A Using a yeast two-hybrid screen, residues 144–463 of GRINL1A C-terminal end were shown to bind to DYNLL1.. With that in mind, we fused the 100 C-terminal amino acids of GRINL1A residue
Trang 1chain 1 and the glutamate channel homolog GRINL1A
Marı´a F Garcı´a-Mayoral1, Mo´nica Martı´nez-Moreno2, Juan P Albar3, Ignacio Rodrı´guez-Crespo2 and Marta Bruix1
1 Departamento de Espectroscopı´a y Estructura Molecular, Instituto de Quı´mica-Fı´sica Rocasolano, Consejo Superior de Investigaciones Cientificas (CSIC), Madrid, Spain
2 Departamento de Bioquı´mica y Biologı´a Molecular I, Universidad Complutense, Madrid, Spain
3 Proteomics Facility, Centro Nacional de Biotecnologı´a, Consejo Superior de Investigaciones Cientı´ficas, Madrid, Spain
Keywords
dynein light chain; glutamate channel
homolog; NMR; protein–protein interactions
Correspondence
I Rodrı´guez-Crespo, Departamento de
Bioquı´mica y Biologı´a Molecular I,
Universidad Complutense, 28040 Madrid,
Spain
Fax: +34 913944159
Tel: +34 913944137
E-mail: nacho@bbm1.ucm.es
M Bruix, Departamento de Espectroscopı´a
y Estructura Molecular, Instituto de
Quı´mica-Fı´sica Rocasolano, CSIC, Serrano
119, 28006 Madrid, Spain
Fax: +34 915642431
Tel: +34 917459511
E-mail: mbruix@iqfr.csic.es
(Received 12 January 2010, revised 11
March 2010, accepted 15 March 2010)
doi:10.1111/j.1742-4658.2010.07649.x
Human dynein light chain 1 (DYNLL1) is a dimeric 89-residue protein that
is known to be involved in cargo binding within the dynein multiprotein complex Over 20 protein targets, of both cellular and viral origin, have been shown to interact with DYNLL1, and some of them are transported
in a retrograde manner along microtubules Using DYNLL1 as bait in a yeast two-hybrid screen with a human heart library, we identified GRINL1A (ionotropic glutamate receptor N-methyl-d-aspartate-like 1A),
a homolog of the ionotropic glutamate receptor N-methyl d-aspartate, as a DYNLL1 binding partner Binding of DYNLL1 to GRINL1A was also demonstrated using GST fusion proteins and pepscan membranes Progres-sive deletions allowed us to narrow the DYNLL1 binding region of GRINL1A to the sequence REIGVGCDL Combining these results with NMR data, we have modelled the structure of the GRINL1A–DYNLL1 complex By analogy with known structures of DYNLL1 bound to BCL-2-interacting mediator (BIM) or neuronal nitric oxide synthase (nNOS), the GRINL1A peptide also adopts an extended b-strand conformation that expands the central b-sheet within DYNLL1 Structural comparison with the nNOS–DYNLL1 complex reveals that a glycine residue of GRINL1A occupies the conserved glutamine site within the DYNLL1 binding groove Hence, our data identify a novel membrane-associated DYNLL1 binding partner and suggest that additional DYNLL1-binding partners are present near this glutamate channel homolog
Structured digital abstract
l MINT-7713396 : DYNLL1 (uniprotkb: P63167 ) and GRINL1A (uniprotkb: P0CAP1 ) bind ( MI:0407 )
by nuclear magnetic resonance ( MI:0077 )
l MINT-7713280 , MINT-7713382 : DYNLL1, (uniprotkb: P63167 ) physically interacts ( MI:0915 ) with GRINL1A (uniprotkb: P0CAP1 ) by two hybrid ( MI:0018 )
l MINT-7713416 , MINT-7713439 : GRINL1A (uniprotkb: P0CAP1 ) binds ( MI:0407 ) to DYNLL1 (uniprotkb: P63167 ) by peptide array ( MI:0081 )
l MINT-7713307 : GRINL1A (uniprotkb: P0CAP1 ) binds ( MI:0407 ) to DYNLL1 (uniprotkb: P63167 )
by pull down ( MI:0096 )
Abbreviations
DTT, dithiothreitol; DYNLL, dynein light chain; GKAP, guanylate kinase-associated protein; GRINL1A, ionotropic glutamate receptor
N-methyl- D -aspartate-like 1A; GSH, reduced glutathione; GST, glutathione S-transferase; NMDA, N-methyl D -aspartate; nNOS, neuronal nitric oxide synthase; PAK1, p21-activated kinase 1; PSD, post-synaptic density.
