Article

ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 7, pp. 1202-1210 © The Author(s) 2024. This article is an open access publication.

1202

Mechanism of PARP1 Elongation Reaction

Revealed by Molecular Modeling

Sergey V. Pushkarev

, Evgeny M. Kirilin

2,a

, Vytas K. Švedas

1,3

, and Dmitry K. Nilov

3,b

Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University,

119234 Moscow, Russia

Abu Dhabi, United Arab Emirates

Lomonosov Moscow State University, Belozersky Institute of Physicochemical Biology,

119992 Moscow, Russia

e-mail: e.kirilin@gmail.com

e-mail: nilovdm@gmail.com

Received April 15, 2024

Revised June 6, 2024

Accepted June 8, 2024

Abstract— Poly(ADP-ribose) polymerase 1 (PARP1) plays a major role in the DNA damage repair and transcrip-

tional regulation, and is targeted by a number of clinical inhibitors. Despite this, catalytic mechanism of PARP1

remains largely underexplored because of the complex substrate/product structure. Using molecular modeling

and metadynamics simulations we have described in detail elongation of poly(ADP-ribose) chain in the PARP1

active site. It was shown that elongation reaction proceeds via the S

1-like mechanism involving formation of the

intermediate furanosyl oxocarbenium ion. Intriguingly, nucleophilic 2′

-OH group of the acceptor substrate can

be activated by the general base Glu988 not directly but through the proton relay system including the adjacent

3′

-OH group.

DOI: 10.1134/S0006297924070046

Keywords: Poly(ADP-ribose) polymerase 1, PARP1, ADP-ribosylation, reaction mechanism, oxocarbenium ion,

molecular modeling, metadynamics

Abbreviations: MD, molecular dynamics; MM, molecular

mechanics; PAR,poly(ADP-ribose); PARP1,poly(ADP-ribose)

polymerase1; QM,quantum mechanics.

* To whom correspondence should be addressed.

INTRODUCTION

Poly(ADP-ribose) polymerase 1 enzyme (PARP1)

plays an important role in regulating gene expression

and maintaining genome integrity. PARP1 is activat-

ed under conditions of stress and synthesizes a neg-

atively charged polymer, poly(ADP-ribose) (PAR), that

performs signaling functions [1-6]. PARP1 is the most

abundant of the PARP family members (PARPs 1-4,

tankyrases1 and 2, PARPs6-16) and accounts for ap-

proximately 90% of PARP catalytic activity [7-9]. Patho-

genesis of the diseases of cardiovascular, nervous,

immune, respiratory, and other body systems is often

associated with PARP1 activation and PAR synthesis

[10-13]. Furthermore, preclinical data on the PARP1’s

involvement in DNA repair led to the development of a

series of novel anticancer inhibitors (including olapar-

ib, rucaparib, niraparib, etc.) [14-17].

PARP1 utilizes NAD

as a donor of ADP-ribose

units, releasing nicotinamide as a by-product. PAR

synthesis involves three ADP-ribosylation reactions:

(i) initiation (attachment of the first ADP-ribose unit to

an acceptor protein), (ii) elongation, and (iii) branching

(Fig. 1) [18-21]. In the elongation reaction, the O-glyco-

sidic bond is formed between the adenine ribose of the

terminal PAR residue and nicotinamide ribose of the

metabolized NAD

, with the 2′

-OH group acting as an

attacking nucleophile. In the branching reaction, the

bond is formed between two nicotinamide ribose rings,

with 2′

-OH acting as a nucleophile (subscriptsA and N

denote adenine and nicotinamide ribose, respectively).

Formation of the negatively charged PAR polymers

(up to 200 units in size) results in modulation of chro-

matin structure and recruitment of a number of cel-

lular proteins [22]. In particular, activity of PARP1

MECHANISM OF PARP1 ELONGATION REACTION 1203

BIOCHEMISTRY (Moscow) Vol. 89 No. 7 2024

Fig. 1. Chemical structure of PAR composed of ADP-ribose units. Branches are formed every 40-50 units.

atthe sites of DNA damage recruits base excision re-

pair proteins XRCC1, DNA polymerase β, and DNA li-

gase III [23,24].

Despite its biological significance, catalytic mech-

anism of PAR synthesis remains underexplored be-

cause of the complex substrate/product structure. Some

assumptions on the PAR and NAD

binding can be

made based on the crystal structure of the complexes

of PARP1 with inactive substrate mimics [18, 25, 26].

