DSpace at VNU: Coarse grained simulation reveals antifreeze properties of hyperactive antifreeze protein from Antarctic bacterium Colwellia sp.

DSpace at VNU: Coarse grained simulation reveals antifreeze properties of hyperactive antifreeze protein from Antarctic...

Title: Coarse grained simulation reveals antifreeze properties

of hyperactive antifreeze protein from Antarctic bacterium

Colwellia sp., Chem Phys Lett (2015), http://dx.doi.org/10.1016/j.cplett.2015.08.042

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(1) We used coarse grained simulation to study the mechanism of Colwellia antifreeze protein (ColAFP)

(2) We found the distribution and conformation of ice crystal network surrounding

ColAFP at low temperature

(3) We identified phase space and located active conformation of water surrounding

ColAFP during the freezing process

Coarse grained simulation reveals antifreeze properties of hyperactive

antifreeze protein from Antarctic bacterium Colwellia sp

Hung Nguyen, 1 Thanh Dac Van, 1,2 and Ly Le 1,2,*

ABSTRACT

The novel hyperactive antifreeze protein (AFP) of Antarctic sea ice bacterium Colwellia sp provides a

target for studying the protection of psychrophilic microgoranisms against freezing environment Interestingly, the

Colwellia sp hyperactive antifreeze protein (ColAFP) was crystallized without the structural dynamic

characteristics Here, the result indicated, through coarse grained simulation of ColAFP under various subfreezing

simulation analyses also revealed the adaptive mechanism of ColAFP in subfreezing environment Our result provides a structural dynamic understanding of the ColAFP

Keywords: Antifreeze protein, ColAFP, coarse grained simulation, subfreezing temperature

I INTRODUCTION

Ice binding proteins or commonly known as antifreeze proteins (AFPs) are specialized glycoproteins for

There are commonly two types of AFPs: moderate AFPs and hyperactive AFPs Moderate AFPs are found in

Yuichi Hanada et al previously reported that an AFP identified from Antarctic bacterium Colwellia sp

strain SLW05 (ColAFP) was homologous to AFPs from a wide variety of psychrophiles They analyzed antifreeze

activity and solved crystal structure of the Antarctic bacterium Colwellia sp and demonstrated ColAFP TH activity

indicated ColAFP has the properties of hyperactive AFP Yuichi’s group further revealed the high resolution

structure of hyperactive ColAFP (PDBID 3WP9) which has an irregular β-helical structure They observed that

ColAFP has an ice-binding site which differs from the established ice-binding sites of other hyperactive AFPs

colleagues used the correspondence analysis as an ordination technique to reveal that evolution of genetic factors is

a selective and continuous process The method doesn’t make assumption about the data falling into discrete clusters

et al found that bacterial and archaeal ice binding proteins (IBPs) have relatively higher average hydrophobicity than

the eukaryotic IBPs Furthermore, bacterial IBPs and archaeal IBPs contain comparatively more strands, and

assumed to undergo higher selection pressure Additionally, molecular docking studies found that the ice crystals

form more stable complex with the bacterial and archaeal AFPs than their eukaryotic orthologs Analysis of the

docked structures showed that the ice-binding sites (IBS) in all of the orthologs facilitate ice-binding activity even

after mutation using the comprehensive IBS model of Typhula ishikariensis Notably, all of the IBS mutations have

been found to prefer polar and hydrophilic amino acids which assist in the ice-binding using “anchored clathrate

mechanism” Horizontal gene transfer studies indicated the strong selection pressure favoring independent evolution

of the IBPs in some Antarctic microorganisms facilitates their adaptation and survivability to the adversities in their

In this study, we investigate the molecular recognition mechanism and interactions of ColAFP in freezing

and subfreezing marine water using coarse grained molecular dynamics simulation method