Trang 2Dynein light chain, which has two isoforms termed
DYNLL1 and DYNLL2 in mammals, is a small
dimeric protein that was initially described as a
mem-ber of the myosin V [1,2] and dynein molecular motors
[3–5] Originally, DYNLL1 was thought to bind to
cer-tain protein cargos and transport them in a retrograde
manner along microtubules, bound to the dynein
machinery However, DYNLL1 can also be found in
its soluble form and not associated with the large
dynein motor or microtubules [3,6] In addition,
cer-tain viruses are known to hijack the dynein machinery
during their infective cycles, and several viral proteins
are known to bind to DYNLL1 directly, such as rabies
virus P protein [7], African swine fever p54 protein [8]
or Ebolavirus protein VP35 [9]
Numerous crystal and NMR atomic structures of
dynein, both in the presence and absence of peptide
ligands, are now available [10–13] Sequence inspection
of DYNLL1-associated proteins followed by yeast
two-hybrid assay, pepscan, site-directed mutagenesis
and pull-down assays have revealed that DYNLL1
associates with its target proteins essentially through
the sequence motifs (R⁄ K)STQT and (K ⁄ R)(D ⁄
E)-TGIQVDR [11,14,15] In both cases, the polypeptide
stretches that associate with DYNLL1 adopt an
extended conformation and form an additional
b-strand that extends a pre-formed b-sheet, with the
glutamine residue occupying an invariant position in
the DYNLL1 binding groove
DYNLL1 and DYNLL2 are also known to
associ-ate on the cytosolic face of the plasma membrane,
mostly at the post-synaptic density, where they seem
to be involved in protein clustering in the proximity of
the N-methyl d-aspartate (NMDA)
receptor–post-synaptic density 95 (PSD-95) complex For instance,
DYNLL2, and to a lesser extent DYNLL1, bind to a
PSD-95-associated protein guanylate kinase
domain-associated protein (GKAP) [16] DYNLL1 also binds
to neuronal nitric oxide synthase (nNOS), another
PSD-95-associated protein also present in the
post-syn-aptic density [17,18] Likewise, at inhibitory synapses,
receptors for either glycine or c-amino butyric acid co-localize with the post-synaptic scaffolding protein gephyrin, another DYNLL1-associated protein [19,20] Our proteomic studies have shown that, in rat brain, DYNLL1 can associate with several NMDA receptors, such as the NR3A-2 isoform [19]
In this paper, we describe the screening of a human heart library using human DYNLL1 as bait Several independent clones of the potential ionotropic gluta-mate receptor-like gene N-methyl-d-aspartate-like 1A (GRINL1A) were retrieved corresponding to potential DYNLL1-associated proteins Then, using the pepscan technique, we narrowed down the DYNLL1 binding site, and showed that it is localized close to the cytosolic C-terminus of GRINL1A Further complementary approaches, including GST fusion, yeast two-hybrid assays and NMR titrations, confirmed the interaction and allowed fine mapping of the DYNLL1 binding site within GRINL1A Finally, all the experimental evi-dence was combined to build a structural model of DYNLL1 bound to a GRINL1A peptide
Results
Identification of GRINL1A as a new DYNLL1-interacting protein
Human DYNLL1 was fused in-frame to the binding domain of GAL4 in the prey vector pGBT9, which was then used to transform yeast After mating with
a yeast pre-transformed human heart library, positive clones were selected and analysed by auto-mated DNA sequencing Several overlapping clones were identified that included residues 144–463 of the human GRINL1A gene (Fig 1A, accession number GI:238064959), which encodes a protein of 463 amino acids homologous to ionotropic glutamate receptors [21] In order to identify potential DYNLL1 binding sites within the GRINL1A sequence, the pepscan assay was used Overlapping dodecapeptides covering resi-dues 156–466 of the prey protein were synthesized on
A
B
Fig 1 Identification of GRINL1A as a
DYNLL1-interacting protein and fine
map-ping of the binding site (A) Using a yeast
two-hybrid screen, residues 144–463 of
GRINL1A (C-terminal end) were shown to
bind to DYNLL1 (B) Pepscan analysis of the
putative DYNLL1 binding site within the
GRINL1A sequence.