TheGlu988 residue in the active site is critical for poly-

mer growth and likely provides the required orienta-

tion of PAR and NAD

by forming hydrogen bonds with

their 3′

-OH and 2′

-OH groups [18, 25, 27, 28]. Some au-

thors consider that Glu988 also forms a hydrogen bond

with the 2′

-OH group of PAR and acts as a proton ac-

ceptor upon nucleophilic attack by the S

2 mechanism

[18, 27]. However, in our molecular dynamics (MD)

simulation of the enzyme–substrate complex with ter-

minal fragment of PAR and NAD

we failed to observe

direct interaction between the Glu988 and 2′

-OH as

well as reactive in-line configuration of the O2′

, C1′

and N1

atoms, typical for S

2 [29]. The present pa-

per is a further attempt to provide details concerning

PARP1 catalysis through molecular modeling and meta-

dynamics simulations. We describe the possible S

like mechanism for the elongation reaction involving

formation of an intermediate oxocarbenium ion.

MATERIALS AND METHODS

Starting model of the PARP1 enzyme-substrate

complex was constructed based on the catalytic do-

main coordinates extracted from the 4dqy crystal struc-

ture (residues 662-1011) [30]. N- and C-terminal ends of

the protein were capped with ACE (acetyl) and NME

(N-methylamide) groups, respectively. Coordinates of

NAD

were transferred from the 6bhv structure [26].

Coordinates of an ADP moiety as a terminal PAR frag-

ment were transferred from the 1a26 structure [18];

its diphosphate chain was capped with amethyl group

(Fig.S1 in the Online Resource1).

Next, the model was optimized using AmberTools20

and Amber20 [31-33] installed on the Lomonosov-2

supercomputer [34]. The protein molecule was de-

scribed with the ff14SB force field [35]. The NAD

mol-

ecule was described with parameters from the Amber

Parameter Database (http://amber.manchester.ac.uk)

[36, 37]. Parameters for the methyl-ADP molecule were

derived as follows. Force constants, equilibrium bond

lengths/angle values, and van der Waals parame-

ters were taken from the NAD

parameter set. Partial

atomic charges were taken from the NAD

set except

for the methoxy group. Charges for this group were

determined using the R.E.D.-III.5 and RESP programs

[38, 39], as shown in Fig.S2 in the Online Resource1.

Hydrogen atoms were added to the protein structure

considering ionization properties of amino acid resi-

dues, and then it was surrounded by a layer of TIP3P

water (12 Å).

Energy minimization included two stages: the

first one with positional restraints on heavy atoms

of the protein and substrates (2500 steepest descent

steps + 2500 conjugate gradient steps), and the sec-

ond one without restraints (5000 steepest descent

steps + 5000 conjugate gradient steps). The obtained

system was heated up from 0 to 300 K with positional

restraints on the protein and substrate atoms (1000 ps,

constant volume) and equilibrated at 300 K (1000 ps,

constant pressure); time step was 0.002 ps. Finally, a

100 ns trajectory of the equilibrium MD simulation was

calculated. All protocols are described in detail in our

PUSHKAREV et al.1204

BIOCHEMISTRY (Moscow) Vol. 89 No. 7 2024

Fig. 2. Collective variables used for metadynamics simulations. a)CV1 describes nucleophilic substitution at the C1′

atom.

b)CV2 describes deprotonation of the nucleophilic 2′

-OH group. This group is activated by Glu988, and the corresponding

proton relay system could include either the 3′

-OH (system A) or 2′

-OH (systemB) group.

previous work [40]. The simulation trajectories were

analyzed using cpptraj5.1.0 [41] and VMD1.9 [42].

To perform metadynamics calculations, 4 frames

were selected from the equilibrium MD trajectory in

which positions of the substrates were close to the

reactive configuration. Each selected frame was then

simulated using a hybrid quantum mechanics/molecu-

lar mechanics (QM/MM) functionality in Amber20 [43];

detailed protocols are given in TableS1 in the Online

Resource 1. The QM region included essential parts

of the substrates and of Lys903 and Glu988 residues,

which were treated using the PM6-D semi-empirical

Hamiltonian [44, 45], and the MM region included the

remaining atoms of the system (Fig. S3 in the Online

Resource 1). Prior to metadynamics, 25 ps simulations

with 0.001-ps time steps were carried out to adjust

structures of the active site after switching from the

MM to QM/MM approximation. Next, well-tempered

metadynamics implemented in Plumed 2.6.2 [46-50]

was used to explore free energy surface of the PARP1-

catalyzed elongation reaction. Free energy landscape

was reconstructed by 8 simulations (2 for each starting

structure) run in parallel with shared metadynamics

potential (so-called “walkers”) [51]. Duration of each

simulation was 300ps, time step was 0.0005ps.

