molecules

Article The Role of Active-Site Plasticity in Damaged- Recognition by Human Apurinic/Apyrimidinic APE1

1,2, 1, 1 Anatoly A. Bulygin † , Alexandra A. Kuznetsova †, Yuri N. Vorobjev , Olga S. Fedorova 1,2,* and Nikita A. Kuznetsov 1,2,*

1 Institute of Chemical Biology and Fundamental Medicine, Lavrentyev Ave. 8, 630090 Novosibirsk, Russia; [email protected] (A.A.B.); [email protected] (A.A.K.); [email protected] (Y.N.V.) 2 Department of Natural Sciences, Novosibirsk State University, Pirogova St. 2, 630090 Novosibirsk, Russia * Correspondence: [email protected] (O.S.F.); [email protected] (N.A.K.) These authors contributed equally to this work. †  Academic Editor: James W. Gauld  Received: 4 August 2020; Accepted: 26 August 2020; Published: 28 August 2020

Abstract: Human apurinic/apyrimidinic (AP) endonuclease APE1 hydrolyzes phosphodiester bonds on the 50 side of an AP-site, and some damaged such as 1,N6-ethenoadenosine (εA), α-adenosine (αA), and 5,6-dihydrouridine (DHU). To investigate the mechanism behind the broad substrate specificity of APE1, we analyzed pre-steady-state kinetics of conformational changes in DNA and the during DNA binding and damage recognition. Molecular dynamics simulations of APE1 complexes with one of damaged DNA duplexes containing εA, αA, DHU, or an F-site (a stable analog of an AP-site) revealed the involvement of residues Asn229, Thr233, and Glu236 in the mechanism of DNA lesion recognition. The results suggested that processing of an AP-site proceeds faster in comparison with nucleotide incision repair substrates because eversion of a small abasic site and its insertion into the do not include any unfavorable interactions, whereas the insertion of any target nucleotide containing a damaged base into the APE1 active site is sterically hindered. Destabilization of the α-helix containing Thr233 and Glu236 via a loss of the interaction between these residues increased the plasticity of the damaged-nucleotide binding pocket and the ability to accommodate structurally different damaged nucleotides. Nonetheless, the optimal location of εA or αA in the binding pocket does not correspond to the optimal conformation of catalytic residues, thereby significantly decreasing the cleavage efficacy for these substrates.

Keywords: ; AP endonuclease; conformational dynamics; active site plasticity; apurinic/apyrimidinic site; 5,6-dihydrouridine

1. Introduction Apurinic/apyrimidinic sites (AP-sites) are regarded as common lesions that occur in DNA spontaneously or owing to the hydrolysis of N-glycosidic linkages by DNA glycosylases [1,2]. It has been estimated that >10,000 AP-sites form in every mammalian cell every day and have both mutagenic and cytotoxic effects [3–5]. The major enzyme of base excision repair (BER), human APE1 (AP endonuclease), initiates the process of removal of AP-sites from the genome [6,7]. It is thought that the key function of this enzyme is phosphodiester bond hydrolysis on the 50 side of an AP-site in DNA, thereby causing the cleavage of the deoxyribose-phosphate backbone and forming termini carrying a 20-deoxyribose-50-phosphate and 30-hydroxyl group [8]. On the other hand, regarding substrates, it has been shown that this enzyme can recognize not only various AP-sites but also some types of damaged bases, for example, oxidatively damaged [9], bulky photoproducts [10], benzene-derived

Molecules 2020, 25, 3940; doi:10.3390/molecules25173940 www.mdpi.com/journal/molecules Molecules 2020, 25, 3940 2 of 13

DNA adducts [11], etheno-derivatives of DNA bases [12,13], α-anomers of 20-deoxynucleosides [14], and 20-deoxyuridine [15]. In addition, the APE1 enzyme has 30-phosphodiesterase, 30-50-exonuclease, and 30-phosphatase activities [16,17]. X-ray crystallography of human APE1 when free [18–20] or in various complexes with DNA [21–24] has revealed that APE1 induces DNA bending and eversion of an AP-site from the double helix into the enzyme’s active site. The structural findings suggest that the rigid core of the APE1 protein causes conformational distortions of DNA because of the formation of specific contacts that are needed for a catalytically competent state. Amino acid residues Arg73, Ala74, and Lys78 come into contact with three successive DNA phosphates in the opposite strand, 50 to the AP-site. Met270 is incorporated into the DNA minor groove, thereby displacing the opposite the AP-site. Gly127 and Tyr128 broaden and span the DNA minor groove. Amino acid residue Arg177 is inserted through the major groove of DNA and enters into a hydrogen bond with the AP-site 30-phosphate. The set of DNA–enzyme contacts makes the extrahelix conformation of the abasic site stable, coordinates the active-site scissile 50-phosphate group, and results in effective enzymatic hydrolysis of the phosphodiester linkage between nucleotides. In spite of good characterization of crystal structures of human APE1 in complex with a DNA substrate containing a stable analog of a natural AP-site devoid of the OH group on the C10 atom of ribose (i.e., an F-site) or a cleaved product (DNA), currently, there are no documented structures of an APE1 complex with substrate DNA carrying a lesion (damaged base). In our previous study [25], an analysis of conformational dynamics during an interaction of this enzyme with DNA containing 1,N6-ethenoadenosine (εA), α-adenosine (αA), 5,6-dihydrouridine (DHU), or an F-site revealed that the DNA distortions induced by enzyme binding and initial-complex formation depend on the nature of the damaged nucleotide. It has been suggested that the key factor responsible for the substrate specificity of APE1 is the ability of a damaged nucleotide to get everted from a double helix and to get inserted into the damaged-nucleotide binding pocket. Nevertheless, it should be mentioned that in addition to the eversion of a target nucleotide from the substrate structure, its insertion into the active site should also be taken into account in such studies. Indeed, on the basis of literature data, we can compare APE1 activities on various damaged DNA duplexes: the recognition and cleavage of an F-site in a complementary duplex proceeds during a 1 s period [26–28], whereas the duration of recognition of such damaged nucleotides as εA, αA, or DHU is much longer and takes up to 1000 s [25,29,30]. Moreover, native nucleotides are processed in the 30-50 exonuclease reaction within 100–1000 s [31], but endoribonuclease cleavage of native ribonucleotides takes place much more slowly, up to hours [32]. Overall, it could be suggested that during the eversion of a small abasic site and its insertion into the active site of the enzyme, the deoxyribose residue does not engage in any unfavorable interactions. On the other hand, insertion of any kind of target nucleotide bearing a native or damaged base into the APE1 active site is far slower than abasic-nucleotide insertion owing to a steric hindrance. Consequently, the main objective of this study was to clarify the major steps in the mechanism underlying DNA–protein interaction that enable specific recognition of structurally varied damaged nucleotides αA, εA, or DHU as compared to an F-site. We analyzed the pre-steady-state kinetics of conformational alterations of APE1 and certain DNA substrates in the course of DNA binding. Changes in fluorescence intensity of residues were recorded to characterize conformational transitions in the protein molecule. Fluorescently labeled DNA duplexes containing a damaged nucleotide and a FRET pair of dyes made it possible to detect conformational changes in DNA that were induced by the enzyme. Molecular dynamics (MD) simulations of enzyme–substrate complexes revealed that destabilization of the α-helix containing Thr233 and Glu236 via a loss of the interaction between these residues increases the plasticity of the damaged-nucleotide binding pocket and the ability to accommodate structurally different damaged nucleotides. Moreover, the obtained data allow us to conclude that the efficacy of cleavage of different substrates is related to the coherence of the optimal location of a damaged base in the enzyme’s binding pocket and optimal distances between catalytic amino acid residues and the scissile phosphate group. Molecules 2020, 25, x FOR PEER REVIEW 3 of 14

