Crystal Structure of an Activity-Enhancing Mutant of DUSP19

ISSN (Print) 1225-9918 ISSN (Online) 2287-3406 Journal of Life Science 2018 Vol. 28. No. 10. 1140~1146 DOI : https://doi.org/10.5352/JLS.2018.28.10.1140

Crystal Structure of an Activity-enhancing Mutant of DUSP19

Da Gyung Ju, Tae Jin Jeon and Seong Eon Ryu* Department of Bioengineering, College of Engineering, Hanyang University, Seoul 04763, Korea Received August 14, 2018 /Revised October 5, 2018 /Accepted October 5, 2018

Dual-specificity phosphatases (DUSPs) play a role in cell growth and differentiation by modulating mitogen-activated protein kinases. DUSPs are considered targets for drugs against cancers, diabetes, immune diseases, and neuronal diseases. Part of the DUSP family, DUSP19 modulates c-Jun N-terminal kinase activity and is involved in osteoarthritis pathogenesis. Here, we report screening of cavity-creating mutants and the crystal structure of a cavity-creating L75A mutant of DUSP19 which has significantly enhanced enzyme activity in comparison to the wild-type protein. The crystal structure reveals a well-formed cavity due to the absent Leu75 side chain and a rotation of the active site- bound sulfate ion. Despite the cavity creation, residues surrounding the cavity did not rearrange significantly. Instead, a tightened hydrophobic interaction by a remote tryptophan residue was observed, indicating that the protein folding of the L75A mutant is stabilized by global folding energy minimization, not by local rearrangements in the cavity region. Conformation of the rotated active site sulfate ion resembles that of the phosphor-tyrosine substrate, indicating that cavity creation induces an optimal active site conformation. The activity enhancement by an internal cavity and its structural information provide insight on allosteric modulation of DUSP19 activity and development of therapeutics.

Key words : Activity enhancement, allosteric modulation, DUSP19, protein tyrosine phosphatase