The first collective variable (CV1) was defined as

-d

, where d

– distance between the C1′

and N1

atoms, and d

 – distance between the O2′

and C1′

atoms (Fig.2a). The second collective variable (CV2)

was defined as d

-d

, where d

– distance between the

O2′

and O2′

:H atoms, and d

– distance between the

O2′

:H atom and either the O3′

or O2′

atom (Fig.2b).

CV1 describes nucleophilic substitution at the C1′

atom, and CV2 – deprotonation of the nucleophilic

2′

-OH group. Gaussian potentials of an initial height

of 6kJ/mol and width of 0.1 Å (CV1) and 0.075 Å (CV2)

were added every 100 simulation steps, bias factor was

set to 16. Upper and lower walls were applied to d

, d

, and d

(to limit exploration in the CV1/CV2 space),

as well as to some other distances to prevent unwant-

ed proton transfer scenarios (e.g., transfer between

O2′

and O2′

in system A, or between O2′

and O3′

in systemB). Gaussian potentials were summed with

metadynminer 0.1.7 (“fes2” function) [52], minimum

energy paths were calculated using the nudged elastic

band method [53].

RESULTS

Elongation of PAR polymers is the most common

reaction catalyzed by PARP1. Two substrates are re-

quired for elongation activity: NAD

(ADP-ribose do-

nor) and terminal PAR fragment (ADP-ribose accep-

tor). Wehave decided to use methyl-ADP molecule as

a structural analogue of the acceptor substrate termi-

nus in molecular modeling experiments. A 100-ns MD

simulation of the PARP1 enzyme-substrate complex

demonstrated that NAD

forms two hydrogen bonds

with Gly863 and π-stacking with Tyr907, which con-

firms the results of previous studies [25,29]. Thecat-

alytic Glu988 residue forms hydrogen bonds with

the2′

-OH group of NAD

and 3′

-OH group of methyl-

ADP, providing reactive orientation of the substrates.

MECHANISM OF PARP1 ELONGATION REACTION 1205

BIOCHEMISTRY (Moscow) Vol. 89 No. 7 2024

Fig. 3. Important molecular interactions observed in the modeled enzyme–substrate complex of PARP1. Hydrogen bonds

of Gly863, Lys903, and Glu988 are shown as dotted lines. Phenyl group of Tyr907 forming π-stacking is shown in yellow.

For clarity, non-polar hydrogen atoms are omitted.

The neighboring Lys903 residue forms hydrogen bonds

with both Glu988 side chain (stabilizing its conforma-

tion) and diphosphate moiety of methyl-ADP (Fig.3).

Nucleophilic 2′

-OH group of the acceptor sub-

strate is presumably activated by the Glu988 carboxyl

group [18, 27]. However, according to the MD model-

ing data, these groups do not interact with each other

directly (Fig.3). We therefore considered two possible

proton relay pathways for further metadynamics sim-

ulations: in system A proton transfer is mediated by

the 3′

-OH of the acceptor substrate, and in systemB –

bythe 2′

-OH of the donor substrate (Fig.2b).

QM/MM metadynamics allowed us to reconstruct

2D free-energy landscapes of the PARP1-catalyzed elon-

gation reaction, in which CV1 describes nucleophilic

substitution at the C1′

atom, and CV2 – deprotonation

of the nucleophilic 2′

-OH group (Fig.4). Theenergy

surfaces demonstrate that in the case of both proton

relay systems, A and B, the reaction proceeds via the

1-like mechanism: an intermediate (oxocarbenium

ion) is formed and next it is attacked by the 2′

-OH

group of the acceptor substrate, with concomitant pro-

ton transfer to Glu988. It is worth mentioning, howev-

er, that the proton transfer is conducted by the 3′

-OH

(SystemA) rather than by 2′

-OH (SystemB), as indicat-

ed by the corresponding free-energy barriers (Fig.5).

Discrepancy in the P (reaction products) energies is

likely due to the proton transfer to different oxygen at-

oms of the Glu988 carboxyl group in systems A andB

(see Fig.2).

Fig 4. 2D free-energy landscapes of the PARP1-catalyzed elongation reaction. a)Proton relay systemA (includes 3′

-OH). b)Pro-

ton relay system B (includes 2′

-OH). Collective variable CV1 describes nucleophilic substitution at the C1′

atom, and CV2–

deprotonation of the nucleophilic 2′

-OH group. An oxocarbenium ion intermediate(I) is formed along the minimum energy

pathway between reactants(R) and products(P).

PUSHKAREV et al.1206

BIOCHEMISTRY (Moscow) Vol. 89 No. 7 2024

Fig. 5. Comparison of the free energy profiles of elongation

reaction reconstructed using proton relay systems A and B

(R,reactants; I,intermediate; P,products). The reaction coor-

dinate represents progress along the minimum energy path-

ways shown in Fig.4. Calculated errors for R, I, and P states

are given in TableS2 in the Online Resource1.