Molecules 2020, 25, 3940 3 of 13 enzyme’s binding pocket and optimal distances between catalytic amino acid residues and the scissile phosphate group. 2. Results and Discussion 2. Results and Discussion 2.1. Relative Cleavage Activity of APE1 2.1. Relative Cleavage Activity of APE1 PAGE analysis of product accumulation in the course of the interaction of APE1 with every X-substratePAGE (X analysi= αA,s DHU, of product F-site, accumulation or εA as damage) in the was course performed of the first.interaction Direct registrationof APE1 with of every product formationX-substrate helped (X = to αA, rank DHU, the effi F-site,cacy or of DNAεA as cleavage damage) as was follows: performed F-site first.> DHU Direct>> registrationαA > εA, in of line withproduct other formation studies [25 helped,30,33 ].to rank the efficacy of DNA cleavage as follows: F-site > DHU >> αA > εA, in line with other studies [25,30,33]. 2.2. Effects of Mg2+ Ions on DNA Binding and Catalysis 2.2. Effects of Mg2+ Ions on DNA Binding and Catalysis The DHU-substrate containing the smallest damaged base is cleaved with greater The DHU-substrate containing the smallest damaged pyrimidine base is cleaved with greater efficiency than αA and εA; therefore, this substrate was chosen for further detailed analysis of efficiency than αA and εA; therefore, this substrate was chosen for further detailed analysis of the the substrate-binding process. It should be mentioned that the activity of APE1 depends on the substrate-binding process. It should be mentioned that the activity of APE1 depends on the concentration of Mg2+ [14,34]. Typically, the AP-site cleavage activity is tested in the presence concentration of Mg2+ [14,34]. Typically, the AP-site cleavage activity is tested in the presence of ≥5 of 5 mM Mg2+ [14,35–38], but the cleavage of DNA bearing a damaged base is tested at a low mM Mg2+ [14,35–38], but the cleavage of DNA bearing a damaged base is tested at a low ≥ 2+ concentrationconcentration of of Mg Mg2+,, within within the the 0.01–0.50.01–0.5 mM range [12,14,35,36,39]. [12,14,35,36,39]. Consequently, Consequently, to to verify verify the the 2+ effiefficiencyciency of of APE1 APE1 binding binding to to the the DHU-containing DHU-containing DNADNA duplexduplex at various various Mg Mg2+ levels,levels, the the kinetics kinetics of of substrate–enzymesubstrate–enzyme complex complex formation formation and and of the of catalysis the catalysis were were investigated investigated by the by stopped-flow the stopped- methodflow 2+ (Figuremethod1a). (Figure Without 1a). Mg Withoutions, Mg only2+ ions, a decreased only a decrease phased of phase Trp fluorescence of Trp fluorescence intensity intensity was recorded, was reflectingrecorded, the reflecting formation the of formationan enzyme–substrate of an enzyme complex.–substrate With complex. an increase With in an the increase concentration in the of Mgconcentration2+, this process of Mg accelerated,2+, this process and the accelerated, growth phase and the appeared growth atphase later appeared reaction at time later points, reaction indicating time thepoints, progression indicating of a the catalytic progression reaction of a and catalytic subsequent reaction dissociation and subsequent of the dissociation enzyme–product of the enzyme complex.– Indeed,product the complex. onset of theIndeed, phase the of onset the increase of the inphase intensity of the at increase late time in points intensity is consistent at late time with points the onset is ofconsistent the product with accumulation the onset of revealedthe product by accumula PAGE (Figuretion revealed1b). by PAGE (Figure 1b).

(a) (b)

FigureFigure 1. The 1. The influence influence of Mg of2+ Mgions2+ onions the APE1on the interaction APE1 interaction with the DHU-substrate. with the DHU- (substratea) Stopped-flow. (a) kineticStopped traces-flow of kinetic Trp fluorescence. traces of Trp The fluorescence. first syringe The contained first syringe 1.0 µcontainedM APE1 1.0 that μM was APE1 incubated that was with incubated with different concentrations of MgCl2. The second syringe held 1.0 μM DHU-substrate different concentrations of MgCl2. The second syringe held 1.0 µM DHU-substrate and an equivalent and an equivalent amount of MgCl2. Final MgCl2 concentrations are indicated beside the kinetic amount of MgCl2. Final MgCl2 concentrations are indicated beside the kinetic trace. (b) Reaction producttrace. ( accumulationb) Reaction product as evidenced accumulation by PAGE. as evidenced by PAGE.

