From the Structural Biology Program, Lerner Research
Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, the
§ Department of Pharmacology, School of Medicine, Case
Western Reserve University, Cleveland, Ohio 44102, and the
Department of Pathology, University of Pittsburgh,
Pittsburgh, Pennsylvania 15261
Received for publication, August 21, 2000, and in revised form, November 10, 2000
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ABSTRACT |
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PINCH is a recently identified adaptor protein
that comprises an array of five LIM domains. PINCH functions through
LIM-mediated protein-protein interactions that are involved in cell
adhesion, growth, and differentiation. The LIM1 domain of PINCH
interacts with integrin-linked kinase (ILK), thereby mediating focal
adhesions via a specific integrin/ILK signaling pathway. We have solved the NMR structure of the PINCH LIM1 domain and characterized its binding to ILK. LIM1 contains two contiguous zinc fingers of the CCHC
and CCCH types and adopts a global fold similar to that of functionally
distinct LIM domains from cysteine-rich protein and cysteine-rich
intestinal protein families with CCHC and CCCC zinc finger types.
Gel-filtration and NMR experiments demonstrated a 1:1 complex between
PINCH LIM1 and the ankyrin repeat domain of ILK. A chemical shift
mapping experiment identified regions in PINCH LIM1 that are important
for interaction with ILK. Comparison of surface features between PINCH
LIM1 and other functionally different LIM domains indicated that the
LIM motif might have a highly variable mode in recognizing various
target proteins.
Proteins often function through domains or recurring motifs. The
LIM domain is a common protein-protein interaction motif that was
originally discovered in the products of the lin-11, isl-1, and mec-3 genes and hence given the
acronym "LIM" (1, 2). The domain consists of a loosely conserved
cysteine-rich consensus sequence
(CX2CX16-23HX2CX2CX2CX16-21CX2-3(H/D/C)) that encodes two separate zinc fingers (shown in underlined and boldface type, respectively) (3-5). Frequently occurring as an array
of one to five copies, the double zinc finger LIM domains have been
found in a variety of proteins with diverse functions, either alone or
associated with other functional domains (5). Based on sequence
similarity, LIM-containing proteins are classified into three groups
(5): Group 1 includes the LHX (LIM homeodomain protein), LMO, and LIMK
(LIM kinase) subfamilies; Group 2 contains the
CRP1 and CRIP subfamilies;
and Group 3 is heterogeneous, and the sequences in this group are very
divergent from those in Groups 1 and 2. Although genetic and
biochemical studies have shown that LIM domains can interact with
diverse target proteins, the molecular basis of how the domains confer
specificity and/or coordinate with other functional domains remains
elusive. Structural studies geared toward understanding the LIM
functions so far have been performed on avian CRPs, related mammalian
intestinal protein (CRIP), and a fragment of Lasp-1 protein (6-12),
which belong to Group 2 of the LIM subfamilies with CCHC and CCCC zinc
finger types. These studies revealed a conserved fold of LIM domains in
which two zinc ions orchestrate the formation of separate zinc fingers
that stack together through a shared hydrophobic core. Each finger consists of two orthogonally packed antiparallel PINCH (particularly interesting new
Cys-His protein) is a widely expressed adaptor
protein comprising five LIM domains that belong to Group 3 of the LIM
protein subfamilies (5). Originally identified from screening of a
human cDNA library with antibodies recognizing senescent
erythrocytes (13), PINCH has become increasingly interesting because of
its involvement in mediating integrin signaling (14, 15). Specifically,
PINCH interacts with a membrane-proximal integrin-linked kinase (ILK),
which colocalizes with In this study, we have determined the solution structure of the PINCH
LIM1 domain and characterized its binding to ILK. The structure not
only serves as a starting template for molecular elucidation of
PINCH-ILK-mediated integrin signaling, but also sheds light upon the
questions of the fold and binding mode of functionally different LIM
domains. Although the PINCH LIM1 domain has a more divergent sequence
than those of the CRP and CRIP families (Fig.
1), we show here that it adopts a
conserved LIM fold. On the other hand, detailed comparison of surface
features in the PINCH LIM1 structure and those of the CRP and CRIP
families indicates that the binding mode of LIM domains may be highly
variable. Gel-filtration and NMR experiments show that PINCH LIM1 and
the ILK ankyrin repeat domain form a tight 1:1 complex, providing
biophysical evidence of the specific PINCH-ILK interaction that is
critical for mediating focal adhesions and integrin signaling. Further
chemical shift mapping analysis has revealed the regions in PINCH LIM1
that may be important in binding to the ILK ankyrin repeat domain.
