Ultrafast Real-Time Visualization of Active Site Flexibility of Flavoenzyme Thymidylate Synthase Thyx

Ultrafast real-time visualization of active site ﬂexibility of ﬂavoenzyme thymidylate synthase ThyX

Sergey P. Laptenoka,b, Latifa Bouzhir-Simaa,b, Jean-Christophe Lambrya,b, Hannu Myllykallioa,b, Ursula Liebla,b, and Marten H. Vosa,b,1

aLaboratory for Optics and Biosciences, Centre National de la Recherche Scientiﬁque Ecole Polytechnique, 91128 Palaiseau, France; and bInstitut National de la Santé et de la Recherche Médicale U696, 91128 Palaiseau, France

Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved April 18, 2013 (received for review November 9, 2012) In many bacteria the flavoenzyme thymidylate synthase ThyX optical techniques. In particular, the fluorescence properties of produces the DNA nucleotide deoxythymidine monophosphate intrinsic or external fluorophores can be exquisitely sensitive to from dUMP, using methylenetetrahydrofolate as carbon donor the protein environment. In flavoproteins, interaction of the and NADPH as hydride donor. Because all three substrates bind in flavin cofactor with the protein environment has been shown to close proximity to the catalytic flavin adenine dinucleotide group, diminish the lifetime of the flavin fluorescence from the intrinsic substantial flexibility of the ThyX active site has been hypothe- nanosecond timescale to the femtoseconds-to-picoseconds time- sized. Using femtosecond time-resolved fluorescence spectros- scale, due to quenching by photooxidation of nearby aromatic copy, we have studied the conformational heterogeneity and the residues (3, 9–15). In the present study, we use ultrafast fluo- conformational interconversion dynamics in real time in ThyX from rescence spectroscopy to investigate conformational flexibility of the hyperthermophilic bacterium Thermotoga maritima. The dy- the flavoenzyme thymidylate synthase ThyX. namics of electron transfer to excited flavin adenine dinucleotide ThyX is a homotetrameric enzyme discovered a decade ago (16), from a neighboring tyrosine residue are used as a sensitive probe which is essential for de novo synthesis of the DNA precursor 2′- of the functional dynamics of the active site. The fluorescence de- deoxythymidine-5′-monophosphate (dTMP) in a large number of cay spanned a full three orders of magnitude, demonstrating a very bacterial systems. ThyX shows no structural homology to thymi- wide range of conformations. In particular, at physiological temper- dylate synthase ThyA, which is used in most eukaryotes (17). Be- BIOPHYSICS AND atures, multiple angstrom cofactor-residue displacements occur on cause the ThyX pathway is used by a number of pathogenic bacteria