2.3.2.3. Analysis Analysis of of DNA DNA Conformational Conformational ChangesChanges in the Course of of Interaction Interaction with with APE1 APE1 ToTo analyze analyze conformational conformational changeschanges in in DNA during the the interaction interaction with with APE1, APE1, we we used used FRET-X-substrates,FRET-X-substrates, where where X X= =C, C, F-site,F-site, oror DHU (Figure 22).). In In the the FRET FRET experiments, experiments, to to prevent prevent the the possiblepossible eff effectsects associated associated with with unsaturation unsaturation of of thethe activeactive site of the enzyme enzyme by by Mg Mg2+2, +we, we employed employed a a high MgCl2 concentration (5.0 mM) in reaction buffer. These conditions clearly illustrated the high MgCl2 concentration (5.0 mM) in reaction buffer. These conditions clearly illustrated the difference between the DNA-binding process alone (absence of Mg2+ in the active site) and DNA binding with a subsequent catalytic step (the active site saturated by the Mg2+ ion). Duplex FRET-C served as a control for conformational changes occurring during the formation of a nonspecific complex. As presented Molecules 2020, 25, x FOR PEER REVIEW 4 of 14

2+ differenceMolecules 2020 between, 25, 3940 the DNA-binding process alone (absence of Mg in the active site) and DNA4 of 13 binding with a subsequent catalytic step (the active site saturated by the Mg2+ ion). Duplex FRET-C served as a control for conformational changes occurring during the formation of a nonspecific complex.in Figure 2 As, the presented interaction in Figureof APE1 2, with the interaction the FRET-C of duplex APE1 (bluewith the trace) FRET leads-C to duplex a slight (blue increase trace) leadsin the to FRET a slight signal increase within inthe the period FRET up signal to time within point the 0.1 periods. When up a tocatalytically time point inactive 0.1 s. When form of a 2+ catalyticallyapo-APE1 interacted inactive withform theof apo FRET-F-substrate-APE1 interacted in the with absence the FRET of Mg-F-substrateions (black in the trace) absence and catalysis of Mg2+ wasions blocked, (black trace) only andthe phase cataly ofsis the was FRET blocked, signal only decrease the phase was recorded. of the FRET The decrease signal decrease in the FRET was recorded.signal indicates The decrease that FAM in and the FRET the BHQ1 signal quencher indicates became that FAM spatially and the close BHQ1 to each quencher other, and became this spatiallyevent may close be associatedto each other, with and bending this event of the may DNA be associated backbone. with Otherwise, bending in of the the presence DNA backbone. of Mg2+ Otheionsrwise, (red trace), in the in presence the region of Mg of the2+ ions kinetic (red curves trace), upin the to 0.02 region s, a of rapid the kinetic decrease curves in the up FRET to 0.02 signal s, a rapidtakes place, decrease reflecting in the the FRET formation signal takes of the place, enzyme–substrate reflecting the complex, formation followed of the by enzyme a growth–substrate phase complex,between timefollowed points by 0.05 a growth and 5 s. phase The increase between in time the FRET points signal 0.05 isand associated 5 s. The with increase an increase in the FRET in the signaldistance is associated between FAM with and an theincrease BHQ1 in quencher, the distance and between this event FAM is possible and the afterBHQ1 the quencher, catalytic stepand andthis eventdissociation is possible of the enzyme–productafter the catalytic complex. step and The dissociation visually obvious of the decrease enzyme in–product FRET signal complex. amplitude The 2+ visuallyfor FRET-F-substrate obvious decrease in the in presence FRET signal of Mg amplitudeions is related for FRET to the-F-substrate overlap of in the the complex presence formation of Mg2+ ionsstage is with related catalytic to the stageoverlap and ofdissociation the complex of formation the enzyme–product stage with catalytic complex, stage thereby and dissociation leading to anof theincrease enzyme in the–product FRET signal.complex, thereby leading to an increase in the FRET signal.

Figure 2. Changes inin thethe FRETFRET signal signal during during the the interaction interaction of of APE1 APE1 with with each each FRET-X-substrate FRET-X-substrate in the in 2+ theabsence absence “ ” or“−” presence or presence “+” of“+” 5.0 of mM 5.0 MgmM .Mg FRET2+. FRET traces traces of the of interaction the interaction of APE1 of with APE1 the with FRET-F- the − 2+ FRETand FRET-DHU-substrates-F- and FRET-DHU-substrates in the absence in the ofabsence Mg haveof Mg been2+ have reported been reported earlier [25 earlier]. [25].

As depicted in Figure 22,, thethe interactioninteraction ofof APE1APE1 withwith thethe FRET-DHU-substrateFRET-DHU-substrate inin thethe absenceabsence ofof 2+ Mg2+ ionsions induces induces a adecrease decrease in in the the FRET FRET signal signal up up to to time time point point 1 s, 1 thus s, thus reflecting reflecting the the formation formation of theof the complex. complex. These These data data revealed revealed that that the the bending bending of DNA of DNA containing containing DHU DHU is ~10 is ~10-fold-fold slower slower in 2+ comparisonin comparison with with an abasic an abasic site site(1 s (1and s and 0.1 s, 0.1 respectively). s, respectively). By contrast, By contrast, in the in presence the presence of Mg of2+ Mgions, theions, growth the growth phase phase characterizing characterizing the thecatalytic catalytic stage stage was was not not detectable, detectable, as asin in the the case case of of the the F F-site,-site, indicating that that the the duplex duplex bending bending alone alone is not is a su notffi cient a sufficient process for process the formation for the offormation the catalytically of the catalyticallycompetent state competent of the active state of site. the active site. 2.4. MD Simulations 2.4. MD Simulations Structures of the complex of APE1 with F-site-containing DNA and a Mg2+ ion (retrieved from Structures of the complex of APE1 with F-site-containing DNA and a Mg2+ ion (retrieved from X-ray structures 1DE8 and 4IEM) were equilibrated within 10 ns to stabilize total potential energy X-ray structures 1DE8 and 4IEM) were equilibrated within 10 ns to stabilize total potential energy of of the complex. As shown in Figure3a, the equilibrated structures of the complex of APE1 with the complex. As shown in Figure 3a, the equilibrated structures of the complex of APE1 with F-site-containing DNA and a Mg2+ ion have very similar positions of the F-site located in the active F-site-containing DNA and a Mg2+ ion have very similar positions of the F-site located in the active site; the same is true for the Mg2+ ion. Equilibrium 100 ns MD trajectories were generated for both APE1–DNA complexes and did not reveal significant differences in the final structures.