Introduction DUSP19, which is also called stress-activated protein kinase (SAPK) pathway-regulating phosphatase 1 (SKRP1), be- Dual specificity phosphatases (DUSPs) which belong to longs to the atypical DUSP and is widely distributed in hu- the protein tyrosine phosphatases (PTPs) family, are classi- man tissues [25]. DUSP19 plays a scaffold role for the c-Jun fied into six subgroups based on sequence similarity: PRLs N-terminal kinase (JNK) signaling pathway [25, 26]. Recent (phosphatases of regenerating liver), CDC (Cell division cy- studies reported that DUSP19 expression was significantly cle) 14 phosphatases, PTENs (phosphatase and tensin homo- reduced in osteoarthritis (OA) cartilage and artificial over- logs deleted on chromosome 10), myotubularin, MKPs expression of DUSP19 restored normal function of chon- (mitogen-activated protein kinase phosphatases) and atyp- drocytes [18]. DUSP19 appeared to inhibit apoptosis of chon- ical DUSPs [14]. Due to their functions in critical cellular drocytes through dephosphorylating JNK [18]. DUSP19 processes, PTPs and DUSPs are considered as drug target up-regulation also was shown to inactivate JAK/STAT3 against cancers, diabetes, immune diseases, and neuronal pathway, resulting in inhibition of IL-1β-induced chon- diseases [2, 9, 14, 15]. Representative drug development ef- drocytes apoptosis and MMPs expression [24]. Thus, modu- forts by utilizing PTPs/DUSPs include those with PTP1B lation of enzyme activity and protein expression of DUSP19 [10], SHP2 [4] and LMPTP [17] for cancers and diabetes. has potential for therapeutics development for OA treatment. DUSP1 and DUSP3 and other DUSPs were also validated Although PTPs/DUSPs are important therapeutic targets, as drug targets for depression and immune response impair- small molecule drug development efforts targeting the phos- ment [6, 11]. phatase active site have not been easy due to the pocket’s shallow depth and amino acid sequence conservation [16, *Corresponding author 20]. Recently, allosteric regulation sites were exploited to *Tel : +82-2-2220-4020, Fax : +82-2-2220-4023 *E-mail : [email protected] overcome difficulties of the active site-targeting approaches This is an Open-Access article distributed under the terms of [4, 17]. The active site of PTPs/DUSPs appears to be flexible the Creative Commons Attribution Non-Commercial License and prone to allosteric regulation [20, 21]. In an effort to (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction understand the effects of allosteric mutations to the enzyme in any medium, provided the original work is properly cited. activity, we created various mutations in the hydrophobic Journal of Life Science 2018, Vol. 28. No. 10 1141 core of the catalytic domain of DUSP19 and analyzed their side (IPTG) concentrations and induction temperatures. The effects on enzyme activity. Hydrophobic core mutations, optimal induction condition for overexpression of the L75A which often create internal cavities, are expected to disturb mutant was 0.1 mM isopropyl β-D-1-thiogalactopyranoside the protein’s structure that may affect enzyme activity at 18℃ for overnight. Cells were harvested and resuspended allosterically. In case of activity-decreasing mutations, the in cell lysis buffer containing 50 mM Tris-Cl (pH7.5), 500 decreased enzyme activity of hydrophobic core mutations mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), (cavity-creating mutations) can be restored by chemicals 0.05%(v/v) 2-mercaptoethanol and 5%(v/v) glycerol and [23]. then sonicated. The His-tagged protein was first purified by We have previously determined the crystal structure of an affinity chromatography (TALON) column equilibrated DUSP19 at high resolutions [19]. The DUSP19 crystals are with a buffer containing 50 mM Tris-Cl (pH7.5), 500 mM of the highest quality among structurally-characterized PTPs NaCl and 0.5 mM PMSF. After a wash with the equilibration [19]. Therefore, we exploited DUSP19 to analyze the atom- buffer, the bound protein was eluted with a buffer contain- ic-level effects of hydrophobic core mutations by determin- ing the equilibration buffer plus 10 mM β-mercaptoethanol ing high resolution structures of various activity and struc- and 0.5 M imidazole. The eluted fractions were dialyzed ture-modulating mutants. In order to produce cavity in against a buffer containing 20 mM Tris-Cl (pH 7.5), 50 mM DUSP19, site-directed mutagenesis was performed in the hy- NaCl, 10 mM β-mercaptoethanol and 0.5 mM EDTA. His-tag drophobic residues (I69A, L73A, L75A, I95A, L96A and was removed by thrombin cleavage. The His-tag-removed I160A). Most of the cavity-creating mutations exhibited ac- sample was further purified by Q-Sepharose ion exchange tivity decrease as we expected. However, one mutant (L75A) and Sephacryl S-100 gel filtration chromatographies. The fi- showed a significant activity increase of 4.2-folds as com- nal gel filtration chromatography was performed with a buf- pared to the activity of the wild type enzyme. Leu75 is dis- fer containing 25 mM Tris-Cl (pH7.5), 200 mM NaCl, and tant from the active site pocket and the effect to enzyme 2 mM dithiothreitol (DTT). Purified protein was con- activity appears to be allosteric. To analyze the structural centrated to 20 mg/ml, frozen in liquid nitrogen and stored mechanism of the activity increase, we determined the crys- at 203 K. tal structure of the L75A DUSP19 mutant at 2.29 Å. The structure reveals subtle disturbances in the cavity region that PTP enzyme assay appears to induce an optimal conformation of the active site The PTP enzyme activity assay was performed using pocket for phosphorylated substrate-binding. The structure 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as a also indicates that the protein folding of the L75A mutant substrate. The assay buffer contained 20 mM Tris-Cl (pH is stabilized by a tightening hydrophobic interaction in a re- 8.0), 0.01% Triton X-100 and 5 mM DTT. Enzyme and sub- mote region. The structural information provides an im- strate concentrations were optimized to obtain linear pro- portant example of enzyme activity modulation by structural gression curves. The optimized enzyme concentration (same modification in locations distant from the catalytically active for the wild type and L75A mutant proteins) and substrate site. (DiFMUP) concentration were 20 μM and 5 μM, respectively. Fluorescence emission was measured using the fluorescence Materials and Methods spectrophotometry (Perkin Elmer, USA) with excitation and emission wavelengths of 335 nm and 460 nm, respectively. Expression and purification Kinetic parameters were estimated by observing initial ve- Site directed mutageneses were performed in the vector locity data and fitting those into the Michaelis-Menten equa- containing the DUSP19 catalytic domain (residues 65-206, tion using the program SigmaPlot 12.0. All measurements C150S) that was used in the crystallization of the wild type were performed in triplicate. protein [19]. The Stratagene mutagenesis kit was used and mutations were verified by sequencing. The recombinant Crystallization and structure determination vector containing the mutated DUSP19 was used to trans- Crystallization was performed at 291 K by using the sit- form an E.coli strain BL21 (DE3). Initial expression tests were ting-drop vapor-diffusion method. Initial trials were made performed with varying isopropyl β-D-1-thiogalactopyrano- under the same conditions as the wild type DUSP19 contain- 1142 생명과학회지 2018, Vol. 28. No. 10 ing the active site cysteine mutation (C150S) [19]. Crystals constructs, only two (I69A and L75A) were found to exhibit were grown by mixing 1.0 μl of protein (20 mg/ml) solution high-level expression of soluble proteins. The I69A mutant and an equal volume of reservoir solution containing 1.9-2.0 showed the highest protein expression in the initial ex- M ammonium sulfate. Crystals were frozen under nitrogen pression test. However, large amounts of the protein ag- stream in a cryo-protective buffer containing 2.0 M ammo- gregated during the dialysis process with the His-tag affinity nium sulfate and 33% glycerol. The DUSP19-L75A mutant purified sample. Although the L75A mutant yielded initial diffracted to 2.29 Å resolution. Diffraction data were col- expression lower than the I69A mutant, the mutant did not lected in Pohang Light Source beamline 7A (SBⅠ) [13] and have the difficulty associated with aggregation, resulting in processed by using the HKL 2000 software. The DUSP19- protein sample sufficient for crystallization.

