Photon Factory Activity Report 2022 #40 (2023)

BL-17A/2022G642

Influence of Glycan Agents on Protein Crystallization with Ammonium Sulfate
1

Tyuji Hoshino1,* Yan Guo1
Graduate School of Pharmaceutical Sciences, Chiba University,
1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan

1 Introduction
The information from the high-resolution crystal
structures is advantageous for understanding themolecular mechanisms of macromolecules and isessential in rational drug design. A limiting factor is
obtaining single protein crystals of good quality since
the crystallization process is influenced by manyparameters such as precipitant agent, pH,temperature, etc. The predictions of the adequate
values of those parameters are difficult. A current
orthodox approach is to optimize the initial
crystallization conditions obtained with the screening
kits. Ammonium sulfate (AS) is a typical salting-typeagent and is the most widely applied precipitant inprotein crystal growth. Additionally, the protein
crystallization with the salt-type precipitants cansometimes be improved by adding specific chemicals
like small compounds. The additives serve toincrease protein stability and mediate crystal latticecontacts. However, finding the appropriate additives
to improve the quality of single crystals is an empirical
procedure and a time-consuming process of trial anderror.
In this study, two kinds of protein, influenza virus
polymerase acidic subunit N-terminal domain (PAN)
and chitosanase, were crystallized by the AS-based
precipitants. The precipitant solutions containtrehalose and 3α,7α,12α-tri-((O-β-D-glucopyranosyl)
ethyloxy)-cholane (Facade-TEG), respectively. Bothcrystals diffracted better than the crystals obtained inour previous works. Multiple MD simulations wereperformed with and without glycans to understand theprotein–glycan interactions at an atomistic scale andto gain deep insight into the conformational dynamics
of glycan-protein complexes. Root mean squaredeviation (RMSD), radius of gyration (Rg), and Bfactors were obtained from simulation trajectories toassess the conformational stability of the complex
structures. Principal component analysis (PCA) wasemployed to examine the conformational dynamics ofprotein structure. The binding energy between protein
and glycan was also estimated using the molecularmechanics Poisson–Boltzmann surface area (MMPBSA) approach. Next, the shapes of electrostatic
potentials around the proteins were investigated andcompared with the distributions of glycan agents. In
the comparison, we examined if the electrostatic
potential correlated with the favorable binding sites ofglycan agents on the protein surface. Further, thecrystal structures obtained by incorporating glycanagents in AS solution were surveyed through theProtein Data Bank (PDB) to assess the relevance ofglycan agents to protein crystal growth from astatistical viewpoint.
2 Experiment
The PAN domain, residues 1-197, of the influenza
H1N1 strain was cloned into the pET50b plasmidvector. A loop region, residues 51-72, was replacedby an Ala-Ser dipeptide linker to enhance the protein
crystallization. The E. coli strain, C41 (DE3),transformed with the plasmid vector, was cultured in
an LB medium. The protein was expressed at 17 °C
for 48 hours after induction at an OD600 value of 0.8
with 0.2 mM isopropyl-β-D-thiogalactopyranoside(IPTG). A cell pellet obtained by centrifugation wasresuspended in the buffer of 50 mM Tris-HCl at pH
8.0, 150 mM NaCl, and 10 mM imidazole. The
bacterial cell membrane was disrupted by sonication.
The protein was purified by a Co metal affinity
column with a linear gradient of the imidazole
concentration. The eluted protein fraction wasdialyzed overnight against a buffer containing 50 mM
Tris-HCl at pH 8.0 and 150 mM NaCl. The 6×Hisfused Nus-tag was cleaved by incubating with humanrhinovirus 3C (HRV-3C) protease for 24 hours at 4 °C.
The 6×His-fused Nus-tag, HRV-3C protease, and theremaining uncleaved protein were removed by NiNTA resin. PAN was further purified by anion
exchange column with a starting buffer A (50 mM TrisHCl at pH 8.0, 50 mM NaCl) and an elution buffer B(50 mM Tris-HCl at pH 8.0 with 1000 mM NaCl),followed by gel filtration. The gel filtration runningbuffer was of 20 mM Tris-HCl at pH 8.0 and 100 mM
NaCl. The protein was finally concentrated at 10.2
mg/mL.
AnN-terminus-truncatedchitosanase
wasexpressed as a 6×His-fused Nus-tag-conjugatedform with a pET50b vector. An Escherichia coli strain,Rosetta (DE3) pLysS, was transformed with theplasmid vector. After pre-culture in an LB medium at37°C, the protein was expressed at 26°C overnightby induction with 0.2 mM IPTG at an OD600 value of
0.6. The conjugated protein was purified by Co metalaffinity chromatography, followed by the cleavage of
Nus-tag with HRV-3C protease. The cleaved Nus-tag,
HRV-3C protease, and uncleaved conjugated protein
were removed by NTA resin. The protein was further
purified by gel filtration with a running buffer of 10 mM
Tris-HCl at pH 8.0 and 150 mM NaCl. Finally, theprotein was concentrated at 7.3 mg/mL.

