Photon Factory Activity Report 2017 #35 (2018)

BL-5A, AR-NW12A/2016G554

Formation of Chain-like Arrays of Ammonium Sulfate Ions in Protein Crystallization
Tyuji Hoshino1,* Mariko Kitahara1 Satoshi Fudo1 Tomoki Yoneda1 and Michiyoshi Nukaga2
1
Graduate School of Pharmaceutical Sciences, Chiba University,
1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan
2
Faculty of Pharmaceutical Sciences, Josai International University,
Gumyo 1, Togane-shi Chiba 283-8555, Japan

1 Introduction
Protein structure is fundamental information tounderstand the property, shape, and function of a protein.
X-ray crystal analysis is one of the most popular
approaches to determine the protein structure. Growth ofsingle crystal is a critical step in the X-ray
crystallography. Ammonium sulfate (AS) is one of themost popular precipitant agents to induce thecrystallization of protein molecules. AS is, however, notalways effective for all kinds of proteins in crystal growth.
Some proteins are readily crystallized by utilizing AS as aprecipitant but some proteins are not. The reason why theprobability of crystal growth by AS is different amongproteins is unknown. Similar to AS, poly-ethylene glycols(PEGs) are frequently utilized in protein crystallization.
Crystal growth of some kinds of proteins are effectivelyinduced by PEGs, instead of AS. The cause for thedependency of protein crystallization on precipitant has
not been studied in the atomistic level so far.
In our previous study [1], a single kind of protein wasgrown with three different precipitant agents, including
AS and PEG. Molecular dynamics (MD) simulations
were carried out for the protein in the presence ofprecipitants in the concentrations equivalent to therespective crystallization conditions. As a result, thedistribution of precipitant molecules was found to be notisotropic around the protein. In the simulations with AS,
AS ions play roles not only of decreasing the protein
solubility but also of restricting the contact sites on theprotein surface, which is reflected in the molecular
packing in crystal growth. The molecular packing islinked to the space group of crystals. Therefore, therelationship between space group and precipitant agent
was also investigated for several kinds of proteins, bysurveying more than a thousand of crystal structures.
In this work, we performed X-ray structure analysis,by growing single crystals for four kinds of proteins;
human carbonic anhydrase II (CAII), horse myoglobin(Mb), hen egg white lysozyme (HEWL), and humanserum albumin (HSA). The former two proteins, CAIIand Mb, are known to be easily crystallized by AS. On
the other hand, the latter ones, HEWL and HSA, are not.
The crystal growth of HEWL by AS is so difficult that nocrystal structure has been deposited in protein data bank(PDB). Only a photo image of HEWL crystal by AS isavailable in literature. The crystal growth of HSA by AS
is also difficult and then no PDB entry is available and nocrystallization has been reported. It should be noted that alarge number of crystal structures are available in PDB
both for HEWL and HSA, but all of them were obtainedby the crystals grown with the precipitants other than AS.
2 Experiment
Recombinant CAII was produced by an E. coli straintransformed with the expression vector. Mb, HEWL, andHSA were purchased from suppliers who provided nonrecombinant proteins extracted from natural products. Allof the proteins were purified before crystallization. X-ray
diffractions were acquired at synchrotron beamlines.
Atom coordinates were determined by molecularreplacement method.
In this work, we also performed MD simulations forthe four proteins with and without AS ions in thecalculation models. Motion of AS ions, representative AS
distribution around the proteins, and atom fluctuation
with respect to protein residues and AS ions werededuced from the MD trajectories. Electrostatic potentials
were drawn from the protein structures. To clarify thereason for readiness and difficulty in crystal growth byAS, these computational results were compared among
the proteins. MD simulations were executed by a graphic
processing unit (GPU) executable code of pmemd moduleof AMBER16. Three 200 ns MD runs were performed forevery protein in the presence of AS ions and a single 200
ns run was also performed for comparison.
3 Results and Discussion
Protein single crystals were grown for CAII, Mb,
HEWL, and HSA in the AS concentrations of 2.7, 3.2, 1.2,
and 1.2 M, respectively. Details in crystallographic data
are shown in Table S1. The max resolutions for therespective proteins were 1.27, 1.56, 1.13, and 2.92 Å. The
major precipitant reported in literature for crystallizationof CAII and Mb is AS and the space groups are P 1 21 1for CAII and P 6 or P 1 21 1 for Mb. Therefore, thecrystals obtained in this work reproduced the previousstudies. The space group of HEWL crystal grown by AS
was P 1, which was different from the most popular spacegroup of HEWL crystals, P 43 21 2. The max resolutionfor HEWL, 1.13 Å, was better than the average resolutionof the crystal structures deposited in PDB, 1.73 Å. Thespace group of HSA by AS was C 1 2 1 and the max
resolution was 2.92 Å. The major space group of the HSAcrystals deposited in PDB is C 1 2 1 and the average
resolution is 2.60 Å. In terms of resolution, the quality ofPhoton Factory Activity Report 2017 #35 (2018)

the HSA crystal grown by AS is inferior to the crystals
obtained by other precipitants.

