Photon Factory Activity Report 2014 #32 (2015) B

BL-1A, 17A/2014G517, 2012G584

Structure of the stomatin operon partner protein
Hideshi Yokoyama1,* and Ikuo Matsui2
1
School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan,
2
Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology(AIST), Tsukuba 305-8566, Japan
1 Introduction
Stomatin, prohibitin, flotillin, and HflK/C (SPFH)
domain proteins are found in the lipid raft microdomainsof various cellular membranes. Stomatin is one of themajor integral membrane proteins of human erythrocytes.
In a form of human hemolytic anemia known ashereditary stomatocytosis, the stomatin protein isdeficient in the erythrocyte membrane due to mistrafficking. Stomatin and STOPP (stomatin operonpartner protein) genes form an operon in more than 350
archaeal and bacterial genomes, and their protein
products may be involved in the quality control ofmembrane proteins. Two sets of STOPP/stomatin genepairs, PH1510 (STOPP)/PH1511 (stomatin) and PH0471(STOPP)/PH0470 (stomatin), have been identified in thehyperthermophilic archaeon Pyrococcus horikoshii. TheC-terminal region of STOPP PH1510 (residues 371-441,1510-C) was found to be homologous to the soluble core
domain of another STOPP PH0471 (residues 72-143,471-C). In cross-linking and blue native polyacrylamide
gel electrophoresis (BN-PAGE) experiments on 1510-C,
the 1510-C domain formed 12- to 24-mer oligomers [1].
However, the multimeric structure of STOPP has not yet
been examined. To understand the multimeric properties
and possible molecular function of 1510-C in more detail,we herein described the crystal structure of 1510-C,
which indicated the formation of a multimeric assembly[2].
2 Experiment1510-C was mostly prepared as described previously [1,3]. Crystallization drops were prepared by mixing equalvolumes of the protein and reservoir solutions. The
protein solution contained 5.7 mg/mL 1510-C in a buffer
containing 50 mM Tris-HCl (pH 7.5) and 50 mM NaCl.
The reservoir solution was the Crystal Screen II(Hampton Research) #31 condition that contained 20%(v/v) Jeffamine M-600 and 0.1 M HEPES-NaOH (pH7.5). Crystals were grown at 20 °C by the sitting-drop
vapor diffusion method. Rod-shaped crystals grew to an
approximate size of 0.20 × 0.02 × 0.02 mm.
A crystal was transferred into a solution of 20% (v/v)
glycerol in the reservoir solution, and flash-frozen at 95 K.
X-ray diffraction data were collected, and were thenintegrated and scaled with XDS and SCALA.
The structure was determined by the molecularreplacement method with the program PHENIX. The
solution structure of 471-C was used as an initial model(PDB code, 2EXD), although the sequence identity
between 1510-C and 471-C was relatively low, 33% [1].

The model was subjected to several cycles of refinementwith REFMAC5 in the CCP4 suite, followed by manualmodel fitting with COOT.
3 Results and Discussion
The crystal structure of 1510-C was determined at 2.4
Å resolution [2]. The structure contained one 1510-C
molecule in an asymmetric unit. 1510-C was organized
into a compact five-stranded β-barrel fold with a 1-2-3-54-1
topology,whichisknownasanoligosaccharide/oligonucleotide-binding fold (OB-fold)domain. Based on the superposition between 1510-C and471-C, a root-mean-square deviation (r.m.s.d.) was 1.69
Å for 57 Cα atoms. The five β strands were superposed
well, whereas the N-terminus and loop region between
the two β strands of 1510-C differed from those of 471-C.
The structure contained one 1510-C molecule in an
asymmetric unit. However, according to our previous
BN-PAGE results, 1510-C (monomeric Mw = 9.2 kDa)
showed a broad band of 110-220 kDa, indicating that1510-C forms 12- to 24-mer oligomers. By calculating
the interface areas between 1510-C and its symmetryrelated molecules, five pairs of 1510-C and its symmetryrelated molecules showing large interface areas wereobserved, and all these pairs were related by
crystallographic two-fold axes. The largest interface areaof 1510-C (interface 1) was 698 Å2, which was 16.3% of
the total surface area (4,280 Å2). Perfectly conserved
residues Gly381, Val421, and Val431 were included ininterface 1.
According to crystal packing, 1510-C could assemble
into multimers based on the dimer (interface 1) as a basic
unit. 1510-C also forms a large cylinder-like structurecomposed of 24 subunits, or a large triangular prism-likestructure composed of 12 subunits. The multimericassembly formed by the crystal packing appears toconform to the cross-linking and BN-PAGE results.
Stomatin has also been shown to form high order homooligomeric complexes comprising 9- to 12-mers. 1510-C
may function as a scaffold protein to form a multimericassembly of STOPP and stomatin.
References[1] H. Yokoyama et al., Biochimie 95, 1494 (2013).
[2] H. Yokoyama and I. Matsui, FEBS Open Bio 4, 804(2014).
[3] H. Yokoyama and I. Matsui, J. Biol. Chem. 280, 6588
(2005).
* h-yokoya@u-shizuoka-ken.ac.jp