Photon Factory Activity Report 2017 #35 (2018)

BL-17A/2015G517

Crystallographic analysis of extracellular fragments of mammalian plexin-A1
1Tsubasa TANAKA1, Makiko NEYAZAKI1 and Terukazu NOGI1,*Graduate School of Medical Life Science, Yokohama City University,
1-7-26 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Japan

1 Introduction
Semaphorin has been identified as an axon guidancecues to transduce a repulsive signal via the interactions
with a cell surface receptor plexin on neurons.
Furthermore, it has been found that the semaphorinplexin signaling is implicated in a wide variety of
biological phenomena such as immune responses, bonehomeostasis, and angiogenesis. It is also known that
semaphorins are relevant to cancer and autoimmune
diseases. In vertebrates, the semaphorin family contains
21 members belonging to 5 distinct classes (3 to 7) while
the plexin family is constitued by nine members, which
are classified into four types (A, B, C and D). All
semaphorins and plexins commonly possess a semadomain, which assumes a seven-bladed β-propeller fold,in the N-terminus of the extracellular region. It isestablished that semaphorins and plexins bind with eachother via their sema domains. Furtheremore, each
semaphorin can bind to specific members of the plexin
family during the signal transduction. For instance,
Sema3A, which is a founding member of the semaphorin
family, can signal through all of the four type-A plexins(A1, A2, A3 and A4) although neuropilin is required as aco-receptor for stable complex formation. In contrast,class 6 semaphorins bind to specific partners belonging tothe type-A plexins without assistance from neuropilins.
Sema6A shows higher affinities for plexin-A2 and A4
while sema6D binds more stably with plexin-A1 thanother type-A plexins. In this study, we have worked on
crystallographic analysis of an extracellular domain ofplexin-A1 to elucidate the structural basis for the binding
specificity.
2 Experiment
The extracellular region of plexin-A1 contains many
post-translational modifications such as disulfide bonds
and glycosylations. We therefore utilized mammalian
expression system to overproduce a recombinant proteinthat was properly folded and glycosylated for structural
analysis. Specifically, we used a mutant cell line that has
deficiency in glycosylation processing pathway, termed as
HEK293S GnT1(-), so as to reduce heterogeneity of Nglycosylations. On the other side, the plexin-A1 fragment
was expressed as a fusion protein with a polyhistidine tag
under the regulation of the CMV promoter.
To obtain a sufficient amount of protein sample forcrystallization, we established a cell line that stably
expresses the plexin-A1 fragment and culture it using ahigh-density cell culture system. As the plexin-A1
fragment was secreted from the cell, we collected culture

supernatant and purified the fragment using the affinity
chromatography and gel filtration. On average, several
hundred micrograms of the plexin-A1 fragment werepurified from one liter of culture supernatant.
The initial crystallization condition was searched using
a commercial screening kit where the solutions weredispensed by a crystallization robot. Obtained crystals
were soaked in cryoprotectant and then flash-frozen byliquid nitrogen. The cryoprotectant was prepared bymixing the crystallization buffer and ethylene glycol at avolume ratio of 4 : 1. X-ray diffraction experiments wereperformed at the beamline PF BL-17A. The diffraction
data were processed using HKL2000. Structure
determination was performed using the CCP4 program
suite.
3 Results and Discussion
We obtained bipyramidal-shaped crystals of the plexinA1 fragment (Fig. 1). According to the diffraction data,
the crystal was estimated to belong to the tetragonal spacegroup I422. We attempted to determine initial phases bymolecular replacement method using the mouse plexinA2 fragment, which was determined by our previous
structural analysis. [1] Surprisingly, the results indicatedthat the solvent content of the crystal was extremely highand estimated over 80%. The high solvent content might
be due to the presence of N-glycosylation, which should
lower the chance of rigid molecular contacts.

Fig. 1: Crystals of plexin-A1 fragment.
Acknowledgement
We are grateful to the staff of beamline BL-17A at
Photon Factory for providing data collection facilities andtheir kind support.
References[1] T. Nogi et al., Nature 467, 1123 (2010).
* nogi@yokohama-cu.ac.jp