fi COMPUTATIONAL BIOLOGY the picoseconds timescale. These experimental ndings are sup- and absent in humans, ThyX is considered a promising antimicrobial ported by molecular dynamics simulations. Binding of the dUMP target (16, 18); it catalyses carbon transfer from N5,N10-methylene- fl substrate abolishes this exibility and stabilizes the active site in 5,6,7,8-tetrahydrofolate (MTHF or CH H folate) to deoxyuridine fi fl 2 4 acon guration where dUMP closely interacts with the avin co- monophosphate (dUMP) using NADPH as a hydride donor and factor and very efficiently quenches fluorescence itself. Our results fi consequently has three substrates (dUMP, MTHF, NADPH) with indicate a dynamic selected- t mechanism where binding of the the flavin adenine dinucleotide cofactor shuttling between the first substrate dUMP at high temperature stabilizes the enzyme fully oxidized (FAD) and fully reduced (FADH ) forms. dUMP in a configuration favorable for interaction with the second sub- 2 binds in close interaction with the flavin group, displacing a nearby strate NADPH, and more generally have important implications for Tyr residue (19). A very recent study shows that folate derivatives the role of active site flexibility in enzymes interacting with multiple may bind to the opposite side of the flavin cofactor with respect to poly-atom substrates and products. Moreover, our data provide the basis for exploring the effect of inhibitor molecules on the active dU(20). The binding site of NADPH has not been determined by site dynamics of ThyX and other multisubstrate flavoenzymes. structural studies, but inhibition studies indicate that folate and NADPH binding sites may partially coincide (21). Furthermore, fl protein dynamics | flavoprotein | ultrafast fluorescence spectroscopy | reduction of avin by NADPH appears gated by the presence of quenching dUMP (21), further pointing at substrate-induced, functional structural rearrangements. Steady-state crystallographic studies of fl fi fl this enzyme also indicate substantial exibility of the active site: in on gurational exibility is essential for enzyme function the substrate-free structure the flavin group and its close environ- Cduring catalysis. Binding of one or more substrates, accom- ment appear disordered (19); for the folate-bound form, multiple modation of the transition state where the actual reaction takes flavin configurations have been suggested (20). place, relaxation to the product state, and release of the product fl fi Using a newly developed, ultrafast uorescence spectrometer (s) is possible because different con gurations of the enzyme are with full spectral resolution, we performed studies on the dynamics continuously sampled, by thermal or reaction-driven motions, of FAD fluorescence in wild-type and genetically modified ThyX on timescales ranging from femtoseconds to microseconds. The fi enzymes from several bacterial species. In the present work we con gurational space sampled in a certain time range will de- focus on the enzyme from the hyperthermophilic bacterium Ther- pend on the local protein flexibility/energy landscape and the fi motoga maritima that allows studies over a wide temperature range. temperature (1). During these con gurational changes, distances We identified a close-lying tyrosine as well as the substrate dUMP between constituents of the enzyme complex change. It has been itself as fluorescence quenchers. The observed fluorescence decay recognized that the fastest (femtosecond to picosecond) local- revealed the presence of a wide range of conformations, whose ized motions exist alongside the slower motions that occur on the (typically millisecond) timescale of catalysis (2–4).

