Photon Factory Activity Report 2017 #35 (2018)

BL-5A, NW12A/ 2016G559

Crystallization and X-ray diffraction study of membrane-bound, oligomeric, and lipidraft associated protein stomatin
Hideshi Yokoyama1,*1
Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba,278-8510, Japan
1 Introduction
Stomatin is one of the major integral membraneproteins of human erythrocytes. In a form of human
hemolytic anemia which is known as hereditarystomatocytosis and is characterized by stomatocytic red
blood cells with abnormal permeability to Na+ and K+, thestomatin protein is deficient in the erythrocyte membrane
due to mis-trafficking during erythropoiesis. Stomatin isalso widely expressed in various tissues and cell lines,
and localized in detergent-resistant membrane domains,which are also termed lipid rafts. Stomatin is organized
into high order homo-oligomeric complexes of about 300
kDa, comprising of 9- to 12-mers.
Stomatin, prohibitin, flotillin, and HflK/C (SPFH)
domain proteins are found in the lipid raft microdomainsof various cellular membranes. Stomatin-like proteins
have been found in most species of eukaryotes, bacteria,and archaea. These SPFH domain proteins could function
as a membrane-bound oligomeric scaffolding protein inlipid rafts.
We previously determined the first crystal structure of
the core domain of stomatin PH1511 fromhyperthermophilic archaeon Pyrococcus horikoshii [1].
In this structure, the SPFH domain was found to form astable trimer, while three C-terminal α-helical domains
extended from the apexes of the triangle. PH1511 has
two membrane-spanning regions at the N-terminus. The
crystal structures of the mouse stomatin-domain wereshown to assemble into a banana-shaped dimer [2]. As an
overall structure of full-length stomatin containing
membrane-spanning regions and C-terminal hydrophobic
region has not been determined, it remains to beelucidated how stomatin proteins oligomerize forthemselves, and how they are associated with themembranes. In order to understand the function ofoligomeric protein stomatin, we tried to elucidate thestructure of full-length stomatin.
2 Experiment
Stomatin PH1511 was mostly prepared as described
previously [1]. The protein was solubilized in thepresence of 1.5% (w/v) dodecyl-β-D-maltoside (DDM,Anatrace), and purified on a nickel affinity column and ananion-exchange Q Sepharose column in 0.05% (w/v)
DDM and other buffer solution. The resultant protein
contains whole residues 1-266 of PH1511, andadditionally contains an initial methionine at its Nterminus and LEHHHHHH at its C terminus.
Firstly, crystallization trials were performed at 20 °C
by the hanging-drop vapor diffusion method.

Crystallization drops were prepared by mixing equalvolumes of the protein and reservoir solutions. Theprotein solution was approximately 1 to 5 mg/mLstomatin PH1511 in a buffer containing 50 mM Tris-HCl(pH 7.5), 50 mM NaCl, and 0.05% (w/v) DDM.
Next, lipidic cubic phase (LCP) methods were tried tocrystallize membrane protein stomatin. Monoolein (MO,Nu Chek Prep) and the protein solution were mixed inmass ratio of 3:2 using two syringes connected with acoupler to produce cubic phase. Then, 0.2 µL of theprotein-MO mixture was dispensed and 1 µL of aprecipitant solution were added to it on a glass sandwich
crystallization plate (Hampton Research), and were kept
at 20 °C.
3 Results and Discussion
Using the hanging-drop vapor diffusion method,
hexagonal plate-shaped crystals appeared, but did notappear reproducibly. On the other hand, using the LCP
method, rectangular-shaped crystals were grown to an
approximate size of 0.1 mm on a side using theprecipitant solution containing 0.1 M sodium chloride,
0.1 M sodium citrate (pH 5.6), and 12% (w/v) 2-methyl2,4-pentanediol in several days. According to thereference [3] about how to harvest the crystals in LCP,crystals were fished out from the cubic phase using a cryoloop, and then flash-frozen at 95 K. X-ray diffraction
images were collected to check the crystal quality. In
almost the diffraction images, concentric ring patterns
around 20 Å resolution or no patterns were detected.
Now, we try to improve methods to harvest the crystals
in LCP, and try to prepare and crystallize the stomatin
PH1511 in the buffer containing several types ofdetergents. We will next try to prepare and crystallize thehuman stomatin. Given the structure of stomatin isdetermined, it will provide useful information about how
stomatin oligomerizes, how it is associated withmembranes.
Acknowledgement
We thank the Photon Factory staffs for assistance in Xray diffraction experiments.
References[1] H. Yokoyama et al., J. Mol. Biol. 376, 868 (2008).
[2] J. Brand et al., EMBO J. 31, 3635 (2012).
[3] D. Li et al., J. Vis. Exp. 67, e4001 (2012).
* yokoyama@rs.tus.ac.jp