase AUM, which we have identified in this study.

Acknowledgments—We thank Andrea Odersky and Beate Vogt fortechnical assistance; Ingrid Tessmer for help with AUC experiments;Christin Schäfer, Petra Hänzelmann, Antje Schäfer, and MariaHirschbeck for discussions; and Heino Prinz for help with phospho-peptide arrays.

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Manhard, Hermann Schindelin, Jörg Schultz and Antje GohlaAnnegrit Seifried, Gunnar Knobloch, Prashant S. Duraphe, Gabriela Segerer, Julia

Different Substrate SpecificitiesPhosphatases AUM and Chronophin Provide Insight into the Basis of Their

Evolutionary and Structural Analyses of Mammalian Haloacid Dehalogenase-type

doi: 10.1074/jbc.M113.503359 originally published online December 13, 20132014, 289:3416-3431.J. Biol. Chem.

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