Purification and NMR Sample Preparation of PINCH
LIM1--
Expression plasmid pMAL-C2x, encoding maltose-binding
protein fused to the N terminus of residues 1-70 of human PINCH
protein via Factor Xa-cleavable linker, was used for
preparation of the NMR sample. Residues 1-70 contain the entire LIM1
domain. Due to cloning artifacts, the C terminus of LIM1 had three
additional residues (WIL), whereas the N terminus contained four
(ISEF). Escherichia coli BL21(DE3) cells harboring
plasmid were grown in LB medium or in M9 minimal medium in the presence
of 100 µg/ml ampicillin. For isotope labeling, M9 minimal medium
contained 1.1 g/liter [15N]NH4Cl and
unlabeled or 3 g/liter 13C-labeled glucose. Three liters of
cultures were induced at A600 = 0.5 for
4 h at 37 °C with 1 mM
isopropyl- Purification of the ILK Ankyrin Repeat Domain and Its Complex
with the PINCH LIM1 Domain--
Residues 1-189 of mouse ILK (99%
identical to human ILK) were fused to GST in pGEX-5X-3 (Amersham
Pharmacia Biotech), and the construct was expressed by growing 4 liters
of culture in LB medium. The induction of GST-ankyrin was performed at
room temperature with 0.5 mM
isopropyl- NMR Experiments and Resonance Assignments of LIM1--
All NMR
experiments described below for resonance assignments and structural
analysis were carried out as reviewed in Refs. 20 and 21. These
experiments were performed at 25 °C on a Varian Inova 500-MHz
spectrometer equipped with a triple resonance probe and shielded
z-gradient unit. Spectra were processed with nmrPipe (22)
and visualized with PIPP (23). HNCACB and CBCA(CO)NH experiments
provided assignments for the HN, N, C Distance and Angle Restraints--
Distance restraints were
derived from a three-dimensional 15N/13C NOESY
(25) experiment (100-ms mixing time). A two-dimensional NOESY
experiment was performed on the unlabeled sample in 100% 2H2O (150-ms mixing time) to obtain NOEs
involving aromatic side chains. 1105 NOE distance restraints and 8 Structure Calculations--
A simulated annealing protocol (26)
was used that employs quadratic potentials for covalent geometry, flat
bottom quadratic potentials for experimental distance, and dihedral
angle restraints and a quadratic repulsion term for van der Waals
non-bonded interactions. CHARMM19 (27) force field was used for protein
covalent geometry. For zinc, the bond lengths Zn-S and Zn-N were set
to 2.30 and 2.00 Å, respectively (28); tetrahedral coordination was
enforced by weak force constant (40 kcal mol Secondary Structure--
The set of the 25 lowest energy simulated
annealing structures of the PINCH LIM1 domain is displayed in Fig.
2A. Structural statistics and
NMR constraints are summarized in Table
I. The secondary structure elements of
PINCH LIM1 are arranged in the order of Tertiary Structure of PINCH LIM1 and Its Comparison with Other LIM
Structures--
The overall fold of LIM1 (Fig. 2B) is
similar to those of previously solved LIM structures from the CRP and
CRIP families (Fig. 1). Sequence alignment (Fig. 1) shows that zinc
coordination residues are the same as those from CRP and CRIP, except
that zinc finger 2 has a CCCH module instead of a CCCC module.
Thr9-Cys35 form zinc finger 1 with
Cys10, Cys13, His32, and
Cys35 coordinated to zinc (CCHC module), whereas
Cys38-His61 form zinc finger 2 with
Cys38, Cys41, Cys59, and
His61 coordinated to zinc (CCCH module). Each finger of
PINCH LIM1 consists of two antiparallel
Among the LIM domains with known structures, CRP2 LIM2 possesses the
most similar sequence to PINCH LIM1: 33% identity and 49% similarity.
