Photon Factory Activity Report 2014 #32 (2015) B

BL-1A,5A,17A/2009G136,2011G585

Structural insights into how Yrb2p accelerates the assembly of the Xpo1p nuclear
export complex
Masako Koyama1, Natsuki Shirai1 and Yoshiyuki Matsuura*1,2
1
Division of Biological Science and 2Structural Biology Research Center, Graduate School ofScience, Nagoya Univeristy, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan
1 Introduction
Rapid exchange of macromolecules between thecytoplasm and the nucleus is a crucial cellular functionthat regulates many physiological processes in eukaryotes.
Nuclear transport occurs through nuclear pore complexes
and is mediated in most cases by soluble transport
receptors that belong to the karyopherin-β superfamily. In
Saccyaromyces cerevisiae, Xpo1p (CRM1 in vertebrates)is the most versatile nuclear export receptor that carries abroad range of cargo proteins and ribonucleoproteinsfrom the nucleus to the cytoplasm. The majority ofcargoes of Xpo1p contain the so-called leucine-richnuclear export signal (NES) that typically harbor four orfive characteristically spaced hydrophobic residues.
Yrb2p (RanBP3 in vertebrates) is a primarily nuclear
Gsp1p (yeast Ran)-binding protein that has a multidomain structure based on an N-terminal domain
containing a nuclear localization signal, a central domain
containing FG-repeats that bind Xpo1p specifically, and a
C-terminal Gsp1p-binding domain (RanBD) that binds
Gsp1p-GTP weakly. Although it has been known for
more than a decade that efficient export of NES-cargoes
requires Yrb2p (RanBP3), the mechanism of action ofYrb2p (RanBP3) remains poorly understood. To dissect
the mechanism of action of Yrb2p (RanBP3), weperformed
detailed
structuralandfunctional
characterization of the interactions between Xpo1p,Yrb2p, Gsp1p, NES, and FG-nucleoporins in this study[1].

bound to PKI (a representative NES-cargo) and Gsp1pGTP at 2.15 Å resolution (PDB code, 3WYG).
The NES-binding cleft, formed between the outerhelices of HEAT repeats 11 and 12 of Xpo1p, is closed in
the crystal structure of Xpo1p-Yrb2p-Gsp1p-GTP
complex. However, biochemical data suggested that
preloading of Gsp1p-GTP onto Xpo1p by Yrb2p,conformational flexibility of Xpo1p, and the low affinityof RanBD enable active displacement of Yrb2p RanBD
by NES to occur effectively.
The structure of Xpo1p-Yrb2p-Gsp1p-GTP complex
also revealed the major FG-repeat binding sites on Xpo1p.

2 Experiment
Crystals of Xpo1p-Yrb2p-Gsp1p-GTP complex andXpo1p-PKI-Gsp1p-GTP complex were grown by hanging
drop vapor diffusion method. Preliminary X-ray
diffraction experiments were carried out at Photon
Factory and the datasets used for final structuredetermination were collected at SPring-8.
3 Results and Discussion
FRET-based kinetic measurements showed that Yrb2p
remarkably accelerates association of Gsp1p-GTP andNES to Xpo1p. To understand the structural basis for how
Yrb2p acclerates the assembly of the Xpo1p nuclear
export complex, we determied the structure of a key
assemblyintermediate(Xpo1p-Yrb2p-Gsp1p-GTPcomplex) by molecular replacement at 2.22 Å resolution(Fig. 1; PDB code, 3WYF). To enable a complete
comparison to be made between different conformationsof Xpo1p, we also determined the structure of Xpo1p

Fig. 1: Crystal structure of Xpo1p-Yrb2p-Gsp1p-GTP
complex (PDB code, 3WYF).
References[1] M. Koyama, N. Shirai, & Y. Matsuura, Cell Reports 9,
983-995 (2014).
* matsuura.yoshiyuki@d.mbox.nagoya-u.ac.jp