4P079

Structure and Stability of the Binding of HNP-1 with DPPC
Lipid Bilayer: A Molecular Dynamics Study(Kanazawa Univ.*, Bandung Institute of Technology**) Nandia Primasari*,**,Hiroaki Saito*, Santi Nurbaiti**, Kazumoto Kawaguchi*, Hidemi Nagao*[Introduction]

Defensins are cationic immune peptides contain three disulfide
bonds, which are in charge of protecting from pathogenic microbes. Mammalian
defensins are classified into α, β, and θ category, based on the size and the disulfide bondpattern. HNPs 1, 2, and 3, are the most abundant forms of human α-defensins. From
many experimental studies, it is known that HNP-3 is less microbicidal than HNPs 1and 2 against most of tested microbes. HNP-1 and HNP-2 showed almost
indistinguishable activities against the gram-positive strains, however HNP-2 appeared
to be more potent than HNP-1 against the gram-negative strains. It leads to theresearch of the interaction of HNP-1 with lipid membrane.
It is known that HNP-1 can kill the pathogen by disrupting its membrane. Thismechanism of the membrane disruption by the HNP-1 has been predicted in somestudies. The details of bind structure, molecular interactions, and the stability of theHNP-1, which are key properties to understand the mechanism, have not been clarified
yet. In this study, we thus carried out the molecular dynamics (MD) simulations of theDPPC lipid bilayer in the presence and absence of HNP-1 dimer and analyzed thedynamical structure of the system in detail. The effects of HNP-1 binding on themembrane structure and the molecular affinity between the polar headgroups of themembrane and the arginine side of HNP-1 were also investigated in this study.
[Experiment]
MD simulations were carried out by using NAMD 2.9 and VMD
1.9.1 simulation package. CHARMM36 force field and TIP3P water model were adopted
for lipid and water molecule, respectively. A structure of the HNP-1 was obtained with
X-ray diffraction experiment and was downloaded from the Protein Data Bank (3GNY)
as the initial structure. The initial structure of DPPC (dipalmitoylphosphatidylcholine)
membrane contains 128 lipid molecules and 4000 water molecules, and this wasconstructed by using Packmol.
Figure 1 shows the predicted membrane disruption mechanism of the HNP/membrane[1]. This model reference [1] shows the process when the arginine side of HNP initiallybinds to the bilayer surface electrostatically. This process is a part of the defensinsmediated pore formation.
In this study, we modeled the binding structure, which the HNP-1 dimer is placed on theequilibrated membrane using VMD, and investigated the dynamical structure of theHNP-1/DPPC membrane by the MD simulation. First, we carried out the MD simulationof the DPPC membrane in the absence of HNP-1 dimer for 50 ns. Langevin thermostat
and barostat were used to control the system temperature (T=323.15K) and pressure(P=1atm), and the oscillation period of thermostat and barostat were 5 ps and 0.2 ps,respectively. After that, we analyzed the membrane structure parameters: area per lipid

Figure 1. Model reference of HNP-1 bound toDPPC lipid bilayer (Reference [1])Figure 2. HNP-1 dimer binds to the DPPCbilayers

molecule (! ! ); membrane thickness; and order parameter (!!" =!
!

3 cos ! ! − 1 ), to get

the appropriate initial condition for the system simulation and see the difference withthe condition when HNP-1 exists. Furthermore, we construct the system by placing theHNP-1 on the equilibrated membrane (Figure 2). Then, we carried out MD simulation ofthis system at 320 K through four stages: melting the lipid tails, minimization andequilibration
withprotein
constrained,equilibration
withprotein
released, and the NPT long production run for 50 ns. The structural stability of theHNP-1 was analyzed by estimating the RMSD, RMSF, and SASA of the peptide.

[Results and Discussion]

The membrane quantities of the DPPC bilayers areevaluated by using the area per lipid molecule, membrane thickness, and the orderparameter. The effect of binding of HNP-1 on the membrane interface was accessed bythese membrane quantities in the absence and presence of the HNP-1. In the absence ofHNP-1, the simulation yielded ! = 61.16  Å! /lipid. This result is close to theexperimental data, ! = 63.0 ± 1.0!"   Å! . The bilayer thickness is found to be 38.8  Å,
being agree with the experimental value of 38.3  Å.
The equilibration MD simulations of theDPPC membrane in the presence of HNP-1
have been done for 1 ns. Figure 3 shows theRMSD as a function of MD time steps. We
found that the structure of HNP-1 could
reach a stable position at the interface
quickly and oscillates around 1.5 Å. This
indicates that the structure of HNP-1 on
the membrane interface is sufficientlyFigure 3. RMSD of HNP-1 at theequilibration with protein released
maintained. Further results and analysis
stage for 1 nswill be displayed in the poster session.

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