L75A crystal belonged to the P212121 space group as that of For the PTP activity assay of the mutants, we screened the wild type crystal. Unit cell parameters were a=40.31 Å, the optimal reaction condition that yields a linear pro- b=47.31 Å, c=50.59 Å, a=b=g=90.00o. The structure of DUSP- gression curve (Fig. 1). In the chosen optimal reaction con- L75A mutant was determined by the molecular replacement dition, the enzyme activity of the L75A mutant was about technique using the wild type structure as the search target. 4.2-fold higher compared to that of the wild type. Other mu- The initial electron density map obtained by the program tant proteins either yielded low protein expression or de- CCP4 [22] confirmed that Leu75 was mutated to alanine. The creased activity (data not shown). Because cavity-creation in structure was further refined by the program PHENIX [1] the protein interior usually accompanies with cavity-filling with rebuildings by using the program COOT [7]. The final rearrangements in the cavity area that often propagate to refinement statistics are shown in Table 1. the active site residues, a cavity-creating mutant is expected to result in decrease of enzyme activity. Thus, the activity Results and Discussion increase observed in the L75A mutant is unusual and we set out to analyze the crystal structure of the mutant. Enzyme activity of the L75A mutant The structure of the wild type DUSP19 [19] was examined Structure of DUSP19-L75A and seven hydrophobic core residues with large side chains The L75A mutant (plus the C150S mutation for crystal- (Ile69, Leu73, Leu75, Ile95, Leu96 and Ile169) were identified lization [19]) was crystallized with the similar crystallization for mutation to alanine. Among the six individual mutant condition to the wild-type protein (see Methods). The C150S mutation was necessary to minimize uncontrolled oxidation Table 1. Data collection and refinement statistics of the reactive cysteine as in the crystallization of the wild Data collection

Space group P212121 Resolution (Å) 36.09 - 2.29 Cell parameter (Å) a=40.31Å, b=47.31Å, c=50.59Å, α=90.0˚, β=90.0˚, γ=90.0˚ Completeness (%) 95.8

Rmerge (%) 7.8 I/σ(I) 19.5 Refinement Number of reflections 32,070 Number of atoms 1,045 / 90 (protein/nonprotein)