Photon Factory Activity Report 2022 #40 (2023)

A crystal of PAN was grown by vapor diffusion at
18°C in hanging drops containing 1.0 µL of protein
solution including 4.0 mM MnCl2 and 1.0 µL of wellsolution consisting of 0.1 mM MES at pH 5.8, 1.275
M ammonium sulfate, 0.1 M potassium chloride and
9% (v/v) trehalose. The crystal was cryo-protected bybrief immersion in a well solution containing 25% (v/v)
glycerol, followed by freezing in liquid nitrogen.
A single crystal of chitosanase was grown by thesitting drop vapor diffusion method at 18°C. The
protein solution was mixed with a precipitant solution
consisting of 0.1 M sodium citrate at pH 5.0, 3.4 M AS,
and 5% (v/v) Facade(R)-TEG at an equal volume.
The crystal was cryo-protected by immersion in asolution containing 25% (v/v) glycerol before freezing.
X-ray diffraction data were collected at 100 K witha wavelength of 0.98 Å at the BL17A beamline ofphoton factory (PF), Tsukuba, Japan. The diffraction
data were indexed, scaled, and merged by XDS.
Intensities were converted into structure factors, and5% of the reflections were flagged for Rfree
calculations. The structures for PAN and chitosanase
were determined by molecular replacement by
MolRep with the crystal structures of PDB codes:
4ZQQ and 7CJU as search models, respectively.
Structure refinement and model building were carriedout using Refmac and Phenix. The molecular
structure and electron density map were visualized byCOOT.
3 Results and Discussion
Trehalose, C12H22O11, is a non-reducing
disaccharide composed of two D-glucose units linked
by an α-1,1-glycosidic linkage (Figure 1). Trehaloseis useful for various applications, including enzyme
catalysis, metabolism, life sciences, and medicine.
The non-reducing glycosidic bonds of trehaloseprevent hydrolysis under weakly acidic and hightemperature conditions.32 Based on its high solubility,
low reactive nature, and antioxidant properties,
trehalose is particularly effective in improving thestability of the protein.
Facade(R)-TEG, C48H84O21, is a sugar-based
facial amphiphile consisting of three β-D-glucoside
units with uncharged polar groups and a steroidbackbone (Figure 1). Three glucoside units areattached to the steroid backbone at the 3α-, 7α- and12α-hydroxy group positions. Facade(R)-TEG
improves protein stability and dispersion. Hence,Facade(R)-TEG is a desirable detergent forsolubilizing membrane proteins.

Fig. 1: Chemical structures of trehalose (left) andFacade(R)-TEG (right).

Single crystals of PAN and chitosanase weregrown in the presence of glycan agents. The crystalsof PAN appeared within 3 days after the setup, andthe crystal sizes reached 0.5 mm at harvest. The rodshaped chitosanase crystals were observed 4–6 days
after setup, and the maximum size was 0.15 mm. The
X-ray crystal structures were obtained for PAN(Figure 2a) and chitosanase (Figure 3a). The
maximum resolutions for the protein crystals were1.60 Å for PAN and 1.50 Å for chitosanase. These
resolutions were 0.15 Å and 0.14 Å better than thoseobtained in our previous studies [1,2].