Various experimental techniques allow monitoring changes in Author contributions: H.M., U.E.L., and M.H.V. designed research; S.P.L., L.B.-S., and J.-C.L. interactions resulting from configurational changes. Long-range performed research; S.P.L. contributed new reagents/analytic tools; S.P.L., J.-C.L., and micro/millisecond domain motions in flexible proteins have been M.H.V. analyzed data; and S.P.L., H.M., U.E.L., and M.H.V. wrote the paper. studied by NMR (5) and FRET techniques (6, 7). Molecular dy- The authors declare no conflict of interest. namics simulations and NMR experiments indicate that more lo- This article is a PNAS Direct Submission. calized structural fluctuations in these flexible regions occur on the 1To whom correspondence should be addressed. E-mail: [email protected]. fl picoseconds-to-nanoseconds timescale (6, 8). These uctuations This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. can only be directly investigated using very high time-resolution 1073/pnas.1218729110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1218729110 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 interconversion accelerates at the physiological temperature of this designed to prevent electron transfer while maximally preserving hyperthermophilic enzyme. Binding of dUMP was found to stabi- the structural environment of the flavin. lize the active site in a configuration allowing close interaction The overall fluorescence decay kinetics of the mutant ThyX between dUMP and FAD and favorable for interaction with protein are much slower than those of WT (Fig. 1B; note the NADPH. Our data have important implications for the role of active logarithmic scaling after 10 ps), implying that ET from Tyr-91 is site flexibility in multisubstrate enzymes and ultimately permit ex- indeed the dominant quenching process. However, in both cases, ploring the effect of inhibitor molecules on the active site dynamics. a fast decay in the order of ∼1 ps is present (see below), which we assign to relaxation processes in the excited state. Furthermore, Results the decay of the mutant protein still takes place faster than that The absorption spectrum of ThyX from T. maritima (TmThyX; of FAD in solution, indicating that aromatic residues other than fl Fig. S1A) is very similar to that published previously (22), and the Tyr-91 also contribute to quenching. The shape of the uores- ∼ fluorescence spectrum (Fig. S1C) typical for oxidized flavins. The cence spectra was found to be constant after 200 ps and to be significantly different from that of FAD in solution (Fig. S3A). enzyme contains four FAD binding sites (closest center-to-center fi distance 27 Å). Because in principle, resonance energy transfer This observation further con rms that free FAD does not con- between identical molecules within the protein (homo-FRET) can tribute to the signal. Analysis of the full data set in terms of multiexponential decay of take place, potentially complicating the analysis, we measured both the WT and the Y91F mutant protein requires at least four fluorescence anisotropy. The anisotropy was close to the theo- decay rates (Fig. S3 A and B). This analysis indicates that red shifts retical maximum of 0.4, implying that no homo-FRET occurs. occur concomitant with fluorescence decay on the timescale <200 This finding is consistent with an estimation of energy transfer fl fl > ps, and presumably re ect charge relaxation processes of the avin times of 3 ns using the Förster formalism and the observation environment (25). The nonexponentiality of the decay can be (see below) that quenching takes place on faster timescales. – fl assigned to a distribution of conformations of the donor acceptor Transient uorescence spectra and kinetics at the emission pairs that do not or only partly interconvert during the excited maximum are shown in Fig. 1; kinetics at different wavelengths state lifetime. The fact that the nonexponentiality is also found fl are shown in Fig. S2, and they show a highly multiphasic uo- in the mutant protein implies that this distribution is not uniquely rescence decay, spanning timescales from ∼1psto∼1 ns. due to heterogeneity specifically of the Tyr-91 conformation. However, all decay occurs substantially faster than the intrinsic nanosecond (∼3 ns) decay of FAD (23). The most probable Temperature Studies. To obtain detailed insight into the confor- origin of this quenching is electron transfer (ET) from close-by mational flexibility of the active site, we measured the fluorescence aromatic residues to the excited flavin cofactor. According to the properties of the enzyme as a function of temperature. Here, we crystal structure of TmThyX (19), Tyr-91, a widely conserved take advantage of the fact that the ThyX enzyme from the residue (24), is the closest aromatic residue to the FAD cofactor, hyperthermophile T. maritima allows studies over a wide range of and therefore likely to act as main fluorescence quencher. To temperatures, up to 70 °C, into the physiological range of the en- test this hypothesis, we investigated the Y91F mutant protein, zyme (at higher temperatures the isolated enzyme starts to pre- cipitate). Fig. S1C shows that the total fluorescence decreases with increasing temperature, and that this decrease is fully reversible upon cooling. The fluorescence decrease rather than increase at A higher temperatures indicates that FAD is not released at high 0 ps temperature (up to 70 °C), as has been observed in flavodoxin (26). 10 ps As shown in Fig. 2, the decrease of the total fluorescence is the 100 ps result of the acceleration of the fluorescence decay kinetics. In 1000 ps a multiexponential global analysis, at all temperatures at least four components (eight fit parameters in total) are required to satisfactorily fit the data. It was possible to describe the data at fluorescence all temperatures with the same set of four rate constants (Fig. S3 C and D). In this case, the amplitudes of the two longest decay components decreased and those of the shortest decay components increased with temperature. Qualitatively, the result of this analysis implies that with increasing temperature the distribu- 475 500 525 550 575 600 625 tion of fluorescence decays shifts toward the shorter-lived side, wavelength, nm B without the faster decays themselves becoming faster. The high number of exponentials (and fit parameters) required to fit the data suggests that a continuous distribution of rates, rather than four distinct rates, may provide a more ade- Y91F quate and simpler description. To explore this possibility, we attempted to fit the integrated fluorescence decay with a power WT law. For all temperatures, a satisfactory fitwasobtained(att >2ps) using Eq. S1, which contains only three fit parameters: the amplitude I0, mean lifetime τ0, and distribution width parameters q. The corresponding lifetime distributions are shown in Fig. 3C. normalized fluorescence The average lifetime τ0 (Fig. 3A) is seen to gradually decrease -5 0 5 10 100 1000 with temperature. Overall, the relative width of the distribution time, ps (Fig. 3B) decreases at higher temperatures and in particular fl at 70 °C, which is in the physiological temperature range for Fig. 1. (A) Transient uorescence spectra of WT TmThyX measured at 20 °C. fl The feature at ∼460 nm in the 0-ps spectrum is due to Raman scattering of T. maritima. Through its in uence on the electronic coupling, the water. (B) Kinetics at 520 nm of WT and Y91F ThyX. The time axis is linear until ET rate k critically depends on the edge-to-edge distance r be- −βr 10 ps and logarithmic thereafter. Solid lines are fits with a four-parameter tween the flavin cofactor and the quenching residue as k = k0e exponential decay; the dashed line with power law. (3, 27). Assuming ET between Tyr and excited FAD is near