However, sequence alignment based on the BLAST program revealed that
zinc finger 1 of PINCH LIM1 contains a single amino acid insertion at
Asn27 compared with qCRP2 LIM2 and other LIM domains with
known structures (Fig. 1). Hence, we superimposed the minimized average
structure of PINCH LIM1 with that of qCRP2 LIM2 by omitting
Asn27, which yielded a root mean square deviation of 2.7 Å. To confirm that Asn27 is indeed the insertion point, we
superimposed the first 30 structured residues of qCRP2 LIM2 with the
homologous 31-residue stretch of PINCH LIM1 while systematically
omitting one residue of the latter. A distinct root mean square
deviation minimum was found to occur in the region of
Pro19-Ile23 (±0.2 Å), which connects
Hydrophobic residues that form the core of the PINCH LIM1 structure are
well conserved. The aromatic ring of Phe17 is buried and
acts as a nucleus of zinc finger 1, whereas Ile23 and
Leu30 partly pack against it. The two zinc fingers are
packed through a central hydrophobic core involving the completely
buried rings of Tyr31, Phe36, and
Phe50. Other residues such as the Val24,
Leu49, and Lys57 methylene side chains form the
periphery of the core and are partly exposed (Fig.
3A). Interestingly,
Lys57 in PINCH LIM1 is substituted for the hydrophobic
Ile/Val/Pro residue at an equivalent position in other LIM domains with
known structures (6-12). The lack of a hydrophobic residue at this
position appears to result in greater stacking of Tyr31 and
Phe36 rings in PINCH LIM1 (Fig. 3A) than of the
corresponding residues in other LIM domain structures. The total
surface of two rings that needs to be covered is 18 Å2
less compared with qCRP2 LIM1 and 69 Å2 less compared with
qCRP2 LIM2 structures (note that qCRP2 LIM2 contains a bulkier
tryptophan at an equivalent position to Thr31 of PINCH
LIM1; see Fig. 1). On the other hand, Lys57 still plays a
similar role to purely hydrophobic residues: its methylenes pack
against the edge of the Tyr31 ring (Fig. 3A),
whereas its primary amino group points toward the surface (see
below).
Surface Features of PINCH LIM1 and Other LIM Domains--
Analysis
of the PINCH LIM1 structure revealed that only 4 out of 13 aromatic
residues are completely buried in the hydrophobic core. These are
Phe17, Tyr31, Phe36, and
Phe50. Of the rest, the two histidines coordinate to
Zn2+, whereas the remaining two tyrosines and five
phenylalanines are at the surface or at least partly exposed to the
surface. Phe67 is disordered, as is the side chain of
Phe45. Other aromatic rings compose parts of surface
hydrophobic patches. There are three distinct hydrophobic regions. The
methyls of Leu30, Ile23, and Ala8
compose one of them near the N terminus (Fig.
4A), and the second hydrophobic patch comprises the exposed Phe42 ring and
Val37. Val37 lies at the bottom of a depression
between the tips of
The overall surface of PINCH LIM1 is predominantly negative since
negatively charged residues are more prevalent than the positively
charged ones (eight Glu + one Asp residue versus three Lys + two Arg residues). However, it was surprising to find that the charges
are distributed asymmetrically. As Fig. 4B illustrates, negative charges dominate on one face of the molecule. The most prominent positively charged region is located on the opposite side of
the molecule right under the Phe42 bulge. It is created by
Arg12 guanido and Lys57 amino moieties being
close to each other with the inclusion of Glu29, which
apparently forms a salt bridge with Arg12. Hence, the
Phe42 region contains both a hydrophobic prominence and a
mosaic of charged spots (Fig. 4, compare A and
B). It is not clear if this site contributes to the
protein-protein interactions.
Characterization of PINCH LIM1 Binding to the ILK Ankyrin Repeat
Domain--
It has been shown by deletion experiments that PINCH LIM1
interacts with the ankyrin repeat domain of ILK (16). In this report,
we have characterized the binding of PINCH LIM1 to the ILK ankyrin
repeat domain using gel-filtration and NMR experiments. Whereas PINCH
LIM1 (residues 1-70) and ILK ankyrin (residues 1-189) eluted at ~8
and ~20 kDa individually, the equimolar mixture eluted at ~30 kDa
(data not shown), indicating a tight 1:1 complex. Fig. 5 shows the two-dimensional
1H-15N HSQC spectrum of free
15N-labeled PINCH LIM1 in free form and
15N-labeled PINCH LIM1 in complex with the ILK ankyrin
repeat domain (unlabeled). Fig. 6 shows
the plot of the change in 1H and 15N chemical
shifts of LIM1 upon binding to the ILK ankyrin repeat domain as a
function of residue number. Although resonance assignments of the
complex are not available, in certain cases (e.g. glycine residues), the proximity of the peaks of free and complexed forms renders the assignment obvious. In other instances, we used the distance to the closest unassigned neighbor in the spectrum of the
complex (36). Most of the residues in zinc finger 1 appeared to be
unperturbed or only slightly perturbed, whereas many residues in zinc
finger 2 underwent larger chemical shift changes as compared with zinc
finger 1; in particular, residues in the C-terminal helix of zinc
finger 2 experienced substantial changes. Fig.
