Research Article: Bacterial Synthesis and Purification of Normal and Mutant Forms of Human FGFR3 Transmembrane Segment

Date Published: , 2011

Publisher: A.I. Gordeyev

Author(s): S.A. Goncharuk, M.V. Goncharuk, M.L. Mayzel, D.M. Lesovoy, V.V. Chupin, 
E.V. Bocharov, A.S. Arseniev, M.P. Kirpichnikov.



The fibroblast growth factor receptor 3 (FGFR3) is a protein belonging to the
family of receptor tyrosine kinases. FGFR3 plays an important role in human
skeletal development. Mutations in this protein, including Gly380Arg or
Ala391Glu substitutions in the transmembrane (TM) region, can cause different
disorders in bone development. The determination of the spatial structure of the
FGFR3 TM domain in a normal protein and in a protein with single Gly380Arg and
Ala391Glu mutations is essential in order to understand the mechanisms that
control dimerization and signal transduction by receptor tyrosine kinases. The
effective system of expression of eukaryotic genes in bacteria and the
purification protocol for the production of milligram amounts of both normal TM
fragments of FGFR3 and those with single pathogenic mutations Gly380Arg and
Ala391Glu, as well as their15N- and
[15N,13C]-isotope-labelled derivatives, were described.
Each peptide was produced inEscherichia coliBL21(DE3)pLysS
cells as a C-terminal extension of thioredoxin A. The purification protocol
involved immobilized metal affinity chromatography and cation- and
anion-exchange chromatography, as well as the fusion protein cleavage with the
light subunit of human enterokinase. The efficiency of the incorporation of
target peptides into DPC/SDS and DPC/DPG micelles was confirmed using NMR
spectroscopy. The described methodology of production of the native FGFR3 TM
domain in norma and with single Gly380Arg and Ala391Glu mutations enables one to
study their spatial structure using high-resolution heteronuclear NMR

Partial Text

The fibroblast growth factor receptor 3 (FGFR3) belongs to the family of receptor
tyrosine kinases (RTKs). This protein consists of an extracellular component with
three immunoglobulin-like domains, a hydrophobic transmembrane (TM) domain, and an
intracellular component with two tyrosine kinase domains. Specific ligands
(fibroblast growth factors) and heparin are bound to the immunoglobulin-like domain
of FGFR3, thus stabilizing the dimer complex consisting of two receptor molecules
and providing signal transduction inside the cell [1, 2]. FGFR3 plays an important
role in the processes of human growth and development (both embryonic/neonatal and
that in an adult organism). Mutations in this protein may result in various
disorders in the development of connective tissues and skeleton [3–5]. FGFR3 has also been known to participate in
tumor formation [5, 6]. In particular, Gly380Arg and Ala391Glu mutations in the TM
region of FGFR3 cause lethal dysplasia [7] and
the Crouzon syndrome with acanthosis nigricans [8], respectively. The Ala391Glu mutation occurs both upon disorders in
skeletal development and upon oncogenesis [6].
The Ala391Glu mutation is considered to stabilize FGFR3 dimerization in the cell
membrane, resulting in uncontrollable signal transduction and the emergence of a
pathology [9, 10]. However, the detailed mechanism of FGFR3 functioning has not been
fully revealed. The approach that has been most frequently used in modern structural
biology assumes the division of the membrane protein under study into components and
studying the individual water-soluble components of the molecule and its TM regions
[11–15]. It is extremely
important to obtain a high-resolution structure of the native TM domain of human
FGFR3 and that of the domain with Gly380Arg or Ala391Glu mutation to understand the
mechanisms that control their dimerization and functioning, because these fragments
act as the linking units between the extracellular and intracellular RTK domains and
directly participate in signal transduction inside the cell.

In this study, we used Escherichia coli strains XL-10-Gold
(Stratagene, United States) and BL21(DE3)pLysS (Stratagene, United States), plasmids
pGEMEX-1 (Promega, United States) and pGEMEX-1/TRX-TMS [16]. Oligonucleotides were synthesized by Evrogen (Russia). DNA
was sequenced at the Inter-institute Center of Shared Use GENOME (Russia). The
reagents purchased from CIL (United States) were used to introduce the isotope
labels 15 N and 13 C. The completely deuterized
dodecylphosphoglycerol (DPG) was produced by enzymatic transphosphatidylation from
completely deuterized dodecylphosphocholine (DPC) and glycerol in the presence of
phospholipase D [17].

System of

The elaborated system of gene expression and purification protocol enables to produce
recombinant transmembrane peptides tmFGFR3, including the isotope labelled
derivatives to milligram amount, which are required for structural and functional
studies. The relatively small size of the peptide complexes in the membrane-like
environment attests to the possibility of obtaining the spatial structure of
tmFGFR3-nat in dimeric state using high-resolution heteronuclear NMR spectroscopy
[12, 24]. The conformation of the tmFGFR3-nat dimer was determined recently,
and the study of the processes accompanying the specific association of tmFGFR3-E
and tmFGFR3-R is now under way. The proposed technology of recombinant peptides
production will help better understand the mechanism underlying the functioning, as
well as signal transduction, with the participation of the FGFR3 receptor, as well
as shed light on the molecular mechanisms of different disorders in human skeletal
development, wich are directly associated with mutations in the FGFR3 TM domain.