Trang 3a cellulose membrane, which was subsequently
incu-bated with purified recombinant DYNLL1 The
bind-ing assay of DYNLL1 to GRINL1A revealed three
potential binding regions, which are all close to the
C-terminus of the protein (Fig 1B) The three stretches
comprised spot C4 [residues ASASLRERIRHL(342–
353)], spot C30 [residues TREIGVGCDLLP(420–431)]
and spot D7 [residues VMPSRNYTPYTR(441–452)]
None of the peptides has a consensus
DYNLL1-bind-ing site such as KSTQT or KDTGIQVDR [14,15], and
no glutamine residues were found in these three
positive spots
We considered spot C4 to be a false-positive, and
discarded it on the basis that peptides enriched in
His, Lys and Arg amino acids typically bind to the
antibody against hexahistidine used in the
develop-ment of the pepscan assay With that in mind, we
fused the 100 C-terminal amino acids of GRINL1A
(residues 363–463) to glutathione S-transferase
(GST), and performed an in vitro binding assay to
DYNLL1 (Fig 2A) Purified
GST–GRINL1A(363-463) was bound to a glutathione–agarose resin, and
a solution of purified recombinant DYNLL1 was
passed through the column Elution of GST–
GRINL1A(363–463) from the column using reduced
glutathione (GSH) allowed us to detect DYNLL1
associated with GRINL1A by Coomassie staining
(Fig 2A, lanes B and C) or by using
DYNLL1-specific antibodies (data not shown) This
observa-tion demonstrates that the binding site of DYNLL1
within GRINL1A is located between residues 363
and 463 DYNLL1 did not interact with GST alone
bound to the glutathione–agarose resin (data not
shown) Next, we analysed this interaction in detail
using a yeast two-hybrid approach, with wild-type
DYNLL1 fused to the bait vector pGBT9 and
vari-ous GRINL1A constructs fused to the prey vector
pGAD Positive interactions were confirmed by
means of the X-Gal assay using double
transfor-mants that were able to grow in the absence of Trp,
Leu and His Sequential shortening narrowed the
binding site of DYNLL1 within the GRINL1A
poly-peptide to residues 365–463 (Fig 2B) We produced
several point mutations, including the glutamine
embedded in the consensus DYNLL1-binding motif
PSQT (Q433G), and residues contained within spots
D7 and D8 (R445G, Y447G and the double mutant
Q433G⁄ R445G), and determined that these mutants
still bind to DYNLL1 Finally, we shortened
GRINL1A and tested the segment 423–463 for
DYNLL1 binding As shown in Fig 2B, this construct
did not result in a positive interaction This indicates
that the binding site is located between residues 365
and 423, or the positive spot C30 in the pepscan assay (residues 420–431) is indeed the binding site and resi-dues 421 and 422 (absent in the yeast two-hybrid con-struct) are necessary for binding
To test this hypothesis, we decided to narrow down the binding site using three partially overlapping syn-thetic peptides in solution that were allowed to bind
A
B
C
Fig 2 Binding of DYNLL1 to a GST–GRINL1A construct and fine mapping of the binding site GST–GRINL1A(363-463) was expressed and purified in Escherichia coli, and extensively dialy-sed (A) Purified protein was loaded onto a GSH–agarose resin Lane A, flow-through (unbound protein) Purified recombinant DYNLL1 was allowed to bind to the immobilized GST– GRINL1A(363–463), and, after extensive washing, the proteins were eluted with GSH (lanes B and C) A control of DYNLL1 only was loaded in lane D (B) Yeast two-hybrid screen of several GRINL1A constructs performed with wild-type DYNLL1 Positive binding is represented by a ‘+’ symbol (C) Comparison between GRINL1A and a set of known peptide sequences from diverse DYNLL1-interacting targets.
Trang 4to purified 15N-labelled recombinant DYNLL1 We
tested peptides with the sequences VETREIGVGCD
LLPS(418–432), LLPSQTGRTREIVMP(429–443) and
SRNYTPYTRVLELTM(444–458) Strong binding
was observed for the first peptide only (see below)
Interestingly, a sequence comparison with the nNOS
stretch known to bind to DYNLL1 revealed a clear
sequence homology, with acidic, basic and b-branched
amino acids occupying similar positions (Fig 2C)
Remarkably, in the GRINL1A sequence, a glycine
residue appears at the position of the glutamine
residue that is present in many DYNLL1-binding
proteins
NMR titration of DYNLL1 with GRINL1A peptides
In order to identify specific DYNLL1-interacting
sequences in GRINL1A, we performed NMR
titra-tions with the three overlapping synthetic peptides
spanning the residues 418–458 of GRINL1A We
recorded 15N-HSQC spectra at increasing peptide:
protein ratios using the 15N-labelled protein and unla-belled peptides The 15N-HSQC spectrum of free DY-NLL1 was used for spectral assignment and to monitor changes upon addition of the peptides The spectral assignment confirmed that, under the experi-mental conditions used, DYNLL1 is a symmetric dimer When DYNLL1 was titrated with peptides LLPSQTGRTREIVMP and SRNYTPYTRVLELTM,
no changes in resonance chemical shifts or in the signal intensity were observed, indicating that no inter-action takes place However, titration with peptide VETREIGVGCDLLPS results in large chemical shift changes for numerous resonances, providing evidence for a specific interaction (Fig 3) For simplicity, we refer to the monomers as A and B, respectively The residues participating in the interaction are: (a) I8– E15, (b) W54–H68 and (c) Y75–F86 of monomer A, and (d) N33–I38 and (e) K43–K48 of monomer B These regions are found mainly in the N-terminal loop (a) and the central b-sheet (b, c) of monomer A, and helix a2 (d, e) of monomer B
Fig 3 NMR titration of DYNLL1 with the
GRINL1A VETREIGVGCDLLPS peptide.