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1218729110 Laptenok et al. Downloaded by guest on September 30, 2021 the FAD concentration, implying that flavin-devoid sites, which 20°C 30°C are also present in ThyX in solution (SI Materials and Methods), 40°C do not bind dUMP in the same affinity range. This finding 50°C 60°C strongly suggests that stacking with FAD is the major binding 70°C determinant of dUMP. A similar affinity was found using the change in the flavin absorption spectrum (Fig. S1A) as a measure for dUMP binding, although the high dynamic range of the fluorescence measurements allows a more precise determination. Detailed binding studies will be presented elsewhere.

normalized fluorescence We also used FAD fluorescence to investigate whether dUMP binding influences the thermal stability of the complex. As men- -4-2 0 5 10 100 1000 time, ps tioned previously, in the absence of dUMP up to 70 °C FAD does not dissociate from the protein. Above 70 °C, the FAD fluorescence fl Fig. 2. Temperature dependence of peak-normalized uorescence decay of rises and redshifts, and at 90 °C an increase corresponding to ∼4% WT TmThyX at 520 nm. The time axis is linear until 10 ps and logarithmic free FAD is observed (Fig. S5). When dUMP is bound, this increase thereafter. amounts to less than 0.5%, and no spectral shift is observed (Fig. S5). Although protein precipitation above 90 °C prohibited the − barrierless (3, 11), and β = 1.36 Å 1 (28), we can convert the determination of full dissociation curves of this hyperthermophilic lifetime distribution (Fig. 3C) to a distribution of distances from enzyme, these data indicate that dUMP binding shifts denaturation which ET takes place (Fig. 3D). In view of uncertainty in k0,we to higher temperatures and thus thermally stabilizes the complex. show the distribution as relative to a mean distance R0.At20°C the distribution is very large (FWHM >2 Å), implying strong Molecular Dynamics Simulations. To investigate the molecular conformational heterogeneity. We note that this distribution parameters underlying the experimentally observed conforma- should be considered qualitatively and does not necessarily di- tional flexibility, we performed molecular dynamics (MD) sim- rectly correspond to the static distribution of flavin–quencher ulations of WT TmThyX in the presence and absence of dUMP pairs, because conformational flexibility within the fluorescence and at two temperatures (27 °C and 70 °C). The models that

lifetime may bias the distribution toward shorter distances. This BIOPHYSICS AND

point is illustrated in Fig. 3D. At higher temperatures, the dis- COMPUTATIONAL BIOLOGY tance distribution shifts to shorter distances, and becomes nar- 100 rower—in particular, close to the physiological temperature range (70 °C). Because warming should enhance the conﬁgura- A B 2.4 tional distribution, the picture that emerges is that (i) at all 80 temperatures a very large distribution of conﬁgurations is pop- ulated, and that (ii) at higher temperatures the interconversion 2.2