7 provides the three-dimensional mapping
of the corresponding changes in LIM1, showing that zinc finger 2 is
mainly involved in interaction with the ILK ankyrin repeat domain. This
is consistent with the above surface analysis that the third
hydrophobic patch involving the C-terminal helix is likely the major
protein recognition site. Mutational analysis also supports the notion
that the second zinc finger of LIM1 is involved in interacting with ILK
(17). Some conformational change may occur in LIM1 upon binding to the
ILK ankyrin repeat domain, as indicated by the large chemical shift changes of some residues. More accurate identification of the LIM1-binding site and how the domain undergoes conformational change
upon binding to ILK require the complete structure determination of the
PINCH LIM1·ILK ankyrin complex, which is in progress in our
laboratory.
In summary, we have determined the NMR structure of the focal adhesion
protein PINCH LIM1 and characterized its binding to ILK. Despite the
loose consensus sequence of the LIM motif, PINCH LIM1 adopts a fold
similar to other functionally different LIM domain structures. Analysis
of the surface features revealed a hydrophobic surface at zinc finger 2 involving the C-terminal helix that appears to be critical for
LIM-target protein interactions, as indicated by the chemical shift
mapping of the HSQC spectrum of the PINCH LIM1·ILK ankyrin complex.
The variation in this surface between the PINCH LIM1 structure and
those from the CRP and CRIP families suggests a highly variable mode
for the LIM domains in recognizing various target proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-sheets, and the
C-terminal CCCC module is terminated by a
-helix. The conserved tetrahedral zinc coordination and hydrophobic core appear to determine the overall fold of LIM domains, whereas other variable regions in the
domains may confer the specificities for binding diverse target
proteins. The exact mode of LIM domain binding to various target
proteins remains essentially uncharacterized.
1-integrin in the focal adhesion
plaques (16, 17). PINCH-ILK interaction is essential for the focal
adhesion localization of ILK (17) and integrin signaling as evidenced
by genetic studies in which loss of PINCH function in
Caenorhabditis elegans resulted in a pat
(paralyzed, arrested elongation at
two-fold) phenotype resembling those of integrin-null
mutants (18). It has recently been shown by deletion experiments that
PINCH-ILK interaction is mediated by the LIM1 domain of PINCH and the
ankyrin repeat domain of ILK (16). Interestingly, the PINCH-ILK
interaction also connects ILK to Nck-2, an SH2/SH3-containing adaptor
protein that interacts with components of growth factor and small
GTPase signaling pathways (19). Such a connection is mediated through
the interaction between the LIM4 domain of PINCH and the third SH3
domain of Nck-2. These findings reveal that PINCH mediates multiple
signaling pathways by using different LIM domains. More important, the
multiple LIM-mediated interactions through a single protein (PINCH)
facilitate communications between different signaling pathways,
i.e. integrin/ILK signaling and growth factor signaling.
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Fig. 1.
Alignment of amino acid sequences of LIM
domains with known structures. Alignment was generated with
BLAST Version 2.0 (37). Sequence numbering is that of the PINCH
protein. Highlighted in black are
Zn2+-coordinating residues. The Lasp-1 structure comprises
only the first zinc finger, half of the LIM domain. The
structure with Protein Data Bank (PDB) code 1cxx is the
R122A mutant of qCRP2 LIM2. cCRP1, chicken
CRP1.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-D-thiogalactopyranoside. Cells were lysed
with a French press, and cleared lysates were fractionated on a
DEAE-Sepharose column (50 mM Tris-HCl, pH 8.0; NaCl
gradient of 0.0-0.8 M). Maltose-binding
protein-LIM1-containing fractions were concentrated, and the buffer was
exchanged to optimize for cleavage (50 mM Tris-HCl, 100 mM NaCl, and 3.5 mM CaCl2, pH 8.0)
and subjected to Factor Xa treatment (Novagen). LIM1 was further purified on a Superdex 75 gel-filtration column. Fractions containing LIM1 were pooled and concentrated to ~0.5 mM
with buffer at pH 7.5 containing 50 mM
Na2HPO4, 100 mM NaCl, and 0.5 mM
-mercaptoethanol. For an amide hydrogen/deuterium
exchange experiment, 15N-labeled LIM1 in the same buffer
was lyophilized and dissolved in 100%
2H2O.