Figure 6 shows the obtained structures of the re-

actants, oxocarbenium ion intermediate, and products

in systemA. The reactants are properly oriented with

respect to each other by the Glu988 residue to main-

tain the reactive conformation (Fig.6a). The oxocar-

benium ion intermediate is formed with the release

of nicotinamide and is stabilized due to the negative

charge of Glu988. Planar configuration of the reactive

center (C1′

atom) facilitates subsequent nucleophilic

attack by the 2′

-OH group (Fig.6b). A new O-glycosidic

bond is formed between the adenine ribose and nic-

otinamide ribose to produce the elongation reaction

product; the Glu988 side chain becomes protonated

due to the proton transfer from 2′

-OH (Fig.6c).

DISCUSSION

Metadynamics is a powerful method to investigate

free energy landscapes of enzymatic reactions [54-58].

It biases the system evolution by a potential construct-

ed as the sum of Gaussian functions deposited along

the trajectory in the collective variable space [46].

The presented metadynamics study describes in detail

the PARP1 elongation reaction mechanism, summarized

in Fig. 7. It shows attachment of the ADP-ribose unit

to the growing PAR chain accompanied with proton

transfer to Glu988 via the 3′

-OH group. Formation of

the intermediate furanosyl oxocarbenium ion confirms

our hypothesis about the S

1-like mechanism [29]. The

main limitation of our methodology is that the acceptor

substrate is modeled using the methyl-ADP molecule.

The negatively charged PAR polymer may electrostati-

cally contribute to formation of the oxocarbenium ion,

whereas methyl- ADP mimics only the terminal PAR unit.

Participation of 3′

-OH in catalysis is consistent with

the results of an experimental study of NAD

analogues

lacking hydroxyl groups at positions 3′

and/or 2′

[59].

Qualitative analysis (SDS-PAGE/Western blotting with

anti-PAR antibodies) showed that absence of the 3′

-OH

group leads to the less efficient PAR formation, but not

completely prevents the reaction (which points to the

existence of an alternative 2′

-OH proton relay system).

The preferable proton transfer via 3′

-OH could pre-

sumably be explained by a shorter distance between

the 2′

-OH and 3′

-OH groups in the enzyme-substrate

complex: the distance O2′

 ∙∙∙ O3′

calculated from the

100 ns MD trajectory was 2.7 ± 0.1 Å, whereas the dis-

tance O2′

 ∙∙∙ O2′

was 3.2 ± 0.5 Å. It might be interesting

to design NAD

analogues lacking hydroxyl group at the

position 2′

or at both positions 3′

and 2′

to confirm

our hypothesis on the proton relay.

Fig. 6. Structures of reactants(a), oxocarbenium ion intermediate(b), and products(c) obtained from modeling of the PARP1-

catalyzed elongation reaction.

MECHANISM OF PARP1 ELONGATION REACTION 1207

BIOCHEMISTRY (Moscow) Vol. 89 No. 7 2024

Fig. 7. Proposed S

1 mechanism of the PARP1 elongation reaction. a)Substrate binding. b)Oxocarbenium ion intermediate.

c)Product formation.

PUSHKAREV et al.1208

BIOCHEMISTRY (Moscow) Vol. 89 No. 7 2024

Using molecular modeling and metadynamics sim-

ulations, we have demonstrated for the first time that

the PARP1-catalyzed elongation reaction proceeds via

the S

1-like mechanism involving formation of an in-

termediate oxocarbenium ion. Intriguingly, our results

show that the nucleophilic 2′

-OH group of the accep-

tor substrate (terminal PAR unit) is activated by the

general base Glu988 not directly but through the pro-

ton relay system including the adjacent 3′

-OH group.

The findings of this study shed light on the PAR synthe-

sis machinery involving complex polymer substrates

and PARP1, a major sensor of DNA damage, and may

be used in further design of new PARP1 competitive in-

hibitors mimicking the substrate and/or oxocarbenium

intermediate structure.

Supplementary information. The online version

contains supplementary material available at https://

doi.org/10.1134/S0006297924070046.

Acknowledgments. The research was carried out

using equipment of the shared research facilities of

HPC computing resources at Lomonosov Moscow State

University.

Contributions. D.K.N. conceptualization and man-

agement; S.V.P. and E.M.K. investigation; D.K.N. and

S.V.P. writing original draft; V.K.Š. review and editing.

Funding. This study was financially supported by

the Russian Science Foundation (project no.19-74-10072).

Ethics declarations. This work does not con-

tain any studies involving human and animal sub-

jects. Theauthors of this work declare that they have

noconflicts of interest.

Open access. This article is licensed under a Cre-

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