Structural studies on the physiologically and
pharmaceutically important PREPL enzyme
Alexander Fullwood*1, Vilmos Fülöp*, Dean Roa*
*School of Life Sciences, University of Warwick,
Gibbet Hill Road, Coventry, CV4 7AL, UK
1A.fullwood@warwick.ac.uk
Introduction
Prolyl endopeptidase-like (PREPL) enzyme is a new member of the S9a subfamily of Serine peptidases, which have a
role in the regulation of neuropeptides; chemical signals that allow nerve cells to communicate with one another.
Chromosomal deletion of the PREPL gene has been associated with a syndrome of multiple symptoms including:
•Cystinuria
•Neonatal seizures
•Hypotonia
•Somatic & developmental delay
•Facial dysmorphism
•Lactic acidaemia
Currently both the
neuropeptide regulated by
PREPL and the mechanism
by which the enzyme acts
are unknown.
Cystinuria
Facial
dysmorphism
Aims
Homology with PREP has identified 2 distinct structural domains; an α/β hydrolase
domain containing a catalytic site and a β-propeller domain. We believe the β-propeller
moves via a hinge mechanism, allowing only small neuropeptides into the catalytic site
of the α/β hydrolase domain, where modification can only occur when the 2 domains
are in direct contact with one another.
PREPL
homolog
PREP, with
α/β hydrolase
domain on
top and βpropeller
domain on
the bottom
The Aim of this project was to observe and compare the structural changes of this βpropeller domain when in the presence and absence of ligand inhibitors using protein
x-ray crystallography, to provide insight into PREPL’s mechanism.
(Rea D & Fülöp V)
Materials & Methods
PREPL was prepared in advance of crystallographic studies using the following series of protocols:
Identification of
PREPL gene from
human cDNA library
Amplification of
DNA using
polymerase chain
reaction (PCR)
Construction of
plasmid vector
containing PREPL,
ampicillin resistance
and glutathione-Stransferase (GST)
tag genes
Selection & further
growth of E. coli
colonies which have
grown on ampicillin
laced agar plates
Insertion of plasmid
vector into
Escherichia coli cells
5 ligand inhibitors were selected for
use in our studies; compound 8
(shown right) and compounds
PP267, PP286, PP287 & PP308,
provided courtesy of Probiodrug.
Structure
and
inhibition
mechanism
of
compound 8
(Lone AM et al)
Harvest expressed
protein contents
from E. coli by
sonication
Purification of
PREPL using GST-tag
affinity
chromatography
and subsequent
cleavage of GST-tag,
producing intact
PREPL protein
4 commercially available crystallisation
screen kits; JCSG+, PACT, MORPHEUS and
PROPLEX where used to screen for
crystallising conditions of PREPL.
Results
The pH/anion/cation-testing (PACT) kit screening identified
potential crystallising conditions for the ligand-associated
PREPL consisting of 0.2 M sodium acetate (NaOAc) and 20%
w/v polyethylene glycol (PEG) 3350, which we attempted to
optimise by combinations of the following:
•Alteration of PEG 3350 concentrations between 15-27.5%
•Crystallisation at 4°C and 18°C
•Addition of glycerol between 10-20%
•Addition of additives using 3 24 additive screens
•Protein:reservoir drop ratios
•Crystal Seeding
Compound 8
18°C
0.2 M NaOAc
17.5% PEG 3350
1:2 drop ratio
Compound 8
4°C
0.2 M NaOAc
15% PEG 3350
1:2 drop ratio
Compound PP287
18°C
0.2 M NaOAc
16% PEG 3350
However crystals produced only
had a resolvable resolution of
around 8 Å, too low for any
meaningful structural studies to
be carried out.
•Szeltner Z, Alshafee I, Juhász T, Parvari R, Polgár L. The PREPL A protein, a new member of the prolyl oligopeptidase family, lacking catalytic activity. Cellular and Molecular Life Sciences. 2005 Oct;62(19-20).
•Rea D, Fülöp V. Prolyl Oligopeptidase Structure and Dynamics. CNS & Neurological Disorders-Drug Targets. 2011 May;10(3).
Further
acquirement of
viable PREPL
crystals capable of
greater resolvable
resolutions are
required to acquire
any meaningful
structural data on
this
pharmaceutically &
physiologically
important enzyme.
Acknowledgements
•Probiodrug, for the provision of ligands
towards this project.
•Lone AM, Bachovchin DA, Westwood DB, Speers AE, Spicer TP, Fernandez-Vega V, et al. A Substrate-Free Activity-Based Protein Profiling Screen for the Discovery of Selective PREPL Inhibitors. Journal of the American Chemical Society. 2011 Aug 3;133(30).