Superposition of 15 N-HSQC spectra of
DYNLL1 recorded at various DYNLL1:
GRINL1A peptide VETREIGVGCDLLPS ratios
during titration The spectra correspond to
free DYNLL1 (red), a 1 : 1 ratio of
protein:peptide (cyan) and excess peptide
(protein:peptide ratio of approximately 1 : 8)
(blue) The inset indicates the chosen
spec-tral region, with selected labelled residues
in the slow exchange regime upon complex
formation One resonance per residue is
observed for free DYNLL1 (red) and in the
presence of excess peptide (blue),
while two resonances appear for the
under-saturated complex at the 1 : 1 ratio
(cyan).
Trang 5The exchange regime for the residues mentioned
above is slow in the NMR time scale Two
reso-nances per residue are observed at peptide:protein
ratios of 0.5 and 1, corresponding to the free and
bound forms, respectively Their intensities vary
according to the population fractions of the free and
bound forms of DYNLL1 at the various peptide:
protein ratios A unique set of resonances is
obser-ved at a molar ratio of 2 : 1, when all the peptide
is bound to the protein dimer, confirming the 2 : 1
stoichiometry of the functional complex under these
sample conditions
Model of the DYNLL1–GRINL1A
VETREIGVGCDLLPS peptide complex
Haddock docking calculations produced structures for
the complex that were classified into four clusters
Analysis of the various clusters allowed us to select
the one with the highest Haddock score as the most
representative model of the DYNLL1–GRINL1A
VETREIGVGCDLLPS peptide complex In this
clus-ter, the GRINL1A peptide is aligned anti-parallel to
the interacting b3 strand of one DYNLL1 monomer,
as observed in all other DYNLL1 complexes reported
so far [10,11,15,22] In two of the other clusters, an
alternative peptide orientation was obtained, and the
anti-parallel orientation was observed in the remaining
cluster, but the interacting surface was shifted, which
disrupted the hydrogen bond network and reduced the
buried interface area
Figure 4A shows the 20 lowest-energy conformers
from the selected cluster modelled using the Haddock
docking calculations The rmsd values for the 20
lowest-energy conformers of the best cluster are
0.71 ± 0.11 A˚ for the backbone and 1.11 ± 0.09 A˚
for the heavy atoms The mean total energy of the
ensemble is )8146 kcalÆmol)1, being )314 kcalÆmol)1
the intermolecular contribution The corresponding
values for the van der Waals’ and electrostatic terms
are )887 and )8523 kcalÆmol)1 for the complex, and
)56 and )259 kcalÆmol)1for the intermolecular
contri-butions, respectively The mean protein–peptide buried
surface area is 1512 A˚2
Biochemical and mutational analysis of several
DYNLL1 partners allowed the identification of two
con-sensus sequences (K⁄ R)XTQT and G(I ⁄ V)QV(D ⁄ E),
which contain a conserved glutamine surrounded
by hydrophobic residues, as DYNLL1-interacting
sequences [14,15] This glutamine forms strong
hydro-gen bonds with two highly conserved DYNLL1
resi-dues, Q35 and K36, at the beginning of helix a2
Structural analysis of our best cluster shows
A
B
C
Fig 4 Structural model of the DYNLL1–GRINL1A VETREIG-VGCDLLPS peptide complex (A) Superposition of the 20 lowest-energy conformers from the selected cluster modelled using Had-dock Had-docking calculations Each DYNLL1 monomer is displayed in a different colour (blue and pink) The VETREIGVGCDLLPS peptide of GRINL1A is shown in green (B) Lowest-energy conformer of the family, showing the electrostatic surface of the protein Positively charged residues are shown in blue, negatively charged residues in red, and uncharged residues in white A stick representation is used for the peptide (C) Lowest-energy conformer of the family, using ribbon and stick representations for the protein and the pep-tide, respectively Selected side chains with important roles in the interactions are shown Residues participating in salt-bridge interac-tions are labelled In all cases, only the peptide that occupies the binding cavity in the front view of the complex is shown.