-D-thiogalactopyranoside. Cells were lysed
with 50 mM Tris-HCl and 1 mM
phenylmethylsulfonyl fluoride at pH 8, and the protein was purified as
described previously (16), yielding 50-100 mg/liter of culture. The
cleavage of the GST fusion protein by Factor Xa was
problematic due to some secondary proteolysis. The cleavage was better
in the presence of LIM1, which tightly binds to ILK ankyrin. Therefore,
100 mg of unlabeled GST-ankyrin were mixed with a slightly excess
amount of 15N-labeled LIM1 (18 mg) for Factor
Xa cleavage (300 units of Factor Xa from
Novagen); the reaction (10 ml) was stopped after 4 h with 1 mM phenylmethylsulfonyl fluoride; and the mixtures were
loaded onto a Superdex 75 gel-filtration column to separate the complex from GST (dimer with a molecular mass of 60 kDa) and other impurities. The fractions containing LIM1-ankyrin complex were collected and concentrated to 0.5 ml for NMR experiments (carried out at pH 6.5 with
50 mM phosphate buffer and 100 mM NaCl).
Precipitation occurred during the concentration process, and the final
sample concentration was ~0.3 mM.
, and C
resonances of
residues 6-68. Residues 1-5 and residues from the vector appeared to
be unstructured, could not be assigned, and thus were excluded from
further analysis and structure calculations. Side chains were assigned
with a combination of HCCH total correlation spectroscopy, HNHA,
C(CO)NH, and H(CCO)NH. Aromatic side chains were assigned with a
combination of two-dimensional total correlation spectroscopy (24),
two-dimensional NOESY (24) performed on an unlabeled sample in
2H2O, and three-dimensional
15N/13C-edited NOESY (25). Three-dimensional
15N/13C-edited NOESY was also used to confirm
assignments across proline gaps and to assign residues
Pro69 and Cys70. Inspection of H-
or H-
NOE cross-peaks to intraresidue H-
and HN enabled us to make
stereospecific assignments of the
-methyls of Val24 and
seven H-
protons.
1 angle constraints from stereospecific assignments were
obtained from these spectra. An HNHA spectrum provided 24 3JHNH
coupling constants that
were converted to
angle constraints. Hydrogen bond distance
constraints were derived from NOE analysis and a hydrogen/deuterium
exchange experiment. Hydrogen bond constraints were only used during
final structure refinement and included only residues comprising
secondary structure elements.
1
rad
2) (29); Zn-N-C angles were the same as
H-N-C in protonated imidazole; and an improper kept zinc in the plane
of the imidazole ring. NOEs were grouped into strong (1.8-2.7 Å),
medium (1.8-3.3 Å), and weak (1.8-5.0 Å). NOEs from equivalent or
non-stereospecifically assigned atoms were treated with
r
6 summation and without correction
for multiplicity. Upper bound corrections for such NOEs were performed
according to the recommendations of Ref. 30. The presence of zinc
necessitated minor modifications of the protocol since convergence with
standard procedure was low, we believe due to covalent bonding of zinc.
First, we used a standard simulated annealing protocol to generate 50 structures of protein in the absence of zinc. We selected the 25 lowest
total energy structures, some of which contained significant
distortions from the final structure. We then added zinc atoms to these
structures, which generated 125 simulated annealing structures (every
zinc-free structure parented five zinc-containing structures). Out of
125 structures obtained in this manner, we selected 25 structures with
the lowest total energy as our final set. The calculation statistics
for this set is shown in Table I. All the structure calculations
were performed with X-PLOR Version 3.851 (31). A Ramachandran plot was
calculated with the program Procheck-NMR to examine the quality of the
structures (32). Surface potentials were calculated (Poisson-Boltzmann
solver) and displayed in Grasp (33). Structures were visualized, and
figures were prepared with InsightII (MSI, Inc.) and MolMol (34).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1-
2-
3-
4-
, which
is consistent with the sequential NOE patterns (24), hydrogen/deuterium
exchange data, and 13C-
/
chemical shift data (35).