Molecules 2020, 25, x FOR PEER REVIEW 5 of 14 site; the same is true for the Mg2+ ion. Equilibrium 100 ns MD trajectories were generated for both APE1–DNA complexes and did not reveal significant differences in the final structures. Specific distances between atoms of the F-site and active-site amino acid residues did not significantly change throughout the MD trajectory (Figure 3b). The phosphate group of the F-site forms stable H-bonds with Asn212 and Tyr171 and engages in a strong interaction with the Mg2+ ion (Figure 3c). The weak unstable contact of the bridging oxygen atom of the scissile phosphate group with His309 features the average distance of 3.5 Å during the final 50 ns of the MD trajectory. The distance between the carboxyl group of the catalytic Asp210 residue and O5′ of the F-site was also very stable. The distances observed in the complex of the enzyme with F-site-containing DNA were used as reference values of the catalytically competent state of the active site for comparisons with Molecules 2020, 25, 3940 5 of 13 other substrates.

(a) (b)

(c)

FigureFigure 3. 3. MolecularMolecular dynamics dynamics (MD (MD)) structure structure and and trajectories trajectories of of the the APE1 APE1 complex complex with with DNA DNA containingcontaining the the F F-site.-site. (a (a) )Comparison Comparison of of the the 1DE8 1DE8 X X-ray-ray structure structure (red) (red) with with equilibrated equilibrated structures structures ofof the the complex complex of of APE1 APE1 with with F F-site-containing-site-containing DNA DNA and and a a Mg Mg2+2+ ionion derived derived from from 1DE8 1DE8 (blue) (blue) or or 4IEM4IEM (green). (green). The The F F-site-site and and Mg Mg2+2 +ionion are are highlighted highlighted in in the the same same color color as as proteins. proteins. (b (b) )The The structure structure ofof APE APE11′s0s binding binding pocket pocket adapted adapted to to an an F F-site.-site. The The F- F-sitesite and and key key amino amino acid acid residues residues o off the the damageddamaged-nucleotide–binding-nucleotide–binding pocketpocket areare shown.shown. ( c()c Changes) Changes in in distances distances between between catalytic catalytic amino amino acid acidresidues residues and and the scissilethe scissile phosphate phosphate group group in the in dynamicthe dynamic trajectories trajectories for the for complexesthe complexes of APE1 of APE1 with withDNA DNA containing containing theF-site. the F-site.

OnSpecific the basis distances of the equ betweenilibrated atoms structure of the of F-site the complex and active-site of APE1 aminowith F- acidsite-containing residues did DNA, not initialsignificantly structures change of the throughout APE1 complex the MD with trajectory DNA containing (Figure3 b).DHU, The αA, phosphate or εA were group obtained. of the F-siteThey forms stable H-bonds with Asn212 and Tyr171 and engages in a strong interaction with the Mg2+ ion (Figure3c). The weak unstable contact of the bridging oxygen atom of the scissile phosphate group with His309 features the average distance of 3.5 Å during the final 50 ns of the MD trajectory. The distance between the carboxyl group of the catalytic Asp210 residue and O50 of the F-site was also very stable. The distances observed in the complex of the enzyme with F-site-containing DNA were used as reference values of the catalytically competent state of the active site for comparisons with other substrates. On the basis of the equilibrated structure of the complex of APE1 with F-site-containing DNA, initial structures of the APE1 complex with DNA containing DHU, αA, or εA were obtained. They were equilibrated also within 10 ns to stabilize the complex’s total potential energy. Next, equilibrium Molecules 20202020, 2525, 3940x FOR PEER REVIEW 6 of 14 13 were equilibrated also within 10 ns to stabilize the complex’s total potential energy. Next, equilibriumMD trajectories MD (100–180 trajectories ns) were (100– obtained180 ns) were at 300 obtained K (room attemperature) 300 K (room for temperature)every complex for of everyAPE1 withcomplex DNA. of APE1 with DNA. The productive MD simulation of the structure of the APE1 complex with DHU after energy optimization and simulated annealing revealed two stable states of the damaged nucleotide in the active site site (Figure (Figure4a). 4a In). the In the initial initial part partof the of MD the trajectory MD trajectory (Figure 4 (Figureb), the Asn229 4b), the residue Asn229 managed residue managedto form an to H-bond form an with H-bond O2 of with DHU, O2 thus of leadingDHU, thus to the leading stabilization to the ofstabilization this state. Then,of this Asn229 state. Then, came Asn229into contact came with into Asn226contact andwith unblockedAsn226 and the unblocked DHU base. the Moreover,DHU base. analysis Moreover, of theanalysis MD trajectories of the MD trajectoriesindicated that indicated the loss that of the the direct loss of Asn229–DHU the direct Asn229 contact–DHU is accompanied contact is accompanied by the loss ofby the the H-bond loss of thebetween H-bond residues between Thr233 residues and Thr233 Glu236, and thus Glu236, resulting thus in resulting significant in loopsignifi reorganizationcant loop reorganization (Figure4a), (Figurethereby 4aallowing), thereby the allowing DHU base the to DHU acquire base mobility, to acquire shifting mobility, the whole shifting nucleotide the whole deeper nucleotide into the deeperactive site into of the the active enzyme. site Interestingly,of the enzyme. no Interestingly, steric hindrance no issteric caused hindrance by the aminois caused acid by backbone the amino of acidthe loop backbone residues, of but the rather loop residues, the loop reorganizationbut rather the leads loop to reorganization an Asn229 shift leads together to an with Asn229 the amino shift togetheracid backbone. with the This amino phenomenon acid backbone. is associated This with phenomenon the rotation is of associated the Asn229 with side the chain rotation and the of loss the Asn229of a direct side contact chain withand O2the ofloss the of DHU a direct base. contact This relocation with O2 of of the DHU DHU leads base. to a This decrease relocation in the of average DHU leadistanceds to a between decrease His309 in the andaverage the scissile distance phosphate between group His309 from and 4.5 the to scissile 3.9 Å, whichphosphate enables group the from catalytic 4.5 tostate 3.9 ofÅ ,the which active enables site. the catalytic state of the active site.

