Photon Factory Activity Report 2005 #23 Part B (2006)
Biological Science

5A, NW12/2003G126

Crystal structures of mouse autocrine motility factor
complexed with various carbohydrate phosphate inhibitors
Nobutada TANAKA*, Noriko NABA, Katsura SHIRAIWA, Yoshio KUSAKABE, Ken-ichi Aoki,
Masaru TSUNODA, Yasumitsu SAKAMOTO, Yasuyuki KITAGAWA, and Kazuo T. NAKAMURA
Laboratory of Physical Chemistry, School of Pharmaceutical Sciences, Showa University,
1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
Introduction
Autocrine motility factor (AMF) was identified
originally by its ability to stimulate directional motility(chemotaxis) and random motility (chemokinetics) of theAMF-producing tumor cells. The AMF stimulates cell
motility via a receptor-mediated signaling pathway
involving receptor phosphorylation, a pertussis toxinsensitive G-protein activation, inositol phosphateproduction, protein kinase C activation, and enhanced
production of a metabolite of arachidonic acid.
Partial amino acid sequencing of mouse AMF(mAMF) and full-length cDNA cloning of human AMF
have identified that the AMF is genetically identical withextracellular cytokines neuroleukin and maturation factor
and, interestingly, with an intracellular enzyme
phosphoglucose isomerase. The PGI is a key enzyme inglycolysis and gluconeogenesis that catalyses the second
step of glycolysis, the interconversion of glucose 6phosphate (G6P) and fructose 6-phosphate (F6P). Sitedirected mutagenesis studies at the sugar binding sites ofhuman AMF/PGI have resulted in abridging cytokine
activity of mutant AMFs suggesting that the regions forthe enzymatic function overlap those for the cytokine
function [1].
Several crystal structures of mammalian (human,rabbit, and pig) AMF/PGIs are available, but highresolution structure of AMF/PGI complexed withcarbohydrate phosphate inhibitor is not available. In theabsence of the high-resolution structure of AMF/PGI
complexed with an inhibitor, a detailed picture of theAMF-inhibitor interactions including water molecules,
which is essential for lead compound design of more
effective AMF inhibitors, is not clear.
Here we report the crystal structure of theinhibitor-free open form and those of various lengths ofinhibitor bound closed forms of mAMF at high resolution(1.4-1.8 Å). This series enabled us to provide the firstcomprehensive
reportof thestructure-activity
relationships of carbohydrate phosphate inhibitors withrespect to the inhibition of the cytokine activity of AMF.
Experimental
Crystallization and X-ray data collection
The expression and purification of recombinantmAMF were performed as described elsewhere [2]. The
data collection for inhibitor-free mAMF was performedby rotation method at 100 K using an ADSC Q210 CCD
detector with synchrotron radiation (λ = 1.00 Å at beamline NW12 of the Advanced Ring of the Photon Factory).
The space group was P21 and the unit-cell dimensions
were a = 69.97 Å, b = 115.88 Å, c = 73.27 Å, and β =101.76°. The current best diffraction data from an
inhibitor-free mAMF crystal were collected up to 1.8 Å
resolution. Data from the crystals of the inhibitor
complexes were collected by procedures similar to thosedescribed above.
Structure determination
Initial phase determination for inhibitor-freemAMF was done by the molecular replacement techniqueusing the coordinate set of inhibitor-free human AMF(PDB code 1JIQ [1]) as a search model. Crystallographicrefinement was performed using the program REFMAC.
The refined structure of inhibitor inhibitor-free mAMF
was used for the structure determination of the inhibitor
complexes by the difference Fourier method.
Results and Discussion
We have determined the high-resolution crystal
structures of the inhibitor-free form and the eight types ofinhibitor [phosphate, erythrose 4-phosphate (E4P),
arabinose 5-phosphate (A5P), sorbitol 6-phosphate (S6P),
6-phosphogluconic acid (6PGA), fructose 6-phosphate(F6P), glucose 6-phosphate (G6P), or mannose 6phosphate (M6P)] complexes of mouse AMF. The
inhibitory activities of the six-carbon sugars (G6P, F6P,M6P, and 6PGA) were found to be significantly higher
than those of the four or five-carbon sugars (E4P or A5P).
A structural comparison revealed that a water-mediatedhydrogen bond between one end of the inhibitor and arigid portion of the protein surface in the shorter-chaininhibitor (E4P) complex is replaced by a direct hydrogenbond in the longer-chain inhibitor (6PGA) complex. Thus,to obtain a new compound with higher inhibitory
activities against AMF, water molecules at the inhibitorbinding site of AMF should be replaced by a functionalgroup of inhibitors in order to introduce directinteractions with the protein surface [3].
References[1] N. Tanaka et al., J. Mol. Biol. 318, 985 (2002).
[2] N. Naba et al., Acta Cryst. D60, 2084 (2004).
[3] N. Tanaka et al., J. Mol. Biol. 356, 312 (2006).
* ntanaka@pharm.showa-u.ac.jp

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