Photon Factory Activity Report 2014 #32 (2015) B

BL-17A/2013G215

Antibody-driven crystallization of sorLA Vps10p domain
1

Yu Kitago1, Junichi Takagi1
Osaka University, Suita, Osaka 565-0871, Japan

1. Introduction
We have recently solved the crystal structureof sorLA Vps10p domain in its ligand-free form and in
two liganded forms with different peptides. SorLA is ahuge single-pass transmembrane protein containing a
multi-domain extracelluar region. It is expressed inneurons, and has been known to be genetically linked
with Alzheimer's disease (AD) [1, 2, 3]. Although its
physiological function has not been entirely clear, we
discovered that sorLA directly binds amyloid-β peptide
through its N-terminal Vps10p domain. Starting fromthis discovery, we conducted a series of investigations
using mouse models and cultured cell system incollaboration with Dr. Thomas E. Willnow's group(Max Delbrück Center for Molecular Medicine, Berlin),
resulting in a proposal that sorLA functions as Aβ
clearance receptor [4].
Following this study, we have tried to unravel
the molecular mechanism of Aβ recognition by sorLAVps10p domain. As a result of biochemical assays andcrystal structure determinations, we concluded thatsorLA Vps10p domain recognizes each ligand peptide
specifically, whereas the amino acid sequences ofligand peptides do not contribute much to the binding
specificity. The cyrstal structure revealed that sorLA
Vps10p accomplishes this moderate binding specificity
by utilizing β-extention mechanism. Finally, wesuggested that sorLA Vps10p may recognize
amyloidogenic peptides while they are monomericinside the cells, and prevent them from becoming
neurotoxic by forming insoluble oligomers [5].
As a next stage of sorLA Vps10p study, we
have tried to crystallize several variants of sorLA
Vps10p domains such as mutants and complexes withcompounds. SorLA Vps10p domain exhibits difficult
crystallization property; it requires very long
crystallization period (several months) with rather low
reproducibility. To improve this poor crystallization
character, we decided to utilize a structure-dependent
antibody as crystallization chaperon.
2. Experiment
Recombinant human sorLA Vps10p domain lacking
propeptide portion with C-terminal His-tag wasproduced using CHO lec 3.2.8.1 [6]. The secreted
protein in the culture medium was purified using NiNTA resin. C-terminal His-tag was cleaved off by TEVprotease, and N-linked glycans were trimmed by
Endoglycosidase H (New England Biolab). Afterchanging the pH value of solution from 8.0 to 4.5,
protein
waspurifiedbycationexchange
chromatography under acidic condition. Finally, the pH
value of solution was reverted to 8.0, and the sample
solution was concentrated by ultrafilteration.

Anti-sorLA Vps10p antibody (Mouse IgG1, κ) was
purified from the hybridoma culture supernatants andthe puriried IgG was digested with papain-agarose(Thermo scientific) to produce Fab fragment. The Fcfragment was removed from the digestion productthough the protein A-Sepharose resin (GE healthcare).
The purified Fab fragment was mixed with sorLA
Vps10p protein, and further purified by size exclusion
chromatography. The complete shift in the elution
position indicated the stable interaction between sorLA
Vps10p and Fab fragment, and the stoicheometriccomplex was isolated. The complex was concentrated
with the ultrafilteration devise to approximately 10.0
mg/mL.
The purified sorLA Vps10p:Fab complex was screened
for its crystallization condition in the presence of 15residue propeptide and its Se-Met derivative (denotedas LPP15 and LPP15Se). The initial condition
including 0.1 M cacodylate buffer pH 6.5, 0.2 M(CH3COO)2Ca, 20 % PEG 8,000 was optimized andfinally we could obtain diffraction quality crystals with
LPP15Se under the condition of 0.1 M MES buffer pH
6.5, 0.2 M (CH3COO)2Ca, 12 % PEG 8,000 forseveral days. Collected images were processed byHKL2000[7] at the resolution of 3.2 Å, and the initial
phases were calculated by molecular replacement withPHSER[8] using our sorLA Vps10p domain structurecomplexed with LPP15 (PDB ID: 3WSY) and antiprogesterone antibody Fab fragment (Mouse IgG1, κPDBID: 1DBA) as search models. Initial rigid-bodyrefinement and subsequent refinment procedure byphenix.refine[8] resulted in the Rwork/Rfree factor of0.208 / 0.260 for two pairs of sorLA Vps10p
complexed with Fab fragment in the asymmetric unit.
3. Result and Discussion
By utilizing specific antibody against sorLA Vps10p
domain, we successfully obtain the crystal of the Fabcomplex which was of appropriate diffraction quality
within several days, in a highly reproducible manner.
This Fab fragment did not interfere with the ligand
binding of sorLA Vps10p, and strongly drove thecrystallization. Using this Fab fragment, it will bepossible to crystallize many kinds of sorLA Vps10p
variants, thereby facilitating our further understandingof the molecular mechanism behind its biologicalfunction.
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[2]
[3]
[4]
[5]

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* kitago@protein.osaka-u.ac.jp[6][7]