(a) (b)

Figure 4. 4. MDMD structures structures and and trajectories trajectories of the of APE1 the APE1complex complex with DNA with containing DNA containing DHU. (a DHU.) The

(structurea) The structure of APE of1′s APE1 binding0s binding pocket pocket adapted adapted to DHU to DHU blocked blocked by Asn229 by Asn229 (reddish) (reddish) or transformed or transformed to theto the catalytic catalytic state state (greenish). (greenish). DHU, DHU, Asn229, Asn229, Thr233, Thr233, and and Glu236 Glu236 are are highlighted highlighted in in brown brown (blocked state) or dark cyan (catalytic state). Key Key amino amino acid acid residues residues of the the damaged damaged-base–binding-base–binding pocket are shown. ( (bb)) Changes Changes in in distances distances between between catalytic catalytic amino amino acid acid residues and the scissile phosphate group in the dynamic trajectories of the complexes of APE1 with DNA containing DHU.DHU.

It is noteworthy that that,, in the case of ααAA (Figure5 5),), thethe decreasedecrease inin thethe distancedistance betweenbetween His309His309 and the the scissile scissile phosphate phosphate group group was was also also accompanied accompanied with with a loss a of loss the of interaction the interaction between between Thr233 Thr233and Glu236 and Glu236 and destabilization and destabilization of the loop. of the Nevertheless, loop. Nevertheless, the subsequent the subsequent turn and turn shift an ofd shift the α ofA thebase αA during base MDduring simulation MD simulation cause an cause increase an increase in the distance in the distance from another from another catalytic catalytic residue, residue, Asn212, Asn212,from 3.4 from to 4.4 3.4 Å, to supporting 4.4 Å , supporting the disruption the disruption of the catalytic of the conformation. catalytic conformation. These data These indicate data indicatethat the optimalthat the optimal location location of the α ofA the base αA in base theenzyme’s in the enzyme’s binding binding pocket pocket does not does correspond not correspond to an tooptimal an optimal conformation conformation of catalytic of aminocatalytic acid amino residue acid Asn212, residue thereby Asn212, correlating thereby with correlating experimental with experimentaldata on reduced data effi onciency reduced of cleavage efficiency of theof cleavageαA-substrate of the in αA comparison-substrate with in comparison the DHU-substrate. with the TheseDHU-substrate. findings also Thes suggeste findings that conformationalalso suggest that instability conformational of the loop instability region may of the be importantloop region for may the berecognition important of for a damaged the recognition nucleotide. of a damaged nucleotide. The MD simulation of the complex of APE1 with DNA containing εA (Figure6) also uncovered the loss of the direct Thr233–Glu236 interaction in the initial part of the trajectory. Moreover, distances between residues Asn212 and His309 and the phosphate group are growing during the initial 30–50 ns of the simulation and stabilize at 4.2 and 5.9 Å, respectively, which supports the full disruption of catalytic-network contacts.

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(a) (b)

Figure 5. MD structures and trajectories of the APE1 complex with DNA containing αA. (a) The structure of APE1′s binding pocket adapted to the initial state of αA (reddish) or in the disrupted final state (greenish). αA, Asn229, Thr233, and Glu236 are highlighted in brown (initial state) or dark cyan (disrupted state). Key amino acid residues of the damaged-base–binding pocket are shown. (b)

Changes in distances(a between) catalytic amino acid residues and the scissile phosphate(b) group in the dynamic trajectories for the complexes of APE1 with DNA containing αA. FigureFigure 5. MD 5. MD structures structures and andtrajectories trajectories of the of APE1 the APE1 complex complex with with DNA DNA containing containingαA. αA. (a) The (a) The structure

of APE1structureThe0 MDs binding ofsimulation APE pocket1′s binding of adaptedthe pocketcomplex to adapted the of initialAPE1 to the statewith initial DNA of αstateA co (reddish) ntainingof αA (reddish) orεA in (Figure the or disruptedin 6)the also disrupted uncovered final state (greenish).the lossfinal state of α the A,(greenish). directAsn229, Thr233αA, Thr233, Asn229,–Glu236 and Thr233, Glu236 interaction and Glu236 are highlighted in are the highlighted initial in partbrown in brown of (initial the (initial trajectory state) state) or .or Moreover, darkdark cyan (disrupteddistancescyan (disrupted between state). Key state).residues amino Key acidAsn212 amino residues acid and residues ofHis309 the damaged-base–binding of and the damagedthe phosphate-base–binding group pocket pocketare are growing shown. are shown. (duringb) Changes(b) the Changes in distances between catalytic amino acid residues and the scissile phosphate group in the initialin distances 30–50 betweenns of the catalyticsimulation amino and acidstabilize residues at 4.2 and and the 5.9 scissileÅ , respectively, phosphate which group supports in the dynamic the full dynamic trajectories for the complexes of APE1 with DNA containing αA. disruptiontrajectories of for catalytic the complexes-network of contacts. APE1 with DNA containing αA. The MD simulation of the complex of APE1 with DNA containing εA (Figure 6) also uncovered the loss of the direct Thr233–Glu236 interaction in the initial part of the trajectory. Moreover, distances between residues Asn212 and His309 and the phosphate group are growing during the initial 30–50 ns of the simulation and stabilize at 4.2 and 5.9 Å , respectively, which supports the full disruption of catalytic-network contacts.