Long range H
-H
, HN-HN, and H
-HN; strong sequential H
-HN
NOE connectivities; and slow amide exchange data revealed that
Thr9-Cys10 and
Gly15-Gly16 constitute
-sheet 1, whereas
Ile23-Ser26 and
Glu29-His32 form
-sheet 2.
-Sheet 3 is
composed of Val37-Cys38 and
Gln43-Gln44, and
-sheet 4 contains
Tyr51-Phe53 and
Arg56-Cys59. Similar to the previously
reported LIM domain structures of the CRP and CRIP families,
rubredoxin-type turns (6) connect strands of
-sheets 1 and 3, whereas individual strands of
-sheets 2 and 4 are connected by tight
turns involving the moderately conserved residues Gly28 and
Gly55. Immediately after
-sheet 4, His61-Lys66 adopt an
-helical structure as
evidenced by characteristic medium range
H
i-HNi+3 and
H
i-H
i+3 as well as strong sequential
HN-HN NOEs.
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Fig. 2.
Three-dimensional fold of the PINCH LIM1
domain. A, best fit superposition of backbone residues
7-67 of 25 final structures, shown as a trace of C- atoms;
B, ribbon diagram of the minimized average structure
(residues 7-67) showing elements of secondary structure.
Zn2+ ions are rendered as black spheres.
Statistics of experimental data and structure calculations
-sheets, i.e.
-sheet 1 versus
-sheet 2 for finger 1 and
-sheet 3 versus
-sheet 4 for finger 2, and the finger 2 is
terminated by an
-helix (Fig. 2B). It is of interest to
note that, in all LIM domain structures determined so far,
-sheets 1 and 3 are shorter than
-sheets 2 and 4. The edge of
-sheet 1 contacts the edge of
-sheet 2; however, these elements do not merge
into a contiguous structure. The amide proton of Glu11 at
the edge of
-sheet 1 has a slow hydrogen/deuterium exchange rate,
apparently due to participation in a hydrogen bond with the carbonyl
oxygen of Lys30 in
-sheet 2 as reflected during
structure calculations; yet the tertiary structure of LIM1 shows that
-sheets 1 and 2 are nearly orthogonal at this site and that the
Glu11 HN-Lys30 oxygen hydrogen bond can be
viewed as a pivot point. In comparison,
-sheets 3 and 4 in finger 2 are less orthogonal to each other and appear to partly merge into a
more extensive four-stranded
-structure.
-sheets 1 and 2, suggesting that the insertion point should be in
this linker region. Superposition of PINCH LIM1 with qCRP2 LIM2 by
sequentially omitting one residue from
Pro19-Ile23 yielded a root mean square
deviation of 2.3-2.4 Å. On the other hand, superposition of PINCH
LIM1 with more sequence-divergent LIM domains such as CRIP and
chicken CRP1 LIM1 yielded larger root mean square deviations
(3.8 and 3.3 Å, respectively). Although individual zinc fingers of
these LIM domains superimpose well with those of PINCH LIM1, there is
significant twisting between the two zinc fingers of the CRIP
(~60°) (7) and chicken CRP1 (~45°) LIM domains,
respectively, what leads to their poor overall superposition with PINCH
LIM1. In the case of PINCH LIM1, zinc finger 2 is twisted only slightly
(~20°) with respect to finger 1. The variable orientations between
the two zinc fingers of different LIM domains are consistent with the
intradomain flexibility of the two zinc fingers as indicated by the
15N backbone dynamics studies of qCRP2 LIM1 compared with
qCRP2 LIM2 (10, 12). It remains to be established how such intradomain mobility might contribute to the LIM-mediated protein-protein interactions.
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Fig. 3.
Hydrophobic features of the PINCH LIM1
domain. A, side chain arrangement of hydrophobic core
residues of the average minimized structure of PINCH LIM1;
B, hydrophobic patch residues near the C terminus of the
average minimized structure. The thin ribbon
represents the backbone of residues 49-67 ( -sheet 4 and
-helix);
side chains are shown as sticks.