Fig. 1: Snapshot structures extracted at 0, 50, and 100 ns
from the simulation trajectories for (a) CAII, (b) Mb, (c)HEWL, and (d) HSA. The right and left columns
represent the simulations with and without AS
precipitants, respectively. The simulations were executedin triplicate in the presence of AS and the snapshots
shown are of the first one for every protein. Protein
molecules are represented by green cartoons. N, S, and O
atoms of AS ions are coloured blue, yellow, and red,respectively. Hem molecule in Mb is depicted in a yellow
stick representation. Sodium and chloride ions and watermolecules are not shown for clarity.

Calculation models for CAII, Mb, HEWL, and HSA
were built from the obtained crystal structures. MD
simulations were performed for 200 ns in the presence ofAS and also for 200 ns in the absence of AS. The
simulations with AS were executed in triplicate forreproducibility. The concentrations of AS ions in thecalculation models were set to be equal to the initial
droplet in the experimental setup for protein
crystallization and other minor ingredients were omittedfrom the models.
The motions of protein and precipitant molecules weremonitored all through the 200 ns simulation. The
snapshot structures at 0, 50, 100 ns were extracted bothfor the models with and without AS (Fig. 1). In every

simulation without AS, protein molecule largely moved
in the solvent and the direction of the principal molecularaxis was ceaselessly changed. Since the molecularweights of Mb and HEWL are small compared to those of
CAII and HSA, the displacement of center of mass wasconsiderably large for Mb and HEWL. Due to thepresence of AS ions, the motion of protein molecule wasmarkedly reduced for every protein. In CAII (Fig. 1a), notonly the center of mass but also the direction of molecularaxis was rarely altered. The initial setup of AS positions,in which AS ions were randomly placed in solvent, wasreflected in the AS distribution at 0 ns. At 50 ns, AS ionswere relocated not to cover all over the protein surface
but to make local contacts with CAII. The AS distribution
is not uniform and instead chain-like arrays were formed.
The chain-like distribution became clear at 100 ns. The
motions of AS precipitants were unexpectedly small. In
Mb (Fig. 1b), AS distribution at 0 ns was random. AS
ions were arranged to form chain-like distribution at 50 ns
and the AS dense areas were connected by the chains.
The chain-like distribution was stable because ASpositions were scarcely changed at 100 ns. In HEWL (Fig.
1c), while AS ions were randomly distributed at 0 ns, AS
ions were strongly localized at a few areas at 50 ns. The
AS localization is, however, instable because thedistribution was rearranged at 100 ns. In HSA (Fig. 1d),the AS initial random distribution at 0 ns was rearranged
at 50 ns. The chain-like AS formation was stable and keptat 100 ns. A comparison of aminations among CAII, Mb,and HSA suggested that most of the AS ions moderately
moved in CAII and Mb but a considerable number of AS
ions largely fluctuated in HSA.
To quantify the motions of AS ions in simulation, root
mean square fluctuation (RMSF) were calculated for theprotein residues and AS ions (Fig. 2). In CAII, RMSFs ofmost of the protein residues were below 20 Å2 while threepeaks over 30 Å2 were observed in the simulation without
AS. The three large peaks were decreased due to thepresence of AS. RMSFs of AS ions were large comparedwith the protein residues and the average RMSF was 38.7
Å2. The number of the peaks beyond 150Å2 was zero. InMb, most of the protein residues showed RMSFs below
10 Å2. The average RMSF of AS ions was 49.9 Å2 andnone of the peaks was beyond 150Å2. In contrast, there
were many peaks over 150Å2 in HEWL and HSA. TheseRMSFs reflect the situations that the restraint on AS ions
are weak in HEWL and HSA. RMSFs in Fig. 2 are theresults of the first simulation of the three calculation trials
for each protein. In CAII, RMSFs of the AS ions in thesecond simulation were considerably low and the average
is below 25 Å2. In Mb, RMSFs in the second simulation
were quite low. In contrast, RMSFs in the second andthird simulations were quite large for HEWL. RMSFs inthe second and third simulations in HSA were also large.
These results clearly suggest that the mobility of AS ion
is small in CAII and Mb but that is large in HEWL andHSA. In HSA, the decrease of RMSFs of the proteinresidues due to the presence of AS was remarkable.