(a) (b)

FigureFigure 6. MD 6. structuresMD structures and trajectories and trajectories of the of APE1 the APE1 complex complex with with DNA DNA containing containingεA. (εaA.) The (a) structureThe structure of APE1′s binding pocket adapted to the initial state of εA (reddish) or in the disrupted of APE10s binding pocket adapted to the initial state of εA (reddish) or in the disrupted final state (greenish).final stateεA, (greenish). Asn229, ε Thr233,A, Asn229, and Thr233, Glu236 and are Glu236 highlighted are highlighted in dark in cyandark cyan (initial (initial state) state) or or brown brown (disrupted state). Key amino acid residues of the damaged-base–binding pocket are (disrupted state). Key amino acid residues of the damaged-base–binding pocket are presented. presented. (b) Changes in distances between catalytic amino acid residues and the scissile phosphate (b) Changes in distances(a) between catalytic amino acid residues and the scissile(b) phosphate group in the group in the dynamic trajectories for the complexes of APE1 with DNA containing εA. dynamicFigure trajectories 6. MD structures for the andcomplexes trajectories of APE1 of the with APE1 DNA complex containing with DNAεA. containing εA. (a) The structure of APE1′s binding pocket adapted to the initial state2+ of εA (reddish) or in the disrupted A comparison of all the structures revealed that the Mg ion2 +engages in a stable interaction with aA nonbridged comparisonfinal state (greenish). oxygen of all theatomεA, structuresAsn229, of the Thr233, scissile revealed and phosphate Glu236 that are the group.highlighted Mg Indeed,ion in dark engages superposition cyan (initial in a stable state) of the or interaction MD withstructures a nonbridgedbrown at (disrupted minimal oxygen state).total atom potential Key of amino the energy scissile acid (Figure residues phosphate 7a of) showed the group. damaged that Indeed, the-base Mg–bi2+ superpositionnding ion is pocket moved are to of the the MD 2+ structuresactivepresented. at site minimal together (b) Changes total wi potentialth in distances the phosphate energy between (Figure catalytic group7a) amino of showed the acid damaged that residues the Mgand nucleotide. the ionscissile is moved Therefore,phosphate to the the active site togetherexperimentallygroup with in the thedetermineddynamic phosphate trajectories dependence group for the ofof complexes thethe enzymatic damaged of APE1 activity nucleotide.with DNA on containingthe Therefore,concentration εA. the of experimentally Mg2+ most determinedlikely is dependencerelated to saturation of the enzymaticof the active activity site by onMg the2+, which concentration is strongly of needed Mg2+ tomost coordi likelynate is the related A comparison of all the structures revealed that the Mg2+ ion engages in a stable interaction with to saturation of the active site by Mg2+, which is strongly needed to coordinate the scissile phosphate a nonbridged oxygen atom of the scissile phosphate group. Indeed, superposition of the MD group.structures The overlap at minimal of alltotal structures potential energy indicated (Figure that 7a both) showed functionally that the Mg important2+ ion is moved loops to containing the Arg177active and site Met270, together which with intercalate the phosphate into DNA group to of stabilize the damaged the extrahelical nucleotide. state Therefore, of the damaged the nucleotide,experimentally are very determined stable in dependence the complexes of the withenzymatic all the activity tested on damaged the concentration nucleotides. of Mg2+ Otherwise, most the looplikely containing is related to Asn229 saturation/Thr233 of the/Glu236 active underwentsite by Mg2+,significant which is strongly damage-dependent needed to coordi reorganization,nate the indicating the important role of this loop in the recognition of the damaged nucleotide (Figure7a). Indeed, the close-up view illustrates the plasticity of this loop depending on the size of the damaged nucleotide (Figure7b). Molecules 2020, 25, x FOR PEER REVIEW 8 of 14

scissile phosphate group. The overlap of all structures indicated that both functionally important loops containing Arg177 and Met270, which intercalate into DNA to stabilize the extrahelical state of the damaged nucleotide, are very stable in the complexes with all the tested damaged nucleotides. Otherwise, the loop containing Asn229/Thr233/Glu236 underwent significant damage-dependent reorganization, indicating the important role of this loop in the recognition of the damaged Moleculesnucleotide2020, 25, (Figure 3940 7a). Indeed, the close-up view illustrates the plasticity of this loop depending on8 of 13 the size of the damaged nucleotide (Figure 7b).

(a) (b)

FigureFigure 7. (a 7.) Superposition(a) Superposition of of the the MD MD structures structures of the the APE1 APE1 complex complex with with DNA DNA containing containing an F- ansite F-site

(red),(red), DHU DHU (dark (dark green), green),εA εA (reddish), (reddish), or orα αAA (green). (green). Damaged Damaged nucleotides nucleotides inserted inserted into into APE APE11′s 0s bindingbinding pocket. pocket. Stable Stable positions positions of of intercalating intercalating loops loops designated designated as asArg177 Arg177 and and Met270 Met270 are shown are shown.. (b) Close-up(b) Close view-up view of theof the damage damage recognition recognition looploop adapted to to different different dam damagedaged nucleotides. nucleotides.