between these is accelerated so that the configurations that give 60 q rise to the fastest quenching rates are more easily reached within 2 the timescale of fluorescence decay. 40 1.8 dUMP Binding. In the absence of the two other substrates, dUMP binds close to oxidized FAD through aromatic stacking against its 20 isoalloxazine ring system (19). Fig. 4 shows that this binding leads 20 30 40 50 60 70 20 30 40 50 60 70 to dramatic quenching of FAD fluorescence: the dominant decay T, °C T, °C phase occurs with a time constant of ∼200 fs in this case, and the slower decay phases are strongly, although not completely, sup- pressed (∼90% decay within the first few picoseconds). C D 20°C The origin of this quenching may be either a direct interaction 30°C between dUMP and the FAD cofactor, or a change in the in- 40°C teraction of FAD and the quenching residues, in particular Tyr-91. 50°C Indeed, the available TmThyX crystal structures suggest that upon 60°C dUMP binding, Tyr-91 moves toward the FAD cofactor (19). 70°C To discriminate between these two possibilities, we investigated the Y91F mutant enzyme, which binds dUMP with comparable normalized P(R) affinity and shows a similar perturbation of the absorption spectrum (Fig. S1B) as WT (see below). We observed (Fig. 4) that the effect of dUMP binding on the fluorescence decay is very similar -2 0 2 4 for WT and Y91F TmThyX, which led us to conclude that it is R-R0, Å dUMP itself that acts as quencher of flavin fluorescence. The minor (∼10%), slower decay phases in the presence of dUMP (Fig. S3E)mayreflect partial and/or heterogeneous dUMP binding. The strong quenching of FAD binding by dUMP can be used as a sensitive probe to determine the affinity of TmThyX for 0 100 200 300 400 500 dUMP. In agreement with the time-resolved data of Fig. 4, the overall fluorescence was found to decrease ∼10-fold upon dUMP Fig. 3. Analysis of the temperature dependence of the fluorescence kinetics binding to WT TmThyX. Analysis of the dUMP titration to 5-μM of WT TmThyX using Eq. S1.(A) Mean value τ0 of the lifetime distribution. (B) FAD binding sites yielded a Kd of 400 nM at 60 °C and in the Heterogeneity factor q of the lifetime distribution. The solid lines are guides presence of 250 mM NaCl (Fig. S4). The apparent binding site for the eye. (C) Lifetimes distribution. (D) Distribution of edge-to-edge dis- concentration deduced from the binding curve was very close to tances between FAD and electron donors normalized to the total integral.

Laptenok et al. PNAS Early Edition | 3of6 Downloaded by guest on September 30, 2021 fi WT temperatures. This nding implies that substrate binding leads WT + dUMP to a rigidification of the active site, and in particular of the FAD Y91F Y91F + dUMP cofactor positioning, and is generally consistent with the better resolution of the active site in the X-ray crystallography studies at cryogenic temperatures (19) and our finding of dUMP- induced thermal stabilization of the FAD–protein complex (Fig. S5). Similar analyses for other residues involved in substrate binding and catalysis also indicate substantial distance variation specifically in the absence of dUMP. For instance Arg-147, Arg-