II MATERIALS AND METHODS

Preparing the protein structure: The 3D structure of Antarctic sea ice bacterium Colwellia sp AFP was collected

visualize the atomistic structure of ColAFP And then, we used MARTINIZE version 2.2 to transform ColAFP

two beads according to their specific sizes, which has been classified into two broad categories: backbone and side

chain beads

Simulation Methods: The GROMACS 4.5.5 package30 with MARTINI force field for coarse grained model were

used to generate MD trajectory The periodic boundary conditions were used throughout the simulation process; the

electrostatic potential was shifted from 0.0 nm to 1.2 nm and the Lennard Jones (LJ) potential was shifted from 0.9

distance of 1.2 nm from the solute and the box boundaries Specialized water model was used for coarse grained

with a concentration of 0.6 mol/L to mimic the condition of sea water The coarse grained water model is described

 The coarse grained water model is a representation for one of the major simplification of the solvent, which

is either implicitly or explicitly modeled as a van der Waals particle The effect of polarization and the proper screening of interactions depending on the local environment is absent The polarizable coarse grained water molecules are represented by three particles instead of one as in the standard Martini force field The central particle W is neutral and interacts with other particles in the system by means of the LJ interactions which is similar to standard water molecules The additional particles WP and WM are bound

to the central particle and carry a positive and negative charge of +q and -q, respectively They interact

with other particles via Coulomb function, and lack any LJ interactions The bonds W-WP and W-WM are

constrained to a particular distance and is symbolized as character l In addition, the interactions between

WP and WM particles inside the same coarse grained water bead are excluded making these particles become “transparent” toward each other As a result, the charged particles can rotate around the W particle

The dipole momentum of the water bead depends on the position of the charged particles and can vary from

zero (charged particles coincide) to 2lq (charged particles are at the maximal distance) A harmonic angle

particles which in turn adjust the distribution of the dipole momentum The average dipole momentum of the water bead is dependent on the charge distribution and is expected to be on average zero in an apolar environment, such as the interior of the lipid bilayer In contrast, some of non-zero average dipole are observed in bulk water or in some other polar environment The mass of the charged particles as well as of the central particle is set to 24 amu and totaling of 72 amu (the mass of four real water molecules)

 Water molecules play an important function as ubiquitous solvent in biological systems, its treatment is crucial to the properties derived from MD simulation In this research, the parameterization of polarizable coarse grained water model was used in combination with the coarse grained MARTINI force field The three-bead model representing four water molecules was used to show that the oriented polarity of real water can be effectively accounted Consequently, the dielectric screening of bulk water is reproduced At the same time, the parameterization was used as a water model with bulk water density and oil/water partitioning data which reproduced a similar level of accuracy as in the standard MARTINI force field

including 263 K, 265 K, 267 K, 269 K, 271 K, 273 K, 275 K, 277 K, 279 K, 281 K, 283 K and 285 K In addition,

mol/l) at 271 K, 273 K, 275 K, 277 K, 279 K, 281 K and 303 K The final MD simulation allowed us to integrate the

Free energy landscape: The free energy landscape along n-dimensional reaction coordinated V = (V1,…,Vn) was

III RESULTS AND DISCUSSIONS

The Fig 1a showed hydrophilic interaction of ColAFP and surrounding water molecules, it was found to

differ with varying temperatures The hydrophilic residues were exposed to liquid water, the interaction between

hydrophilic residues and liquid water generates enthalpy lead to the formation of hydrogen bonds and subsequent

cage-like structure; meanwhile hydrophobic residues interact with ice crystal to produce entropy As enthalpy and

entropy change, ColAFP ice-binding site switches it interaction to adapt with variation in temperature as was found

ColAFP and water molecules is subjected to reduce as ice crystal expanding and vice versa in order to interact with

ice crystal Evidently, our simulation results were able to reveal this interaction mechanism in term of hydrophilic

interaction between ColAFP and various phases of subfreezing water (ice crystal, ice-liquid hybrid, and liquid

water) The results of hydrophilic interaction demonstrate this adaptation mechanism of ColAFP in subfreezing

water environment It shows that the hydrophilic interaction energy of ColAFPs complexes has a tendency to

increase as temperature increases And when temperature reaches above 275 K, the hydrophilic interaction increases

to maximum, this was found to be caused by conformational expansion of ColAFP which subsequently affects the

hydrophilic interaction of the system Moreover, we found that the hydrophilic interaction between ColAFP and its

assume that ColAFP can deploy it ability in prohibiting ice formation by adapting its ice binding site conformation

to specific subfreezing temperature condition

environments This function measures the distance of surrounding water molecules At subfreezing temperatures, the

and 303 K It reveals that the water molecules formed ice crystal from 271 K to 279 K, ice-liquid hybrid at 281 K

and liquid solution at 303 K These results indicate fundament states of subfreezing water: ice crystal, ice-liquid

hybrid, and liquid

Fig 1 The hydrophilic interaction of ColAFP complexes (Fig 1a) and the radial distribution function of