Trang 6that, although this glutamine is not present in the
GRINL1A target, the interaction still occurs in
the canonical mode (Fig 4B) The peptide lies along the
grooves at each side of the DYNLL1 dimer interface,
extending the central b-sheet through a hydrogen bond
network that connects residues H68–F62 in the swapped
b3 strand of DYNLL1 monomer A with residues
R421–C427 of the peptide in an anti-parallel manner,
similar to that described for other complexes (Fig 4C)
Due to symmetry constraints, both peptides bind to
each half-site in a parallel manner, which contributes to
enhanced specificity, and interactions are similar if
monomers A and B are exchanged; consequently
inter-actions concerning only one peptide are discussed
The residue structurally equivalent to the glutamine
present in the consensus sequences, G426, is close to
the side chain of K36 in monomer B; however, the
different nature of glycine does not allow interactions
with the residues capping helix a2 Stabilizing
salt-bridge interactions are found between the carboxylate
group of E422 and the amine groups of K43 and K44
of monomer B, and the side chains of R421 and D12
of monomer A These residues point towards the
inte-rior of the peptide binding groove, facing opposite
sides of the central b-sheet (Fig 4C) As mentioned
previously, the construct starting at residue 423 did
not bind to DYNLL1, and these results corroborate
the importance of residues 421 and 422 for binding
Discussion
Biological significance
Human GRINL1A was initially identified as an
iono-tropic glutamate receptor-like gene that mapped to
chromosome 15q22.1 [21] Subsequent analysis of the
transcription unit revealed that the gene comprises at
least 28 exons, and its organization proved to be much
more complex than previously anticipated [23]
Sequence comparison has revealed that some
tran-scripts of GRINL1A are significantly similar to those
of both the neuromuscular junction protein yotiao and
the N-termini of the NR2 and NR3 NMDA receptor
subunits [23] In addition, GRINL1A is severely
down-regulated in patients with sporadic Alzheimer’s disease
[24] It has been recently reported that GRINL1A and
subunits of the NMDA receptor associate at the
plasma membrane and are able to
co-immunoprecipi-tate [25] Thus, GRINL1A might be enriched in the
post-synaptic density, a specialized electron-dense
structure underneath the post-synaptic plasma
mem-brane of excitatory synapses, co-localizing with the
NMDA receptors and close to proteins such as
PSD-95, nNOS, shank and GKAP The fact that GRINL1A may associate with the NMDA receptor subunits and that its C-terminus can bind DYNLL1 tightly shows that this protein member of the dynein machinery can
be targeted to the post-synaptic density through its association with at least three independent proteins (GKAP, nNOS and GRINL1A), and agrees with the laminar organization revealed by negative staining and immunogold labelling [26]
A non-consensus sequence within GRINL1A is responsible for DYNLL1 binding in the canonical mode
In this study, we have identified GRINL1A as a novel interacting physiological partner of DYNLL1 Using various biochemical and biophysical approaches, we have delimited the protein fragment responsible for this interaction, and found that it spans residues V418– S432 This sequence does not contain the glutamine resi-due conserved in canonical type DYNLL1-interacting sequences So far, structural information is only avail-able for two DYNLL1 complexes with peptides that lack the conserved glutamine, the X-ray structure of p21-activated kinase 1 (PAK1) [22] and the docked model of myosin Va [2], which is involved in actin-mediated intracellular transport However, despite this distinctive feature compared with many DYNLL1 tar-gets, sequence alignment of GRINL1A peptide VET-REIGVGCDLLPS with the interacting portion of the nNOS peptide, which belongs to the GIQVD type of consensus sequence, reveals a high degree of similarity (Fig 2C) Our NMR data provide clear evidence for direct interaction between DYNLL1 and GRINL1A peptide VETREIGVGCDLLPS, and confirm a binding stoichiometry of 2 : 1 (peptide:DYNLL1 dimer) This result, and the fact that only one set of resonances is observed in the spectra for free and fully bound DYNLL1, prove that, under the experimental condi-tions used in this study, DYNLL1 is a symmetric dimer Moreover, we have mapped the interaction surface, which is similar to that reported for other DYNLL1 complexes that do or do not include the conserved glu-tamine The exchange regime of the resonances indicates that the interaction is strong and in the sub-micromolar range Estimated dissociation constants in the low and sub-micromolar range calculated by NMR titration
or isothermal titration calorimetry (ITC) have been reported for a few DYNLL1–target peptide complexes, ranging from 0.6 to 1 lm for Bim and swallow to 3 lm for dynein intermediate chain, 10 lm for nNOS and
100 lm for PAK1 [22,27,28] In addition, a sub-micro-molar dissociation constant of 0.9 lm has also been
Trang 7reported for the dynein–intermediate chain complex
based on changes in the Trp fluorescence [29]
Comparison of the DYNLL1–GRINL1A peptide
complex with other DYNLL1–peptide complexes
The 3D model for the DYNLL1–GRINL1A