-sheets 1 and 3. The Phe42 ring
packs underneath in parallel to the plane of
-sheet 3. As a
consequence, the bulge is created, and what would be a shallow depression becomes deeper groove (Fig. 4A). The third
hydrophobic patch is at the opposite end of patch 1, where the aromatic
rings of Tyr51, Phe53, and Tyr58
protrude upwards from
-sheet 4 and arrange themselves in a nearly coplanar manner (Fig. 3B). The aromatic ring of
Phe63 covers part of this platform, as it stacks against
the tyrosine rings. The other side of the Phe63 ring
contacts the Leu66 side chain. Therefore, this hydrophobic
surface is rather extensive, including the Leu66 aliphatic
group, the Phe63 aromatic ring, and several edges of
aromatic residues Tyr51, Phe53,
Tyr58, and Phe67 (ring-disordered) (Figs.
3B and 4A). Examination of the surface features
in the most homologous qCRP2 LIM2, qCRP2 LIM1, and highly divergent
CRIP proteins revealed that this patch is conserved, involving mostly
the C-terminal helix (Fig. 4C), what may be important for
LIM-target protein interactions (see below). The size and hydrophobic
residues in this patch vary significantly, which may confer specificity
for diverse target proteins.
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Fig. 4.
Surface features of PINCH LIM1 and comparison
with other LIM domains. A, hydrophobic features
of the average minimized structure of the PINCH LIM1 surface. The view
in middle panel corresponds to the slightly rotated view in
Fig. 2. The indicated residues comprise hydrophobic patches
(green) discussed under "Results and Discussion."
The three panels are related by 90° rotation in page plane
as shown for both A and B. B,
electrostatic features of the average minimized structure of the PINCH
LIM1 surface. The bar below shows color mapping of the
electrostatic energy spectrum from 10 to +10 in
kBT units. The potential was calculated with the
program Grasp. C, hydrophobic features of three
representative LIM domains in orientations similar to the middle
panel of A. The indicated residues are conserved
counterparts of the residues of PINCH LIM1 that compose patch 3 near
the C terminus.
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Fig. 5.
NMR spectra of PINCH LIM1 and its interaction
with the ILK ankyrin repeat domain. Shown is an overlay of the
1H-15N HSQC spectra of PINCH LIM1 in its free
form (black) and in a complex with the ankyrin domain of ILK
(light gray). Residue labels correspond to free PINCH
LIM1.
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Fig. 6.
Chemical shift changes in PINCH LIM1 upon
binding to the ILK ankyrin repeat domain. The changes were plotted
as a sum of absolute values of 1H and 15N
chemical shift changes upon binding to the ankyrin domain as a function
of residue number. For Ala39, Gln40,
Phe63, Met65, Leu66, and
Phe67, we were unable to find any tentative assignments;
thus, we assigned the largest observed change in chemical shift (138 Hz
for Ala68) to these residues. Digital resolution was 14 Hz
in the 15N dimension and 8 Hz in the 1H
dimension.
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Fig. 7.
Three-dimensional view of chemical shift
mapping for the LIM1 structure upon binding to the ILK ankyrin repeat
domain. Changes calculated in Fig. 6 were mapped onto the surface
of the minimized average structure of PINCH LIM1. The color
bar at the bottom shows the correspondence of color to the
observed change in chemical shift: white, no or little
change in chemical shift; blue, moderate change;
red, largest change (or peak was not observed in an HSQC
spectrum of the complex), in hertz. Orientations of the three views are
the same as in Fig. 4.
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ACKNOWLEDGEMENTS |
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We thank Frank Delaglio for nmrPipe software; Dan Garrett for PIPP software; and Olga Vinogradova, Sambasivarao Nanduri, and Shoukat Dedhar for useful discussions.
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FOOTNOTES |
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* The work was supported by National Institutes of Health Grants HL58758 (to J. Q.) and DK54639 (to C. W.) and American Cancer Society Research Project Grant 98-220-01-CSM (to C. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1G47) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
Chemical shifts for this protein were deposited in the BioMagResBank under entry number 4884.
¶ These authors contributed equally to this work.
** To whom correspondence should be addressed: Structural Biology Program, NB20, Lerner Research Inst., Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-5392; Fax: 216-445-1466; E-mail: qinj@ccf.org.
Published, JBC Papers in Press, November 14, 2000, DOI 10.1074/jbc.M007632200
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ABBREVIATIONS |
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The abbreviations used are: CRP, cysteine-rich protein; qCRP2, quail cysteine-rich protein-2; CRIP, cysteine-rich intestinal protein; ILK, integrin-linked kinase; GST, glutathione S-transferase; NOESY, nuclear Overhauser effect correlation spectroscopy; NOE, nuclear Overhauser effect; HSQC, heteronuclear single quantum correlation spectroscopy.
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