normalized fluorescence 174, and Ser-88, which all H-bond to dUMP, were all found to be able to intermittently H-bond to FAD in the absence of dUMP. 0 2 4 10 100 1000 time, ps Discussion In this work, the quenching of flavin fluorescence in an enzyme Fig. 4. Effect of dUMP binding on fluorescence decay of WT and Y91F TmThyX at 520 nm. The time axis is linear until 4 ps and logarithmic there- involved in DNA synthesis, associated with MD simulations, was after. A global analysis in terms of DAS of WT TmThyX fluorescence spectra used to probe the dynamic properties of the active site. Experi- in the presence of dUMP is shown in Fig. S3F. mentally, we exploited the fact that photoexcited FAD (FAD*) can accept an electron from the aromatic residues Tyr and Trp on the femtosecond-to-nanosecond timescale, and showed that included all four subunits were based on the X-ray structure of in the case of TmThyX from, Tyr-91 is the principal electron − + dUMP-bound TmThyX that was obtained at −173 °C (19). Fig. donor. It has been argued that the FAD*Tyr→FAD Tyr° re- S6 shows that after warming and equilibration of the model, the action occurs with a driving force in the range of 0.7–1 eV and in rmsd of the backbone atoms of the model from the X-ray a near-activationless regime (3, 11). Indeed, the rates of this structure remained roughly constant and ∼1 Å for the 2.5-ns free electron transfer reaction were found to be temperature in- dynamics trajectories. In the absence of dUMP, the deviation is dependent in flavoproteins with a much less heterogeneous significantly larger for the trajectories at 70 °C than at 27 °C, fluorescence decay distribution than TmThyX (11, 13). More- indicating a larger conformational heterogeneity. Interestingly, over, in TmThyX, increase in temperature leads to a shift of such a difference is not observed in the absence of dUMP, in- slow-decaying populations to fast-decaying populations rather dicating that this substrate effectively rigidifies the protein. than an increase of the rates itself. For these reasons we interpret We now focus on the vicinity of the FAD. Fig. 5 compares the temperature dependence observed in this work as originating time-averaged simulated structures of the four active sites; from changes in the distance-dependent electronic coupling be- a corresponding full tetramer is shown in Fig. S7. Though the tween the reactants, rather than from activation barriers. We structures in the presence of dUMP are similar to the X-ray note that temperature-dependent fluorescence decay times have structure (19), in the absence of dUMP the flavin is rotated in all been reported for the flavoprotein lipoamide dehydrogenase subunits by ∼50° with respect to the X-ray structure and shows (29). The FAD cofactors in this protein have an extremely high substantial orientational freedom (Fig. 5B; Fig. S8). Similar observations were made with models based directly on the dUMP- devoid structure. Interestingly, these rotated conformations are similar to those observed recently in structural studies in some of A His53 the active sites of mutant TmThyX (20). As in the crystal struc- Tyr91 tures of the TmThyX WT proteins (19), in all subunits, the presence of dUMP results in a shorter FAD–Tyr-91 distance. Fig. 6 shows the fluctuations of the FAD–Tyr-91 distance during the free dynamics in each of the four subunits. The shortest edge- to-edge distance is thought to be relevant for the electronic cou- FAD Arg147 pling determining the electron transfer rate. Therefore, at each instant, this distance is plotted rather than the distance between two fixed atoms. In the crystal structure the shortest distance is dUMP between the C6 atom of FAD and the Ce1 atom of Tyr-91. In our simulations, other atom pairs, such as FAD N5-Tyr-91 Cδ1, also occur as shortest distance. The active site structures of Fig. 5 in- B dicate that the distance fluctuations in the dUMP-devoid model are rather the result of fluctuations of the flavin cofactor than of Tyr-91. His53 Tyr91 In the dUMP-devoid model, the FAD–Tyr-91 distance fluc- tuates substantially in all four subunits, at 27 °C from ∼3to∼8Å. Larger fluctuations of 2 Å or more appear infrequently (intervals of several nanoseconds) on the simulation timescale, and the statistics, especially at 27 °C, are clearly undersampled, in- Arg147 dicating that substantial conformational changes also take place FAD on a longer timescale. These observations are in agreement with our analysis of the broad range of fluorescence decay times that indicate that FAD–Tyr-91 distance changes take place on the ∼ fl Fig. 5. Superimposed structures of elements of the four active sites, time timescale of 1 ns or more. At 70 °C the uctuations become averaged over the last 500 ps of the free trajectories of MD simulations of substantially more frequent, both the smaller, high-frequency fl ThyX in the presence (A) and absence (B) of dUMP. The superposition is on and the larger, low-frequency uctuations. the entire backbone of each subunit. The His53 residues associated with the Interestingly, our simulations also indicate that the presence active sites belong to different subunits as the other elements. The carbon of dUMP drastically reduces the distance fluctuations for both atoms are color-coded with the respective subunits.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1218729110 Laptenok et al. Downloaded by guest on September 30, 2021 Å 10 o reductase (3), broader and more heterogeneous lifetime dis- 8 27 C with dUMP o tributions have been interpreted in terms of ground-state hetero- 6 27 C without dUMP 4 geneity. The lifetime distribution in TmThyX is considerably larger chain 1, than in the abovementioned ﬂavoproteins, and the associated do- 10 Å o nor–acceptor distance distribution is substantially wider (∼2Å 8 70 C with dUMP o ∼ 6 70 C without dUMP FWHM at 20 °C; Fig. 3D)thanthe 1 Å FWHM distribution in Yang et al. (3).