VET-REIGVGCDLLPS peptide complex obtained using the
Haddock docking calculations is consistent with the
titration data and in agreement with the DYNLL1
complexes of known structure The peptide occupies
the two identical concave channels on each side of the
dimer surface, and extends the b-sheet dimer interface Residues R421–C427 of the GRINL1A peptide adopt
a b-strand conformation as assumed by residues K229–V235 of nNOS peptide, and the pattern of hydrogen bonds that link the peptide b-strand anti-parallel to the swapped b3 strand of DYNLL1 is that expected from sequence alignments with peptides con-taining the GIQVD consensus motif
Figure 5A shows the superposition of our model and that of the DYNLL1–nNOS complex determined
by NMR spectroscopy The backbone rmsd obtained when the dimer is used for the superposition is 0.88 A˚,
a value that indicates a high conformational similarity, and no important differences in peptide side chain con-formations are detected The PAK1 crystal complex (Fig 5B) is also quite similar (rmsd value 1.41 A˚)
In addition to the main backbone b-sheet hydrogen bonds, the DYNLL1–GRINL1A peptide docked structure shows several intermolecular salt-bridge inter-actions, particularly involving the N-terminus of the peptide These interactions may increase the stability and binding affinity and play important roles in bind-ing specificity Similar electrostatic interactions to those established by E422 of the GRINL1A peptide are maintained by the aspartate residues of nNOS and Bim peptides, and mutation of this residue in the Bim pep-tide significantly reduces the binding affinity [15,30] Additionally, D230 of nNOS has been found to form a hydrogen bond with the hydroxyl group of T67 This interaction is also present in other related complexes Several studies have shown that the aspartate at position i)4 relative to the conserved glutamine is important for binding, and its substitution by other res-idues significantly decreased DYNLL1 binding [22,31] Charge–charge interactions with D12 are similarly maintained by K229 of nNOS and K5 of Bim peptides Hydrophobic interactions play a crucial role in high-affinity binding of DYNLL1 to most of its targets [11] Some important contacts are: I423 with V66, L84 and F73, V425 with F62, F73, Y75 and L84, and C427 with G63, Y75, Y77 and A82 The latter are also pres-ent in the nNOS–DYNLL1 complex for the equivalpres-ent I233 and V235 residues of nNOS Other interactions, although probably less important, are also expected to contribute to the binding affinity, for example those between T420 and the backbone atoms of H68, L429 and Y77 in strand b4, and between P431 and the aliphatic part of Q80 side chain in the loop connecting strands b4 and b5
As mentioned above, conserved Q234 in nNOS and other GIQVD- and KXTQT-containing peptides is replaced by a glycine in GRINL1A Structural analyses
of known DYNLL1complexes have established the
A
B
Fig 5 Comparison of the DYNLL1–GRINL1A VETREIGVGCDLLPS
peptide modelled complex with other DYNLL1 complex structures.
(A) Superposition of the lowest-energy conformers of the DYNLL1–
GRINL1A VETREIGVGCDLLPS peptide modelled complex and the
solution structure of the DYNLL1–nNOS peptide complex (B)
Superposition of the lowest-energy conformers of the DYNLL1–
GRINL1A VETREIGVGCDLLPS peptide modelled complex and the
crystallographic structure of the DYNLL1–PAK1 peptide complex In
both cases, the DYNLL1 dimer from the modelled complex is
shown in pink and the DYNLL1 NMR ⁄ crystal structure is shown in
blue The GRINL1A VETREIGVGCDLLPS peptide is shown in green,
and the nNOS ⁄ PAK1 peptides are shown in purple Selected side
chains with important roles in the interactions are shown.
Trang 8importance of this glutamine, and a role in binding
specificity has been proposed [11] As shown here, the
Q⁄ G substitution does not impair the interaction,
indi-cating that the glutamine residue is not absolutely
required for binding and is most likely involved in
increasing binding affinity G426 of GRINL1A
occu-pies the equivalent position to this glutamine, which is
typically involved in hydrogen bonds with the
nitro-gen of K36 and the carboxylate of E35 that form an
N-terminal cap for helix a2 in monomer B Glycine,
with its minimal side chain, cannot form such
interac-tions in the complex described here However, the short
distance between G426 and G63⁄ K36 (< 5 A˚) enables
van der Waals’ contacts to be established similarly to
those maintained by G424 (G232 in nNOS) with K36
and the Y65 ring The intrinsic flexibility of the glycine
residue may facilitate subtle structural rearrangements
that enhance binding, compensating for interactions
lost through the Qfi G substitution
In summary, this study has identified a peptide
sequence within the GRINL1A protein that adds to
the growing list of DYNLL1 target sequences lacking
the conserved glutamine that is the usual hallmark of
DYNLL1 binding sequences, yet binds to DYNLL1 at
the same binding site and in similar fashion
A hierarchy in the binding affinity of DLC8 targets
has been proposed, with a decreasing order of affinity
depending on the presence of both the conserved
gluta-mine and the aspartate at position i-4, the presence of
the conserved glutamine only, or the presence of the
