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Eect of the SH3-SH2 domain linker sequence on the
structure of Hck kinase
Heike Meiselbach, Heinrich Sticht
To cite this version:
Heike Meiselbach, Heinrich Sticht. Eect of the SH3-SH2 domain linker sequence on the structure of
Hck kinase. Journal of Molecular Modeling, Springer Verlag (Germany), 2010, 17 (8), pp.1927-1934.
�10.1007/s00894-010-0897-z�. �hal-00645110�
1
Effect of the SH3-SH2 domain linker sequence on the structure of
Hck kinase
Received: 30.06.2010 / Accepted: 23.10.2010
Heike Meiselbach and Heinrich Sticht
Bioinformatik, Institut für Biochemie, Friedrich-Alexander-Universität, Erlangen-Nürnberg,
Fahrstraße 17, D-91054 Erlangen, Germany
Tel.: +49 - 9131 / 85 24614; Fax: +49 - 9131 / 85 22485; E-Mail: h.sticht@biochem.uni-
erlangen.de
Abstract
The coordination of activity in biological systems requires the existence of different signal
transduction pathways that interact with one another and must be precisely regulated. The
Src-family tyrosine kinases, which are found in many signaling pathways, differ in their
physiological function despite their high overall structural similarity. In this context, the
differences in the SH3-SH2 domain linkers might play a role for differential regulation, but
the structural consequences of linker sequence are yet poorly understood. We have therefore
performed comparative molecular dynamics simulations of wildtype Hck and of a mutant
Hck, in which the SH3-SH2 domain linker is replaced by the sequence from the homologous
kinase Lck. The simulations reveal that linker replacement does not only affect the orientation
of the SH3 domain itself, but also leads to an alternative conformation of the activation
segment in the Hck kinase domain. The sequence of the SH3-SH2 domain linker thus exerts a
remote effect on the active site geometry and might therefore play a role in modulating the
structure of the inactive kinase or for the fine-tuning of the activation process itself.
Keywords Src-family kinases SH3 SH2 Molecular dynamics simulations Domain
motions
2
Introduction
The Src family of non-receptor tyrosine kinases to date comprises nine members (Src, Fyn,
Lck, Hck, Yes, Fgr, Yrk, Blk, and Lyn) which play critical roles in eukaryotic signal
pathways that control a diverse array of processes such as cell growth, differentiation,
activation and transformation [1-3]. These proteins have a common molecular architecture
that includes five distinct regions [4, 5]: a unique N-terminal region with sequences for lipid
attachment, the regulatory SH3 and SH2 domains and a kinase domain, followed by a
negative regulatory C-terminal tail (Fig. 1b). The SH2 and SH3 domains bind specific
phosphotyrosine and proline-rich motifs, respectively. The catalytic domain consists of two
lobes, which form a cleft that serves as a docking site for ATP and other substrates
representing the active site of the kinase.
The crystal structures of the inactive („closed‟) forms of c-Src [6-8] and Hck [9, 10] reveal
that intramolecular interactions involving the SH2 and SH3 domains are essential for
suppression of kinase activity despite being far away from the active site. In fact, these
regulatory domains are bound to the distal side of the catalytic domain, restricting its
conformational flexibility to hinder productive ATP binding. The SH3 domain is docked to
the N-terminal lobe of the kinase domain via a proline-containing motif in the SH2-kinase
linker (Fig. 1b).
Activation of Src-family kinases (SFKs) requires the disruption of the intramolecular
interactions, the dephosphorylation of the C-terminal tail by phosphatases, or the binding of
high-affinity ligands to the SH2 or the SH3 domain [9-12]. Additionally, the active
conformation becomes stabilized upon autophosphorylation of Tyr416 (in Src) in the
activation segment and is required for maximal kinase activity [13, 14]. In this case, the
phosphorylated activation segment, which contains the highly conserved aspartate-
phenylalanine-glycine (DFG) motif, becomes less ordered and moves out of the active site,
thereby allowing substrate access [15].
An additional feature, which turned out to be important for the activation process, is the
sequence of the linker that connects the SH3 and SH2 domain. In wildtype Src and Hck, a
short rigid linker joins the regulatory SH3 and SH2 domains and leads to dynamic coupling of
the two domains in the closed conformation, thereby forming a stiff clamp [16, 17].