chain 2, 4 Our results show that a very broad range of configurations is 10 Å 8 accessible for TmThyX in the absence of substrate, and that these 6 do not fully interconvert on the fluorescence timescale and at sub- fi

chain 3, 4 catalytic temperatures. These ndings can be considered consistent

10 with an early crystallographic study of substrate-free TmThyX, Å ﬂ 8 where fractions of the protein, and in particular the avin ring, 6 appear disordered, in contrast to the dUMP-containing protein

chain 4, 4 (19), and appear general for ThyX enzymes (38, 39). More specifically for TmThyX, our MD simulations indicate the presence of 1500 2000 2500 3000 3500 24681012 time, ps distance TYR91-FAD, Å substantial variation in the distance between FAD and the Tyr-91 quencher, with large changes (up to ∼4 Å) occurring infrequently on Fig. 6. Dynamics (Left) and distribution histograms (Right) of the distance the timescale of up to several nanoseconds. This flexibility is no between FAD and Tyr-91 (shortest distance between an atom on the iso- longer apparent in the presence of dUMP. Single-molecule studies alloxazine ring and the tyrosine aromatic ring) in the four subunits during of flavin reductase have demonstrated that interconversion of the the 2.5-ns free dynamics. lifetime-distinguishable configurations for a substantial part takes place on the timescale of milliseconds and beyond (3). Our tem- fluorescence quantum yield and are not quenched; here, the perature dependence studies of ThyX show that the distribution activation of the fluorescence decay was due to distinct confor- does not broaden at higher temperature, as could be expected from pure increase of accessible configurational space, but narrows and mational changes that are static on the nanosecond timescale (29). shifts to shorter effective distances. We assign this finding to ac- The analysis in the present work by contrast highlights dynamic celeration of the interconversion rates of configurations on the same BIOPHYSICS AND conformational changes occurring on the picosecond timescale.