aspartate only [22] The GRINL1A peptide
VET-REIGVGCDLLPS target lacks both of these residues,
and still binds to DYNLL1 with high affinity,
suggest-ing that bindsuggest-ing specificity is not as strong as
previ-ously thought This is in agreement with the wide
variety of DYNLL1-interacting partners and its
bio-logical role as a multi-functional protein Further
structural work is needed to shed light on the
molecu-lar basis leading to DYNLL1 target recognition
Experimental procedures
Materials
[15N]-labelled (NH4)Cl was purchased from Cambridge
Iso-tope Laboratories Inc (Andover, MA, USA) Glutathione
Sepharose 4 Fast Flow resin was obtained from Amersham
Pharmacia Biotech (GE Healthcare Europe GmbH,
Barce-lona, Spain) l-leucine, l-tryptophan, l-histidine, l-lysine,
uracil, adenine, X-b-Gal
(5-bromo-4-chloro-3-indolyl-b-d-galactopyranoside) and gluthatione were purchased from
Sigma-Aldrich (Barcelona, Spain) 3-aminotriazole was
obtained from FLUKA (Sigma-Aldrich) The
pre-trans-formed MATCHMAKER library, the yeast nitrogen base without amino acids (SD medium) and yeast transforma-tion system 2 were purchased from BD Clontech (Moun-tain View, CA, USA) Pure synthetic peptides with the GRINL1A C-terminus sequences VETREIGVGCDLLPS (418–432), LLPSQTGRTREIVMP(429–443) and SRNYT-PYTRVLELTM(444–458) were purchased from Thermo Scientific
Yeast two-hybrid screen
Saccharomyces cerevisiae strain Y190 (MATa, ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3, 112, gal4D, gal80D, cyhr2, LYS2::GAL1UAS-HIS3TATA-HIS3,MEL1; URA3::GAL1UAS-GAL1TATA-lacz) was used for all yeast two-hybrid assays The pre-transformed MATCHMAKER library is a high-complexity cDNA library cloned into a yeast GAL4 activation domain (AD) vector and pre-trans-formed into Saccharomyces cerevisiae host strain Y187 (MATa, ura3-52, his3-200, ade2-101, trp1-901, leu2-3, 112, gal4D, met), gal80D, URA3::GAL1UAS-GAL1TATA -lacz,-MEL1) All cloning was performed in DH5a Escherichia coli cells, and protein expression was performed in the protease-deficient BL21 (DE3) E coli strain DYNLL1 pro-tein was used as bait in a yeast two-hybrid screen in order
to identify new DYNLL1-interacting proteins We ampli-fied full-length DYNLL1 cDNA by PCR, introducing EcoRI and SalI sites at the 5¢ and 3¢ ends of the cDNA This PCR product was ligated into pGBT9 plasmid (BD Clontech) in-frame with the DNA binding domain of the yeast transcription factor GAL4 This construction was used to transform the yeast strain Y190 (Mata) in order to identify positive interactions with proteins of a human heart cDNA library (MATCHMAKER pre-transformed library) The library comprised > 2· 106 independent clones inserted in the pACT2 plasmid in yeast strain Y187 (Mata) Yeasts were allowed to mate, and positive colonies were selected by plating on Leu)⁄ Trp)⁄ His)⁄ SD plates in the presence of 10 mm 3-aminotriazole (TDO plates) Approximately 150 positive colonies were picked, and the interaction was confirmed by white⁄ blue screening using X-b-Gal as the substrate The DNA of yeasts that dis-played a positive interaction was isolated and amplified by PCR using oligonucleotides that annealed in the pACT2 plasmid The PCR fragments were subsequently sequenced, and the data were analysed using public databases
b-galactosidase assay
In order to confirm the interaction between DYNLL1 and various fragments of GRINL1A, we performed a colony lift filter assay [32] Colonies grown on plates without dine (TDO plates) were re-grown on fresh plates with histi-dine at 30C for 2 days, and then replicas of the plates were taken using with sterile Whatman filters, and yeasts
Trang 9were allowed to grow over the filter in the same conditions.
For the b-galactosidase assay, cells were subjected to a
free-ze⁄ thaw cycle in liquid nitrogen to cause lysis Then lysates
were incubated with the substrate of the b-galactosidase,
X-b-Gal, at 30C, and the appearance of blue spots was
observed after 1–6 h
Recombinant expression of DYNLL1 and NMR
sample preparation
Cloning of DYNLL1 in pET-23, with a 6· His tag at the
C-terminus of the protein, and expression of the
recombi-nant protein were performed as described previously [18]
Three samples of 15N-labelled DYNLL1 were prepared
to final concentrations of approximately 50 lm in 90%
H2O⁄ 10% D2O aqueous solutions in 100 mm potassium
phosphate buffer, 1 mm dithiothreitol (DTT), pH 7.0 DTT
was added to prevent disulfide-mediated protein
aggrega-tion Concentrated solutions of the three GRINL1A
pep-tides were prepared by dissolving the peppep-tides in 100 mm
potassium phosphate buffer solutions containing
approxi-mately 16 mm DTT to prevent cysteines from forming
intermolecular disulfide bridges The final concentrations of
the peptide solutions were 4.6, 6.0 and 2.7 mm for
peptides VETREIGVGCDLLPS, LLPSQTGRTREIVMP
and SRNYTPYTRVLELTM, respectively
Cloning and expression of GST–GRINL1A
We cloned various fragments of GRINL1A into the
pGEX-2T plasmid by digestion of pACT2-GRINL1A with
EcoRI⁄ XhoI, and ligation into pGEX2T digested with the
same restriction endonucleases These constructions were
used to transform BL21 (DE3) E coli competent cells We
purified the recombinant proteins using the
GSH–Sepha-rose resin, according to the manufacturer’s instructions All