Fig. 2: (a) Crystal structure of PAN. Protein structures arerepresented in cartoons, where the residues of the Nterminal sides are indicated in blue and the C- terminalsides are shown in red, and manganese ions are shown inpurple spheres. (b) Initial calculation model of PAN without
glycan. Protein molecules are represented by green
cartoons, water molecules are represented by red (oxygen)
and white (hydrogen) thin lines, sodium ions are indicatedby purple dots, and chloride ions are indicated by green
dots. (c) Time evolution plot of RMSD (blue) and the radiusof gyration (red) for the PAN without glycan. (d) Twodimensional projection of PCA. The last 0.1 μs trajectory isextracted from the 1 μs MD simulation, where the color
graduation from red to blue represents the time evolution
for 0.1 μs. (e) B-factors of main-chain atoms of theindividual amino acid residues for the last 0.1 μs simulation
trajectory (blue) and B-factors of the crystal structure (red).
The residues with prominent peaks in B-factor are labeledin blue and red, respectively. (f) Superposition of the crystal
structures for PAN obtained with (cyan) and without (green)
trehalose, viewed in the same orientation as (a).

Photon Factory Activity Report 2022 #40 (2023)

X-ray structure analysis revealed that themolecular packing in the crystals grown with glycan
was almost the same as those of the crystals without
glycan. Hence, the crystallographic symmetry was
equal between the crystals with and without glycan.
The number of protein molecules in the asymmetricunit was one for the trehalose-facilitating PAN crystal
and two for the Facade(R)-TEG-facilitating
chitosanase crystal. The space group was P41212 forthe trehalose-facilitating PAN crystal and P212121 forthe Facade(R)-TEG-facilitating chitosanase one.
These space groups were the same as the crystals
grown without glycan. According to our previous work[3], the precipitant utilized for crystallization
significantly impacts on the space group of crystals.
Since the inclusion of glycan hardly changed thespace groups of crystals, the effect of additives on thecrystal structure is relatively less than that caused byprecipitant agents. A superposition of the crystal
structures with and without glycan demonstrated fineoverlaps. The root-mean-square deviation (RMSD)
was 0.128 Å for PAN and 0.123 Å for chitosanase(Figures 2f and 3f).

Fig. 3: (a) Crystal structure of chitosanase. Protein
structures are represented in cartoons. (b) Initial calculation
model of chitosanase without glycan. (c) Time evolution plotof RMSD and radius of gyration for the chitosanase without
glycan for the whole simulation time. (d) Two-dimensional
projection of PCA. The last 0.1 μs trajectory is extracted
from the 1 μs MD simulation, where the color graduationfrom red to blue represents the time evolution for 0.1 μs. (e)B-factors of main-chain atoms of the individual amino acidresidues for the chitosanase without glycan and B-factorsof the crystal structure. (f) Superposition of the crystal
structure of chitosanase crystallized by AS with and without
Facade(R)-TEG, viewed in the same orientation as (a). The
color and representation in the drawing are the same as
those in Figure 2.

A reasonable initial model is a prerequisite for
accurate computer simulation. In this study, the initialprotein structures were obtained from the respective
crystallographic experiments above. One trehalosemolecule was included in the PAN calculation model,and one Facade(R)-TEG was in the chitosanase one.
Eight independent 1 μs MD simulations wereperformed to enhance the reliability of simulation
analysis. Hence, the glycan molecule was randomly
placed at eight different positions in the solvent
around the protein. The total simulation time of 8 μs
(8 × 1 μs) will ensure adequate sampling of binding
poses. For comparison, one 1 μs MD simulation wasalso performed for the model without including glycan(Figures 2b and 3b). Therefore, 18 independent
trajectories were analyzed to monitor the glycanprotein interaction.
The electrostatic potentials were depicted for PAN
and chitosanase (Figure 4). The iso-surface of theelectrostatic potentials around PAN showed that theseparation of the positive and negative areas isdistinct. Their contact area was restricted to thevicinity of protein (Figure 4a). The negative areas had
a spherical lobe, whereas the positive area slightly
deviated from a spherical shape. Protein molecules
are positioned at the boundary area between two
spherical lobes. The distributions of glycan moleculesin the PAN-trehalose complex showed that trehalosemolecules made contact with the protein surface atthe boundary region of two areas of the electrostatic
potential in five models. In contrast, trehalose waslocated in the positive area in the three models. No
glycan molecule was observed in the negative areaof the electrostatic potential. The electrostatic
potential for chitosanase in Figure 4b showed that thepositive and negative areas are almost equivalently
separated in volume. Each area has a spherical lobe
shape expanding in the opposite direction. The
positive and negative areas clearly split even at theboundary, and the contact region of the two areas islimited in the vicinity of protein. Facade(R)-TEG
molecules are likely to be located at the boundary
region of the two spherical lobes. With three
exceptions, glycan molecules are positioned at theboundary regions for chitosanase, where theabsolute value of the electrostatic potential energy islow. No Facade(R)-TEG is positioned at the positive
area of the electrostatic potential [4].
The function of ammonium sulfate in protein
crystallization is considered to vary salt
concentration-dependent solvation with changing the
local solute-solvent interactions. Sulfate ion is astrongly hydrated anion, and it competes for watermolecule with the solute. The ions tended to gather at
the positive and negative areas of the high absolutevalues of the electrostatic potential.12 A strong
anisotropy induced by AS ions is effective for making
solute molecules aligned and perturbs their exposurePhoton Factory Activity Report 2022 #40 (2023)