271 K, 273 K, 275 K, 277 K, 279 K, 281 K and 303 K (Fig 1b) The snapshots of waters-ions complexes simulated

at 271 K, 281 K and 303 K indicates different water states in the form of ice crystal, ice-liquid hybrid, and liquid

accordingly were shown in Fig 1b

Based on previous findings from Fig 1b, we were able to fundamentally establish temperature regions in

which the surrounding water molecules of ColAFP can be classified (ice crystal, ice-liquid hybrid and liquid) and

temperature regions that ColAFP is most likely to maintain its active conformation Fig 2a shows the radial

distribution function of water - water in ColAFP complexes From initial observation of the radial distribution, we

divided our analysis based on the behavior of subfreezing water into three cases: ice crystal, ice-liquid hybrid, and

liquid In the first case, our observation, from four temperature dependent systems of 263 K, 265 K, 267 K and 269

were fluctuated around 4.75 for the first peak, 2.5 for the second peak, 1.75 for the third peak and 1.5 for the fourth

peak accordingly It means that the water molecules surrounding ColAFP were found to exhibit ice crystal structure

at subfreezing temperatures of lower than 269 K In the second case, temperature dependent systems of 271 K and

we found that the water molecules exhibited ice-liquid hybrid form and that the water molecules near antifreeze

protein was not forming crystal but distant water molecules were found to form crystal cage At 271 K, the ice part

in complex was found to be dominant compared with liquid part Interestingly, this phenomenon was reversed when

the system was at 273 K which revealing antifreeze property of ColAFP In the case of liquid water, the last six

for the second peak and 1.25 for the final peak In this case, the water molecules were entirely in liquid state

Fig 2b, which describes the distance between water and protein changed according to simulated temperatures Like

could be found with the ice crystal formation surrounding ColAFP We identified that ColAFP inhibits the ice

ColAFP was homologous to AFPs from a wide variety of psychrophiles They analyzed antifreeze activity and

strongly agreement with previous reports of Yuichi’s group This finding is described in greater detail in later

sections

Fig 2 The radial distribution function of water-water (Fig 2a) and water-protein (Fig 2b) in ColAFPs complexes

The values reveal the activity of ColAFPs under temperature variations

The mean square displacement (MSD) is a method used to describe fundamental dynamic quality, while the temperature dependence of average MSD is used to characterize the internal flexibility of water molecules As seen

from Fig 3a, the MSD values were increasing in accordance with their corresponding temperatures The MSDs

reached highest at 285 K and lowest at 263 K Indicatively, MSDs can be used to establish subfreezing temperature

regions that water molecules exist in the form of ice crystal, ice-liquid hybrid, and liquid

Fig 3 Behavior of water at subfreezing temperatures reveals various phases that lead to ice crystal formation The

Fig 3a and Fig 3b illustrate mean square displacement (MSD) and totally energy results of system as a function of

100 ns simulation time accordingly

the variation in total energy of ColAFP systems at subfreezing temperatures to determine their dependence on

systems was found to reach stability at 1 ns for 263 K and 265 K, 5 ns for 267 K, 20 ns for 269 K, 73 ns for 271 K

and 98 ns for 273 K From this result, we found that the changes in total energy of ColAFP systems indicate physical

state transformation from liquid to solid Especially, at 265 K, the existence of three energy states: the liquid state

which ranged from 0 ns to 1 ns, the first solid state from 1 ns to 40 ns and the second solid state from 40 ns to 100

subfreezing temperatures but not for the remaining temperatures Collectively, these results describe the behavior of