TakenTaken together, together, the the obtained obtained data data mean mean thatthat the movement movement of of the the recognition recognition loop loop is associated is associated with optimal accommodation of the damaged base in the pocket of the active site. For instance, in the with optimal accommodation of the damaged base in the pocket of the active site. For instance, in the case of DHU, two stable conformational states exist, in which DHU is blocked by Asn229 or fully case ofinserted DHU, into two the stable active conformationalsite. The hydrolysis states of the exist, phosphodiester in which bond DHU is is possible blocked only by in Asn229 the second or fully insertedstate into due the to active the formation site. The hydrolysis of optimal of distances the phosphodiester to catalytic bond residues is possible Asn212 only and in His309. the second stateExperi due tomental the formation data revealed of optimal that D distancesHU-substrate to catalytic cleavage residues (Figure 1bAsn212) reaches and a His309. plateau Experimental at ~30%, dataindicating revealed the that saturation DHU-substrate of the second cleavage catalytic (Figure state1 atb) this reaches level and a plateau suggesting at~30%, that the indicating transition the saturationbetween of these the second states is catalytic associated state with at energy this level costs and and suggesting proceeds slowly. that the On transition the other betweenhand, the these statescleavage is associated of αA- with and εA energy-substrates costs andis much proceeds less efficient slowly. because On the the other optimal hand, position the cleavage of these of basesαA- and εA-substratesin the binding is much pocked less doesefficient not because correspond the to optimal optimal position distances of between these bases the in scissile thebinding phosphate pocked doesgroup not correspond and catalytic to amino optimal acid distances residues. Indeed, between in the case scissile of the phosphate αA base, Asn212 group moves and catalytic away and amino cannot come into contact with the phosphate group, but in the case of the biggest base (εA), the acid residues. Indeed, in the case of the αA base, Asn212 moves away and cannot come into contact contacts with both Asn212 and His309 are lost. ε with the phosphateOf note, alignment group, but of amino in the acidcase sequencof the biggestes of AP base ( A), the contacts from Danio with rerio both, Xenopus Asn212 and His309laevis are, and lost. Drosophila melanogaster indicates that Thr233 and Glu236 are fully conserved among these Ofspecies, note, whereas alignment Asn229 of amino is substituted acid sequences by Thr only of in AP the endonucleases AP endonuclease from fromDanio Xenopus rerio laevis, Xenopus. laevisTherefore,, and Drosophila our data melanogaster on active-siteindicates plasticity thatseem Thr233 to reflect and a common Glu236 feature are fully of AP conserved endonucleases among of these species,the APE1whereas structural Asn229 family. is substituted Moreover, by because Thr only of the in X the-ray AP data endonuclease analysis, it is from knownXenopus that AP laevis . Therefore,endonuclease our data APE1 on active-sitehas a rigid core plasticity that slightly seem differs to reflect between a common the free feature enzyme of and AP the endonucleases enzyme of thecomplexed APE1 structural with an abasic family. DNA. Moreover, Because there because are no of structural the X-ray data data on the analysis, complexes it is of known APE1 with that AP endonucleaseother types APE1 of damaged has a rigid nucleotides, core that we slightlyassumed di thatffers this between rigidity theis a free common enzyme feature and of the APE1 enzyme proteins. The results obtained in the present study indicate that during recognition of various complexed with an abasic DNA. Because there are no structural data on the complexes of APE1 with damaged nucleotides, the DNA-binding site of APE1 must undergo conformational changes to other types of damaged nucleotides, we assumed that this rigidity is a common feature of APE1 proteins. The results obtained in the present study indicate that during recognition of various damaged nucleotides, the DNA-binding site of APE1 must undergo conformational changes to accommodate the nucleotides containing the damaged base. Therefore, these findings constitute the evidence of high plasticity of the damaged-nucleotide–binding pocket of APE1.

3. Materials and Methods

3.1. Protein Expression and Purification Wild-type APE1 was expressed and purified as described previously [40,41]. The protein concentration was measured by the Bradford method [42]; the stock solution was stored at 20 C. − ◦ Molecules 2020, 25, 3940 9 of 13

3.2. Oligodeoxynucleotides (ODNs) Sequences of the ODNs employed in this study are given in Table1. These ODNs were prepared by widely accepted phosphoramidite methods by means of an ASM-700 synthesizer (BIOSSET Ltd., Novosibirsk, Russia) from phosphoramidites that were bought from Glen Research (Sterling, VA, USA). α-20-Deoxyadenosine phosphoramidite was purchased from ChemGenes Corp. (Wilmington, MA, USA). The synthesized ODNs were separated from solid support by means of ammonium hydroxide in keeping with the manufacturer’s instructions. The deprotected ODNs were purified via high-performance liquid chromatography. Concentrations of the ODNs were computed from A260. ODN duplexes were generated via annealing of modified and complementary strands in a molar ratio of 1:1.

Table 1. DNA sequences and structures of modified nucleotides *.

Shorthand Sequences of DNA Duplexes

X-substrate 50-TCTCTCXCCTTCC-30 X = F-site, DHU, εA, or αA 30-AGAGAGGGGAAGG-50

FRET-X-substrate 50-FAM-GCTCAXGTACAGAGCTG-30 Y = F-site, DHU, εA, αA, or C 30-CGAGTGCATGTCTCGAC-BHQ1-50 * FAM is 6-carboxyfluorescein, BHQ1 is black hole quencher.

3.3. PAGE (Polyacrylamide Gel Electrophoresis) and Enzymatic Analyses The endonuclease assay was carried out in reaction buffer (50 mM Tris-HCl pH 7.5, 50 mM KCl, 1.0 mM DDT, 1.0 mM EDTA, 7% [v/v] of glycerol, and various levels of MgCl2 within 0.0–1.0 mM). The reaction mixture contained 1.0 µM DNA substrate and 1.0 µM APE1. To increase enzyme stability during the experimental procedures, all the experiments were conducted at 25 ◦C. The reactions were allowed to proceed at 25 ◦C and were stopped by a gel-loading dye containing 50 mM EDTA and 7 M urea and were next loaded onto a 20% polyacrylamide gel (w/v) including 7 M urea. Formation of the product and disappearance of the substrate were investigated by autoradiography and quantitated via scanning densitometry using Gel-Pro Analyzer software v.4.0 (Media Cybernetics, Rockville, MD, USA).