to the solvent. Then, solute molecules are contacted
each other in the areas where the absolute value ofthe electrostatic potential is low. Glycan molecules
were likely to be positioned at the boundary region of
the two spherical lobes with a low absolute value ofelectrostatic potential. Glycan molecules will alter theintramolecular interactions between proteins topromote the formation of a crystal lattice. The loose
binding of trehalose to PAN is advantageous forcrystal growth because the release of attachedmolecules will induce the ordered association ofmacromolecules. Although Facade(R)-TEG is stablybound to chitosanase in MD simulations, the glycan
molecule is not observed in the crystallography.
Therefore, Facade(R)-TEG is also released from theprotein surface in crystal growth, which will assist theregular packing of macromolecules. Furthermore, thelattice order depends on precipitant concentration, in
which high concentrations of AS may improve thestabilization of charged residues. The compatibility
between the shape of the electrostatic potential andthe distribution of glycan molecules suggested theirsubstantial influence on the crystal packing.

obtained in the previous studies. MD simulations
were performed for the glycan-included models toexamine the behavior of glycans on a protein surface.
The shape of the electrostatic potential for bothproteins revealed that many glycan molecules werepositioned at the boundary region of the positive andnegative regions, where the absolute value ofelectrostatic potential is low. The findings of this work
will help the understanding of the role of glycan inprotein crystallization and provide a hint for theoptimization of additive agents.
Acknowledgement
Calculations were performed at the ResearchCenter for Computational Science, Okazaki, Japanand at the Information Technology Center of theUniversity of Tokyo. A part of this work was supported
by a grant for Scientific Research C from the Japan
Society for the Promotion of Science.
References[1] Fudo, S., Qi, F., Nukaga, M., Hoshino, T. Cryst.
Growth Des. 17, 534-542 (2017).
[2] Guo, Y., Qu, L., Nishida, N., Hoshino, T.
Electrostatic Potentials around the Proteins
Preferably Crystallized by Ammonium Sulfate.
Cryst. Growth Des. 21, 297– 305 (2021).
[3] Kitahara, M., Fudo, S., Yoneda, T., Nukaga, M.,
Hoshino, T. Cryst. Growth Des. 19, 11, 6004-6010(2019).
[4] Guo, Y., Hoshino, T. Cryst. Growth Des., 22, 6751–6765 (2022).
* hoshino@chiba-u.jp

Fig. 4. (a) Positive and negative iso-surfaces of electrostatic
potentials for PAN with trehalose molecules. Protein isrepresented by a white surface, and eight trehalosemolecules are in sticks (yellow, magenta, green, white, blue,
dark blue, pink, and red). The positive and negative isosurfaces are drawn in blue and red meshes, with theircontour values at +0.5 and −0.5 in units of kT/e,respectively. (b) Electrostatic potentials for the chitosanasewith Facade(R)-TEG molecules. Chitosanase isrepresented by a surface of white, and eight molecules ofFacade(R)-TEG are in stick (magenta, pink, green, blue,dark-blue, red, yellow, and white). The drawing forelectrostatic iso-surface is the same as in (a).

In conclusion, X-ray structure analysis was carriedout for PAN and chitosanase, in which crystals weregrown by the AS solution containing glycosideglycans. The maximum resolutions were 1.60 Å for
PAN and 1.50 Å for chitosanase. These resolutions
were better than those of the crystal structures