timescale as fluorescence decay. This interpretation is supported by COMPUTATIONAL BIOLOGY Our analysis is based on the widely used (3, 11, 14) notion that our MD simulations that indicate that substantial fluctuations in excited-state quenching in flavoproteins is due to electron transfer donor–acceptor distance do occur on the picosecond-to-nanosec- from aromatic residues. The validity of this notion has been ond timescale, and that these, and in particular the less-frequent demonstrated for tryptophan quenchers (9, 30) and strongly sug- large amplitude fluctuations, occur substantially more frequent at gested for tyrosine quenchers (31, 32). In both cases, the radical higher, physiologically relevant, temperatures. pair can be subsequently stabilized by proton transfer from the We suggest that the flexibility in the active site of this enzyme, as residue. Simultaneous proton and electron transfer, avoiding reflectedinthepicosecond–nanosecond multiangstrom conforma- high-energy charge-separation intermediates, has been observed tional fluctuations, is related to its capacity of binding multiple for phenol oxidation reactions in aqueous solutions (33, 34). different aromatic substrates, of which at least two (NADPH/dUMP Though we cannot fully exclude that such simultaneous pro- and MTHF/dUMP) simultaneously. The picture that emerges is cesses (and hence motions between Tyr-91 and potential proton that the extremely rapid conformational sampling of the protein acceptors) could play a role in FAD* quenching in ThyX, we fl allows dUMP to enter the active site and be accommodated close to consider it unlikely in view of the evidence from other a- the flavin. The binding of dUMP then stabilizes the flavin cofactor in voproteins and the fact that the high-driving force generated by the active site, as indicated by our MD simulations, presumably in a FAD* formation (see above) energetically allows formation of − + configuration favorable for NADPH binding and subsequent flavin the FAD Tyr° intermediate by electron transfer only. reduction. This mechanism is in agreement with the reported drastic We observed that not only aromatic residues, but also the sub- acceleration of flavin reduction by NADPH in the presence of fl strate dUMP quenches FAD uorescence, strongly suggesting that dUMP (21). We stress that our observations have further functional bound dUMP also donates an electron to FAD*. dUMP indeed consequences. Indeed, low stereospecificity and a very high Km for binds very close to FAD, and the isoalloxazine and uracil rings are NADPH have been observed for the TmThyX protein at 37 °C (40, ∼ actually in, or close to, van der Waals contact (distance 3.5 Å in 41). Moreover, this protein turns over substantially faster at 65 °C the crystal structure). The relevant redox properties of dUMP have (within the physiological range) than at 37 °C (41, 42). not been determined, but the light-induced oxidation of uracil in Our results indicate that a selected fit-type mechanism applies, the presence of a photoreducing material has been reported (35), where the substrate binding does not necessarily change the suggesting that the reaction is energetically possible. We will fur- enzyme configuration, but rather stabilizes a favorable configu- ther investigate this hypothesis by transient absorption spectros- ration among many sampled. The accelerated and more exten- copy of the photoproducts formed in the ThyX–dUMP complex. sive sampling at higher temperature does not substantially The FAD* decay dynamics of ThyX are strikingly multiphasic, influence the equilibrium binding of the substrate, but may allow spanning three orders of magnitude in time. We assign the large faster exchange with the solvent of substrate and product. and continuous lifetime distribution in ThyX fluorescence to con- In conclusion, we exploited ultrafast time-resolved FAD fluo- formational heterogeneity in the active site; this assignment is rescence quenching by a neighboring aromatic residue in ThyX strongly supported by the MD simulation that shows large varia- and show that, in combination with temperature dependence tions of active site configurations. In itself, nonmonoexponential studies, it is a sensitive probe for conformational fluctuations. decay of the excited state of quenched flavin cofactors in fla- We have shown that the active site of ThyX, which has to ac- voproteins is frequent (15). Biexponential decay has been docu- commodate multiple substrates, is highly flexible and, in particular, mented spanning shorter time spans (14, 36, 37), and in flavodoxin samples configurations with very high speed, with large, multiple multiexponentiality has been associated predominantly with partial angstrom displacements occurring on the picoseconds timescale in flavin dissociation from the protein (26). In blue light-sensing using the physiological temperature range. We suggest that this struc- FAD (BLUF) photosensor proteins (13, 31, 32) and in flavin tural plasticity allows efficient binding of the dUMP substrate.

Laptenok et al. PNAS Early Edition | 5of6 Downloaded by guest on September 30, 2021 Binding of dUMP, which acts as a very efficient FAD fluorescence motions is likely to be a general feature for flexible protein domains quencher itself, arrests these movements and stabilizes the enzyme and important for understanding enzyme substrate reactivity (2). in a configuration allowing FAD reduction by the NADPH substrate. We conjecture that the rapid large amplitude motions of the Materials and Methods active site monitored in ThyX enzymes may be related to their ca- Proteins were expressed using a codon-optimized expression vector, and fi fl pacity of interacting with multiple different aromatic and relatively puri ed following standard procedures. Time-resolved uorescence experiments were performed using a recently developed spectrally resolved Kerr- rigid substrates and products. The method that we have explored gate femtosecond fluorometer (43). The models for the MD simulations here can be used to study the effect of other substrates and, in (performed with CHARMM) in the presence and absence of dUMP shown in particular, inhibitors, including potential antimicrobial agents (18), the main text were based on the structure of the dUMP-containing protein on the dynamics of the active site of ThyX. More generally, our (PDB ID code 1O26) (19); a model based on the substrateless structure (PDB results emphasize the important role of enzyme dynamics in the ID code 1O2A) gave similar results (Fig. S9). A detailed description of the interaction with the substrates. Whereas flavoenzymes provide an experimental procedures, data analysis, and simulation protocols is given in SI Materials and Methods. intrinsically well-suited tool for real-time monitoring of configurational evolution, extensive high-speed sampling of active site con- ACKNOWLEDGMENTS. This work was supported by the Agence National de figurations on the intrinsic picosecond timescale of global protein la Recherche Grant ANR-09-PIRI-0019.

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