Computational studies and site-directed mutagenesis experiments have shown that the
3
disruption of the structural properties of the rigid linker by introduction of glycine mutations
reduces the strength of the clamp and facilitates activation of the kinase [17].
In Hck and most other SFKs the connecting linker stabilizes the SH3-SH2 domain orientation
by a salt-bridge between a conserved glutamate of the linker (E147 in Hck) and a lysine
(K104 in Hck) of the SH3 domain. In the homologous kinase Lck, E147 is replaced by proline
and therefore the salt-bridge cannot be formed. Accordingly, the isolated Lck SH3-SH2
domain pair shows an ill-defined domain orientation in NMR-spectroscopic studies [18, 19].
The effect of the Lck linker sequence on the intact kinase, however, is yet poorly understood
due to the lack of an intact Lck crystal structure containing both regulatory and kinase
domains.
To assess whether the differences in linker sequence also affect the structure of inactive
SFKs, we have investigated the effect of linker replacement using Hck as a model system.
Comparative molecular dynamics simulations were performed for wildtype Hck and for a
mutant Hck (Hck*), in which the SH3-SH2 domain linker was replaced by the respective
sequence from Lck. To address the effect of the inhibitor PP1 on kinase dynamics,
simulations were performed in the presence and absence of PP1 totalling up to four systems
studied. Our results show that an altered linker sequence does not only result in local changes,
but also has a remote effect on the geometry of the active site.
Materials and methods
Preparation of starting structures
The crystal structures of inactive Hck complexed with the nucleoside analog PP1 (Protein
Data Bank ID 1QCF, [10]), was used to generate the starting structures. For consistency, the
sequence numbering from entry 1QCF was also adapted in the present work. To obtain the
model of mutant Hck (termed Hck*), the wildtype SH3-SH2 linker sequence (
140
V-D-S-L-E-
T-E-E
147
) was replaced with the linker sequence form Lck (A-N-S-L-E-P-E-P) (Fig. 1a) using
the Sybyl 7 [20]. Inspection of the model revealed no steric clashed indicating that the Lck-
linker sequence can be readily accommodated in the Hck scaffold. The systems Hck and Hck*
were simulated each with and without inhibitor PP1.
4
The force field parameters for PP1, which are not available in the AMBER package, were
obtained as follows: The initial coordinates of PP1 were extracted from the 1QCF pdb file.
ArgusLab [21] was utilized to add missing hydrogen atoms. Then, the structure was subjected
to two consecutive geometry optimizations with Gaussian03 [22] using the ab initio methods
HF/MIDI!, and HF/6-31G(d). For all quantum mechanical geometry optimizations the
stationary points found were ensured to be true minima by the calculation of the vibrational
frequencies. The atomic charges for PP1 were then obtained following the established
procedure [23, 24] by fitting the charges to the HF/6-31G(d) computed electrostatic potential
using the Antechamber tool from the AMBER program suite.
Molecular dynamics (MD) simulations
All MD simulations were performed using the PMEMD module of AMBER 8.0 [25, 26] with
the parm99 force field [27, 28]. For the organic compound PP1 the general AMBER force
field (gaff) [29] was used. The parameters for the phosphotyrosine present in the C-terminal
kinase tail were assigned according to Homeyer et al. [30].
An appropriate number of Na+ counter ions was added to neutralize the system and
afterwards the molecules were solvated in a box of water using the TIP3P water model [31]
with at least 10 Å of water around every atom of the solute. All structures were minimized in
a three-step procedure by using the SANDER module of AMBER following a previously
established protocol [32].
After energy minimization, the system was equilibrated for 0.1 ns, raising the temperature
gradually to 298 K by coupling to a temperature bath with a time constant of 0.2 ps, as
described by Berendsen [33]. Subsequently, 30-ns MD simulations with standard NPT
conditions were performed for data collection. Classical equations of motion were propagated
numerically with a time step of 1 fs. A cutoff distance of 10 Å was used for the non-bonded
interactions, and the particle mesh Ewald method [34] was employed to calculate the long-
range electrostatic interactions. All bonds involving hydrogen atoms were constrained using
the SHAKE procedure [35].
The convergence of temperature, pressure, and energy of the systems, as well as the atomic
root mean square deviations of the structure, were used to verify the stability of the systems.