recombinant proteins were dialysed against NaCl⁄ Pi to
remove GSH, and these proteins were used directly to test
the interaction with DYNLL1 in vitro
Binding of recombinant DYNLL1 to peptide
libraries synthesized on cellulose membranes
Purification of recombinant DYNLL1 has been described
previously [18] Mapping studies were performed using
over-lapping dodecapeptides corresponding to a fragment of
GRINL1A prepared by automated spot synthesis (Abimed,
Langenfeld, Germany) on an amino-derivatized cellulose
membrane, with their C-termini immobilized via a
poly-ethylene glycol spacer and their N-termini acetylated We
performed the DYNLL1 binding as previously reported
[14,33] The cellulose membranes were coated with 1%
non-fat dried milk in TBS (50 mm Tris, pH 7.0, 137 mm
NaCl, 2.7 mm KCl) for 4 h at room temperature
Incuba-tion with recombinant DYNLL1 (0.13 lm) was performed overnight at room temperature Subsequently, the mem-brane was incubated for 2 h at room temperature with a commercial antibody against the hexahistidine tag present
in the recombinant protein (1 : 100 000 dilution in TBS) Development of the membrane was performed by enhanced chemiluminiscence according to the manufacturer’s instruc-tions The intensity of each spot was quantified using a UVI-tec digital image analyser (UVItec, Cambridge, UK) and the software UVIband V97 In all cases, spots corre-sponding to the dodecapeptides synthesized onto the same membrane were compared with each other Controls with antibody in the absence of recombinant DYNLL1 were performed in order to be able to subtract non-specific binding due to reactivity of the antibody against certain synthetic peptides
NMR experiments
Each 15N-labelled DYNLL1 protein sample was titrated with an unlabelled GRINL1A peptide Titrations were per-formed by recording series of15N-HSQC spectra at 25C in
a Bruker Avance 800 MHz spectrometer (Bruker, Rheinstet-ten, Germany) equipped with a z-gradient cryoprobe for the free protein and for increasing peptide:protein ratios (0, 0.5, 1.0, 2.0, 4.0 and 8.0) The spectral assignment of DYNLL1 amide proton resonances was determined from published data obtained under similar sample conditions [11,12] Chemical shift perturbation analysis was performed using weighted average values for 15N and 1H chemical shifts according to the following equation:
Ddav¼
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðDd1H)2þ ½ðDd15N)2=10
q
:
Haddock modelling
Docking of DYNLL1 with interacting GRINL1A peptide VETREIGVGCDLLPS was performed using the Haddock software web portal (available at http://haddock.chem.uu nl/services/HADDOCK/) using ambiguous interaction restraints derived from the NMR titration experiments The coordinates for DYNLL1 were taken from the PDB struc-ture of DYNLL1 bound to nNOS peptide (PDB ID 1F96) The coordinates for the GRINL1A VETREIGVGCDLLPS peptide were modelled using the alignment mode within Swiss Model (http://swissmodel.expasy.org/) with the PDB structure of the nNOS chain D peptide complexed with DYNLL1 (PDB ID 1F96) The two-first residues of the target GRINL1A VETREIGVGCDLLPS peptide were excluded from this alignment and were not modelled Similar docking calculations were run with the full-length GRINL1A VETREIGVGCDLLPS peptide structure built from an anti-parallel b-sheet conformation using Pymol software tools (pymol.org) In order to simplify and speed
Trang 10up the calculations, and due to the twofold symmetry axis
of the complex, only one peptide was docked in the
DYNLL1 dimer
During the first step of rigid-body energy minimization,
1000 structures were generated, of which 200 were kept for
the second semi-flexible simulated annealing step and final
flexible water refinement Semi-flexible residues were
automatically defined from an analysis of intermolecular
contacts Active and passive residues for the protein
inter-action interface were selected on the basis of the chemical
shift perturbation data and mean residue solvent
accessibil-ity Residue solvent accessibilities were calculated using the
molmolprogram [34], and a 30% cut-off value was chosen
to define the solvent-accessible surface Selected active
resi-dues for the protein were R60, N61, G63, Y65 and T67
from monomer A, and N33, K36, K44 and K48 from
monomer B Passive residues were I8, K9, N10, D12, E15
and Q80 from monomer A All residues in the peptide
T420-S432 were considered active In the case of the
full-length peptide, residues V418-S432 were considered active
Half of the ambiguous interaction restraints were randomly
deleted for each docking trial Final cluster analysis was
performed to evaluate the structure quality of the docked
complexes The patterns of intermolecular interactions for
the full-length peptide and the modelled peptide were very
similar, and no frame shift is induced by addition of two
residues at the N-terminus
Acknowledgements
This work was supported by grants from the
Minis-terio de Ciencia e Innovacio´n BFU2009-10442,
CTQ2008-00080⁄ BQU and CSD2006-00023 We would
like to thank Fernando Roncal (CBM, Madrid) for
synthesis of the pepscan cellulose membranes and
Peter Rapali (Budapest, Hungary) for careful revision
of the manuscript
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