3.4. Stopped-Flow Measurements with Fluorescence Detection These measurements were taken mostly as described before [43,44]. In short, we utilized a SX.18MV stopped-flow spectrometer (Applied Photophysics Ltd., Leatherhead, UK) equipped with an optical cell with 2 mm path length and a 150 W Xe arc lamp. The dead time of this equipment is known: 1.4 ms. Trp fluorescence was excited at 290 nm (λex) and monitored at >320 nm (λem) as transmitted by the WG-320 filter (Schott, Mainz, Germany). 6-Carboxyfluorescein (FAM) residue fluorescence was excited at 494 nm and detected at >515 nm, as transmitted by the OG-515 filter (Schott, Mainz, Germany). The experiments on Trp fluorescence detection were carried out at 25 ◦C with catalytically active APE1 in a buffer composed of 50 mM Tris-HCl pH 7.5, 1.0 mM DTT, 50 mM KCl, 7% of glycerol (v/v), and various levels of MgCl2 within 0.0–1.0 mM. Detection of the process of DNA binding using 2+ a FRET signal was performed at 25 ◦C with Mg -free catalytically inactive apo-APE1 in a buffer composed of 50 mM Tris-HCl pH 7.5, 1.0 mM EDTA, 1.0 mM DTT, 50 mM KCl, and 7% of glycerol (v/v). In all the experiments, when the concentration of Mg2+ was declared to be zero, first of all, the catalytic activity of APE1 was abrogated by the treatment with EDTA. For this purpose, the solution of APE1 was incubated with 1.0 mM EDTA for 5 min to chelate any divalent metal ions and to obtain catalytically inactive apo-APE1. The reaction solution was supplemented with a certain concentration of MgCl2 when required. Of note, our previous data [34] have revealed that supplementation of the solution of apo-APE1 and 1.0 mM EDTA by MgCl2 (even at 0.05 mM) restores catalytic activity to some Molecules 2020, 25, 3940 10 of 13 extent with subsequent saturation of the enzyme by the Mg2+ ion with the increasing concentration of MgCl2. Therefore, in the present report, we used the same design of experiments. APE1 was added into one of the syringes of the instrument and was mixed rapidly in a reaction chamber with a DNA substrate that came from the other syringe. Concentrations of APE1 and the DNA substrate were 1.0 µM in all the assays. The reported concentrations of reactants correspond to the concentrations in the reaction chamber upon mixing. As a rule, each trace depicted in the figures is the mean of 4 fluorescent traces acquired in individual experiments. ≥ Protein conformational alterations were examined via changes in fluorescence intensity of Trp. FRET-X-substrates modified at 50 ends with the dye–quencher couple FAM/BHQ1 (Table1) were analyzed by Förster resonance energy transfer (FRET) measurements. The latter detected changes in the distance between the quencher and dye during DNA helix distortion in the course of APE1–DNA complex formation. In figures, for clarity, the curves were moved apart by hand. This approach does not influence the results of fitting: background fluorescence is fitted for each curve individually.

3.5. MD Simulations Molecular modeling of the complex of APE1 with each DNA duplex containing DHU, εA, αA, or an F-site (Table1) involved the following stages: (i) building up the initial atomic structure of the complex, (ii) atomic-structure refinement for the complex through simulated annealing and energy optimization, (iii) productive MD modeling of every complex and collection of a representative series of instant structures throughout an equilibrated MD trajectory. A comparison of several X-ray crystal structures was performed to choose an initial structure for the simulations of APE1 with damaged DNA. We analyzed the 2.95 Å resolution structure (Protein Data Bank [PDB] ID 1DE8) of the APE1 complex with DNA containing an F-site without a Mg2+ ion [21], the 1.63 Å resolution structure (PDB ID 5DFI) of the APE1 complex with DNA containing 20-O-methyl 2+ phosphorothioate backbone modification 50 to an F-site without a Mg ion [24], and 2.39 Å resolution structure (PDB ID 4IEM) of the APE1 complex with product DNA with a Mg2+ ion [23]. A comparison of the 1DE8 and 5DFI structures shows a close common overlap of protein and DNA molecules. Nevertheless, in the 5DFI structure, the phosphorothioate backbone modification has two isomers, Sp and Rp. Even though both isomers are everted into the active site, the Rp isomer is shifted away from the active site by 2.1 Å. This observation indicates that the modification in question has some influence on the structure of DNA bound to the active site of the enzyme; therefore, this structure was not appropriate for MD simulation. Then, for the 1DE8 structure, the Mg2+ ion was added, and for the 4IEM structure, product DNA with a hydrolyzed phosphodiester bond was replaced by an F-site. Both modified structures were refined by energy optimization and simulated annealing. The final structures of the complex of APE1 with F-site-containing DNA and a Mg2+ ion were very similar and were chosen for productive modeling of the MD of APE1 complexes with each tested damaged nucleotide. The simulations were performed by means of GROMACS 2019.5 [45] and the AMBER ff99SB-ILDN force field [46]. Hydrogen atoms were placed onto the structure using pdb2gmx, and the solvent consisted of TIP3P [47] water in periodic dodecahedron boxes containing a protein–DNA complex and at least a 1 nm layer of the solvent. Solvent molecules were replaced with counterions until the system was neutralized. Simulations were performed under 300 K with protonated residues. The Verlet cutoff approach [48] was employed with a cutoff of 1.2 nm for both van der Waals and electrostatic interactions, whereas LINCS was utilized to constrain bonds [49]. Electrostatic interactions were computed in PME [50]. The solvated systems were minimized next (through steepest-descent minimization). After that, the systems were equilibrated over two stages with positional restraints on DNA and protein atoms. First of all, the systems were equilibrated for 400 ps in the NVT ensemble with subsequent equilibration for additional 400 ps in the NPT ensemble. At last, production dynamics were realized with a 2 fs time step in the NPT ensemble, and every 10 ps, the coordinates were saved. Molecules 2020, 25, 3940 11 of 13

All simulations were conducted for at least 100 ns. To prevent DNA despiralization at the ends, 16 kJ/(mol nm2) restraints on the terminal nucleotide atoms were applied [51]. · Author Contributions: Conceptualization, N.A.K.; methodology, N.A.K. and O.S.F.; software, Y.N.V.; validation, A.A.B., A.A.K., N.A.K. and O.S.F.; formal analysis, A.A.B. and A.A.K.; investigation, A.A.B. and A.A.K.; resources, N.A.K. and O.S.F.; data curation, A.A.B., A.A.K., N.A.K. and O.S.F.; writing—original draft preparation, N.A.K. and O.S.F.; writing—review and editing, N.A.K. and O.S.F.; visualization, N.A.K.; supervision, N.A.K.; project administration, N.A.K. and O.S.F.; funding acquisition, N.A.K. and O.S.F. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported partially by a Russian-State-funded budget project (No. AAAA-A17- 117020210022-4) and a grant from the Ministry of Science and Higher Education, No. MD-3775.2019.4. The part of the work with stopped-flow kinetics was specifically funded by Russian Science Foundation grant No. 18-14-00135. Conflicts of Interest: The authors declare no competing financial interest.

Abbreviations AP-site apurinic/apyrimidinic site; APE1 human AP endonuclease; ODN oligodeoxyribonucleotide.

References

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Sample Availability: Samples of the compounds are not available from the authors.

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