Rwork/Rfree (%) 22.86 / 29.71 Rms deviations Fig. 1. Progression curves for PTP enzyme reaction. Progression Bond distances (Å) 0.0071 curves of the L75A mutant (blue diamonds, the curve Bond angles (˚) 0.814 above) and the wild type (red rectangles, the curve be- Ramachandran plot low) proteins. Concentration of both enzymes were the Most favored region (%) 94.74 same (20 mM). X-axis is the reaction time in minutes Disallowed region (%) 0.00 and Y-axis is the relative fluorescence unit. Journal of Life Science 2018, Vol. 28. No. 10 1143 type [19]. The structure of the L75A mutant was determined The cavity formed by the L75A mutation faces the catalytic by the molecular replacement technique and refined to a res- P-loop and results in subtle changes in the loop. The side olution of 2.29 Å (Table 1). In the electron density map, the chain of Val154 is rotated by 180 degrees affecting the active cavity due to the absence of the Leu75 side chain was pocket-bound sulfate ion (Fig. 3). In comparison to the wild evident. Unlike the previously-reported I187A mutant struc- type structure, the active site sulfate ion of the L75A mutant ture, which also was designed to create an internal cavity is a little displaced towards the pocket entrance and the ori- [8], there was no major cavity-filling rearrangement of main entation of oxygen atoms are reversed. These differences in chain atoms. In the structure of the I187A mutant, the the sulfate ion appears due to the rotation of Val154. I187A-containing loop moved significantly towards the cav- Superposition of the L75A structure with the phosphory- ity to reduce the size of cavity [8]. In comparison, the cav- lated substrate-bound DUSP3 structure revealed that the sul- ity-facing residues of the L75A structure did not show sig- fate ion orientation matches with that of phosphate moiety nificant movements. When the L75A mutant structure was in the phosphorylated substrate, indicating that the active superposed with the wild type structure, the displacement site of the L75A is better suited for dephosphorylation between the two structures was negligible (Fig. 2). This may reaction. The similar conformation changes in Val154 and be due to the different interactions of the two mutated resi- sulfate ion were observed in the I187A mutant, too [8], sug- dues with neighboring main chains (Fig. 2): I187A is in the gesting that the Val154 conformation is critical to align the loop between two helices, and there are little main chain phosphate group of the phosphorylated protein substrates. hydrogen bonds restricting the loop conformation. In com- However, in the I187A structure, the main chain movement parison, L75A is located in a β-strand that is a part of the caused by the cavity rearrangement appears to induce other central b-sheet and the main chain of the L75A-containing differences in the P-loop residues that may result in decrease strand makes numerous hydrogen bonds with neighboring of enzyme activity of the mutant [8]. β-strands for the β-sheet formation. Thus, it would be diffi- cult for the L75A containing region to move towards the Stabilization of a remote region cavity. Although conformation changes of residues surrounding Due to the lack of main chain movements in the L75A the cavity are minimal and subtle in the L75A mutant, sig- mutant, the cavity size is significantly larger than that of nificant conformation changes were observed in a region re- the I187A mutant. In the L75A mutant, there was a rotation mote from the cavity. A careful examination of superposed in the side chain of Val161 that placed a C-gamma atom of the residue a little towards the cavity (Fig. 2). However, A B the cavity-filling effect by the Val161 rotation is minimal.

A B

Fig. 3. Comparison of the active site-bound sulfate ion conformation. (A) The active site pockets of the L75A mutant (green) and the wild type (magenta) structures are 2- Fig. 2. Conformation and location of the mutated region. (A) superposed. The sulfate ion (SO4 ) of the L75A mutant The L75A mutant structure (green) is superposed with structure is represented in cyan. (B) The active site pock- the wild type structure (grey). The Fo-Fc omit map of et structure of the phosphorylated substrate-DUSP3 residue 75 is presented for the L75A mutant structure. complex (blue) is superposed with that of the L75A mu- (B) Locations of the Leu75 (L75) and Ile187 (I187) are tant (green). As in (a), The sulfate ion of the L75A mu- indicated as a side chain representation (magenta) on tant structure is represented in cyan. To show the phos- a ribbon diagram of the wild type structure. Position phor-tyrosine (P-Tyr) better, the active site pocket re- of the active site cysteine is indicated with a red circle. gion was slightly rotated compared to (a). 1144 생명과학회지 2018, Vol. 28. No. 10 structures of the L75A mutant and the wild type revealed analyze the folding process and dynamics of the L75A mu- that the side chain of Trp72 moved significantly towards the tant in future studies. hydrophobic core of the protein (Fig. 4). The Trp72 movement did not occur in the structure of the I187A mutant Cavity-induced activity modulation (PDB code: 4D3P) [8]. In the wild type structure, the Trp72 Creation of a cavity in proteins triggers conformational side chain is loosely packed onto the hydrophobic core con- changes of cavity-proximal residues to fill the internal va- sisting of Val146, Leu173 and Ile136. The closest distance be- cancy [8]. The movement of cavity-proximal residues results tween the neighboring side chains of Trp72 and Val146 is in another vacancy in the opposite side that induces other 5.06 Å in the wild type, whereas the distance is shortened movements of next residues. These successive movements to 4.19 Å, resulting in a tighter hydrophobic interaction in can eventually affect conformation of residues participating the L75A mutant. The distance between Trp72 and Ile136 in enzyme reaction. For example, the previously analyzed side chains is also shortened (Fig. 4). cavity-creation mutant of DUSP19 (I187A) revealed cavity- It is striking to observe a large movement of Trp72 that filling rearrangements involving movement of the mutated is remote from the cavity when the cavity-facing hydro- Ala187-containing loop towards the cavity and consecutive phobic residues including Val146, Leu173 and Ile136 did not movement of Val154 and other residues in the P-loop. As move. In general, allosteric switches in protein structures oc- a result of those movement, the PTP enzyme activity of the cur through sequential changes in the intervening residues DUSP19-I187A mutant decreased by about 40%[8]. [8]. In the case of Trp72 movement in the L75A mutant, there The 4.2-fold activity increase observed in the case of the was no movement or relay of the intervening residues. Thus, L75A mutant is different from what one usually would the Trp72 movement is likely due to a global energy mini- expect. There may be two possible explanations for the activ- mization rather than an interaction energy-driven transition. ity increase. The first is the conformational changes of Because the internal cavity in proteins destabilizes protein Val154 in the P-loop resulting in the shift of the sulfate bind- structure [3], the tighter interaction between hydrophobic ing geometry towards a more suited one for catalysis as. residues due to the Trp72 movement may offset the destabi- The effect of the shift of the sulfate ion binding geometry lization by the L75A cavity formation. The energy compen- appears complex because the activity-decreasing I187A mu- sation by stabilization of a remote region seen in the L75A tant also showed the similar geometry of sulfate ion [8]. In structure indicates that proteins function as a global entity the case of the I187A mutant, however, large cavity-filling where many parts of the protein communicate with each rearrangements are likely to induce subtle differences in con- other by a global energy balance. It will be interesting to formation and dynamics of the P-loop, which could cause decrease the enzyme activity. The next possible explanation for the increased activity may be the induced dynamics of the enzyme due to the internal cavity. Protein flexibility is required for optimal activity of enzymes [12]. The enzyme catalysis by protein tyrosine phosphatase 1B (PTP1B) also involves structural dynamics of several regions of the protein [5]. Thus, the cavity-induced activity increase of the L75A mutant of DUSP19 may be caused by the cavity-induced dynamics of the P-loop regions. In the case of the activity-decreasing I187A mutant, the cavity volume was decreased by the cavity-filling rearrangements to lessen the cavity-induced dynamics [8]. Fig. 4. Movement of a remote tryptophan. The region of the Thus, the activity increase of the L75A mutant appears to tryptophan movement is shown with superposition be- be originated from induction of the optimal active site con- tween structures of the L75A mutant (green) and the wild type (magenta). Interatomic distances between formation as well as the cavity-induced dynamics in the ac- closest atoms of Trp72 and Val146 are indicated for the tive site region. The L75A mutant had a larger fold-differ-

