parameters for lipids and the ligand were derived using
The periodic box contained about 135000
atoms. The s imulations were conducted at a constant
temperature of 298 K using the Berendsen thermostat and a
constant pressure of 1 bar. The simulation time step was set to
2.5 fs. During the ﬁrst 0.25 ns of the MD simulation each of the
complexes (wild-type and mutant) was kept frozen to allow the
membrane to relax and to adapt to the embedded protein
complex without disrupting its structure. During the next 3 ns
of the simulation we imposed weak restraints on the
experimentally predicted interactions between the ligand and
residues 134, 137, 142, and 144 in the adjacent protein chain.
Restraints were included in diﬀerent combinations (Table S1
and S2): 1. hydrogen bond between the C7-OH of archazolide
and the backbone or side chain of I134 and E137, respectively; 2.
hydrogen bond between the C15-OH of archazolide and the
side chain of Y142; 3. hydrophobic contact between the i-butyl
side chain of archazolide and the side chain of I144. The
restraints were imposed in the form of the SoftSquare poten-
(a hydrogen−acceptor distance equal to 2.0 ± 1 Å and
for the van der Waals interaction with L144 a distance of: 3.0 ±
1.0 Å). We assessed the MD runs by monitoring hydrogen
bond formation (Table S1 and S2). The hydrogen bonds
statistics was computed using the VMD software
acceptor distance: 3.5 Å, angle cutoﬀ: 30 degrees). The MD run
with the restraints set no. 3 (Table S1 and S2) was extended to
5 ns, and the representative snapshot with the lowest energy
was provided as a ﬁnal result.
To check the stability of the V-ATPase model we further
extended the MD simulation with restraints set no. 3 of the
wild type protein to 10 ns and computed the rmsd with respect
to the starting homology model (Figure S4).
Synthesis of Archazolide Derivatives. The structures of
15-dehydro-archazolide (4) and 1′-descarbamoyl-archazolide
(5) were synthesized according to the published procedure.
Biological Assays. Puriﬁcation of yeast vacuoles and
following activity assays were carried out as described in ref
11. The concentration of inorganic phosphate in the samples
was determined according to ref 40.
Protein sequence alignments, yeast homology model, electro-
statics of yeast V-ATPase and archazolide A, computational
details for the molecular docking as well as the molecular
dynamics simulations, additional material of all docking-derived
binding modes, and hydrogen bond analyses of the MD
trajectories. This material is available free of charge via the
Internet at http://pubs.acs.org.
*Phone: +49 6221 3878552. Fax: +49 6221 3878519. E-mail:
International Institute of Molecular and Cell Biology, 4 Ks.
Trojdena, 02-109 Warsaw, Poland.
-Institute of Organic Chemistry and Biochemistry,
The authors declare no competing ﬁ nancial interest.
We thank the German Science Foundation (DFG, Graduate
College 850:’Modeling of Molecular Properties ’ ) for generous
ﬁnancial support (stipend to S.D.) and cluster usage as well as
ger, Alexander Metz, and Doris Klein (Heinrich-
sseldorf) for assistance in using Drugscore
and fruitful discussions. T.C. acknowledges ﬁnancial support
from EMBL and Grant I/81 637 from the Volkswagen Stiftung.
D.L. acknowledges the computational grant G35-6 from ICM
Warsaw. S.B., M.H., and H.W. acknowledge ﬁnancial support
from the Volkswagen Stiftung (Grant I/82 801).
DCCD = N,N′-dicyclohexylcarbodiimide; RDC = residual
dipolar coupling; GA = genetic algorithm; MD = molecular
dynamics; PEA = phosphatidylethanolamine
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