Solution Structure of the Focal Adhesion Adaptor PINCH LIM1 Domain and Characterization of Its Interaction with the Integrin-linked Kinase Ankyrin Repeat Domain*

Algirdas VelyvisDagger §, Yanwu YangDagger , Chuanyue Wu||, and Jun QinDagger §**

From the Dagger  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



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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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 beta -sheets, and the C-terminal CCCC module is terminated by a alpha -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.

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 beta 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.

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.



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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.



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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-beta -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 beta -mercaptoethanol. For an amide hydrogen/deuterium exchange experiment, 15N-labeled LIM1 in the same buffer was lyophilized and dissolved in 100% 2H2O.

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-beta -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.

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, Calpha , and Cbeta 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-gamma or H-beta NOE cross-peaks to intraresidue H-alpha and HN enabled us to make stereospecific assignments of the gamma -methyls of Val24 and seven H-beta protons.

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 chi 1 angle constraints from stereospecific assignments were obtained from these spectra. An HNHA spectrum provided 24 3JHNHalpha coupling constants that were converted to phi  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.

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-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
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EXPERIMENTAL PROCEDURES
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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 beta 1-beta 2-beta 3-beta 4-alpha , which is consistent with the sequential NOE patterns (24), hydrogen/deuterium exchange data, and 13C-alpha /beta chemical shift data (35). Long range Halpha -Halpha , HN-HN, and Halpha -HN; strong sequential Halpha -HN NOE connectivities; and slow amide exchange data revealed that Thr9-Cys10 and Gly15-Gly16 constitute beta -sheet 1, whereas Ile23-Ser26 and Glu29-His32 form beta -sheet 2. beta -Sheet 3 is composed of Val37-Cys38 and Gln43-Gln44, and beta -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 beta -sheets 1 and 3, whereas individual strands of beta -sheets 2 and 4 are connected by tight turns involving the moderately conserved residues Gly28 and Gly55. Immediately after beta -sheet 4, His61-Lys66 adopt an alpha -helical structure as evidenced by characteristic medium range Halpha i-HNi+3 and Halpha i-Hbeta 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-alpha atoms; B, ribbon diagram of the minimized average structure (residues 7-67) showing elements of secondary structure. Zn2+ ions are rendered as black spheres.


                              
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Table I
Statistics of experimental data and structure calculations

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 beta -sheets, i.e. beta -sheet 1 versus beta -sheet 2 for finger 1 and beta -sheet 3 versus beta -sheet 4 for finger 2, and the finger 2 is terminated by an alpha -helix (Fig. 2B). It is of interest to note that, in all LIM domain structures determined so far, beta -sheets 1 and 3 are shorter than beta -sheets 2 and 4. The edge of beta -sheet 1 contacts the edge of beta -sheet 2; however, these elements do not merge into a contiguous structure. The amide proton of Glu11 at the edge of beta -sheet 1 has a slow hydrogen/deuterium exchange rate, apparently due to participation in a hydrogen bond with the carbonyl oxygen of Lys30 in beta -sheet 2 as reflected during structure calculations; yet the tertiary structure of LIM1 shows that beta -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, beta -sheets 3 and 4 in finger 2 are less orthogonal to each other and appear to partly merge into a more extensive four-stranded beta -structure.

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 beta -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.

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).



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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 (beta -sheet 4 and alpha -helix); side chains are shown as sticks.

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 beta -sheets 1 and 3. The Phe42 ring packs underneath in parallel to the plane of beta -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 beta -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.

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.



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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.

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.


    ACKNOWLEDGEMENTS

We thank Frank Delaglio for nmrPipe software; Dan Garrett for PIPP software; and Olga Vinogradova, Sambasivarao Nanduri, and Shoukat Dedhar for useful discussions.


    FOOTNOTES

* 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


    ABBREVIATIONS

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES


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