two structures. ence in Kcat increase (2.7-fold) than KM decrease (2.3-fold) Journal of Life Science 2018, Vol. 28. No. 10 1145

Table 2. Enzyme kinetics

-1 -1 -1 Vmax (RFU min-1) KM (mM )Kcat (RFU min mM )Kcat/KM Wild type 8,477±373 51.9±6.0 423 8.1 L75A 23,521±822 22.8±2.6 1,176 51.5

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초록：효소활성 증가 돌연변이를 함유한 DUSP19의 결정구조

주다경․전태진․류성언* (한양대학교 공과대학 생명공학과)

이중탈인산화효소(DUSP)는 성장인자활성 단백질키나제(MAPK)를 조절해서 세포성장과 분화에 관여하며 암, 당뇨병, 면역질환, 신경질환의 신약개발표적이다. DUSP 단백질군에 속하는 DUSP19는c-Jun N-말단 키나제(JNK) 를 조절하며 골관절염의 질환화과정에 관여한다. 우리는 야생형 DUSP19 에 비하여 상당히 활성이 증가된 cavity 형성 돌연변이인 DUSP19-L75A의 결정구조를 규명하였다. 결정구조는 Leu75의 곁가지가 없어진 결과로 cavity 가 잘 형성되어 있는 것을 보여주며, 활성부위에 결합한 황이온이 회전된 형태로 존재하는 것을 보여준다. Cavity 형성에도 불구하고 cavity를 둘러싸고 있는 잔기들은 그다지 재조정되지 않은 것으로 나타나며 그 대신에 멀리 떨어진 트립토판 잔기가 소수성결합을 강화하고 있는 것으로 나타나서 L75A 돌연변이의 접힘은 cavity 부위의 재조정이 아니라 글로벌 접힘 에너지 최소화 기작에 의해 안정화 되었음을 발견할 수 있었다. 회전된 활성화부위 황이온의 구조는 인산화티로신 잔기와 유사함이 발견되어 L75A 돌연변이가 최적의 활성화형태를 유도했다는 것 을 알 수 있었다. 내부 cavity에 의한 활성증가현상과 이에 대한 구조적 정보는 DUSP19의 알로스테릭 조절과 치 료제 개발에 정보를 제공한다.