Photon Factory Activity Report 2017 #35 (2018)

In order to explain the chain-like distribution of ASions, the iso-surfaces of electrostatic potential around theproteins were depicted (Fig. 3). In CAII and Mb, theelectrostatic potential was clearly separated into thepositive and negative areas almost in half and both areas
had hemispherical shapes. AS chains are positioned toconnect the positive and negative areas. Hence, AS ionsare arranged along the electric field lines that run
perpendicular to the iso-surfaces of electrostatic potential.
In contrast, the polarity of the electrostatic potential isseriously in partial in HEWL and HSA and there is amarked difference between the positive and negative
areas in volume. In HEWL, the whole protein iscompletely covered with the positive potential surface
and then the chain-like arrays of AS ions are unlikely to
be formed. In HSA, the electrostatic potential is greatly
biased to negative and then many of the chain arrays are
entirely within the negative potential area and only a shortarray manages to connect the positive and negative areas.
Consequently, the distribution of AS ions are stabilized in
the situation that the electrostatic potential around theprotein is equally separated into the positive and negative
areas and the AS chain arrays are likely to be formed toconnect the two areas as seen in CAII and Mb.

Fig. 2: RMDFs with respect to the protein residues andthe AS ions in the simulations for (a) CAII, (b) Mb, (c)HEWL, and (d) HSA. The simulations with and without
AS are represented by blue and red lines, respectively.
The green bar on the x-axis corresponds to the protein
residues. RMSF with AS in every protein is of the firstone of the three simulations in the presence of AS.

Fig. 3: Positive and negative iso-surfaces of electrostatic
potential around the proteins for (a) CAII, (b) Mb, (c)HEWL, and (d) HSA. The positive and negative isosurfaces are shown in the blue and red meshrepresentation. The contouring values of iso-surfaces are(a) +0.43 and -0.14 for CAII, (b) +0.36 and -0.13 for Mb,(c) +1.75 and -0.004 for HEWL, and (d) +0.43 and -1.96
for HSA, respectively. Colorings for molecules are thesame as Figure 1. Every electrostatic potential wasobtained by the presentative structure of the first one of
the three simulations in the presence of AS.

It will be expected from the findings of this study thatan introduction of amino mutations can enhance thePhoton Factory Activity Report 2017 #35 (2018)

crystallization even for a protein whose experimental
condition for the crystal growth with AS is not wellestablished. The optimal zone of precipitant concentration
is narrow for crystallization with AS. The electrostatic
potential will be utilized as a guide to modify
recombinant proteins adequate for crystal growth with AS.
For example, when a protein crystal is obtained with
precipitant other than AS but the resolution of X-ray
diffraction is not satisfactorily high, a better quality ofprotein crystal is sometimes required. Modeling of theprotein structure from the low-resolution X-ray
diffraction, modification of the model by introducing
amino mutations, and calculation of the electrostatic
potential for the modified model will provide a goodsuggestion on the choice of amino mutations to promote
the crystal growth with AS. The electrostatic potential
significantly depends on the pH of solution. Multiple
calculations of the electrostatic potentials for the models
changing the protonation states of the titratable residues
will provide the information on the appropriate pH
condition to promote the protein crystallization by AS.
Therefore, the analysis of the electrostatic potential
enables us to take the rational strategy in experiments.
In summary, protein crystals were grown for CAII andMb, which are easily crystallized by AS, and also for
HEWL and HSA, which are not. X-ray diffractions wereacquired from the respective protein crystals andstructures were determined by the molecular replacement.
Based on the determined protein structure, 200 ns MD
simulations were performed in triplicate with the model
including AS ions in a concentration same as theexperimental condition in crystal growth for every protein.
A 200 ns simulation was also performed without AS ionsfor comparison. For all the four proteins, the presence of
AS ions greatly reduced the motion of protein during
simulation. RMSF analysis indicated that the motions ofAS ions were also restricted especially for CAII and Mb.
This means that the AS ions are likely to be stabilizedaround the proteins that are easily crystallized by AS. AS
ions are not randomly distributed and instead they arearranged in the chain-like formation. The chain-likeformation is caused by the electrostatic potential aroundthe protein, in which AS ions are aligned along theelectric field lines. In CAII and Mb, the positive andnegative areas of the electrostatic potential are almost
equally separated and both areas have hemisphericalshape. Hence, a large electric field line connecting bothareas can be generated. In contrast, one of the positive
and negative areas is enlarged in HEWL and HSA. Hence,long electric field line is difficult to be generated. That is,
the shape of the iso-surface of the electrostatic potential isstrongly responsible for the readiness in crystal growthwith AS. If the shape of the electrostatic potential can becontrolled by adjusting pH condition or introducing
amino mutations, the quality of crystallization by AS will
be improved even for the proteins which are not easily
crystallized by AS.

Acknowledgement
Calculations were performed at Research Center for
Computational Science, Okazaki, Japan and atInformation Technology Center of the University ofTokyo. This work has been performed under the approvalof the Photon Factory Program Advisory Committee(proposal no. 2016G554).
References[1] S. Fudo et al., Cryst. Growth Des.. 17, 534 (2017).

* hoshino@chiba-u.jp