Application of cross-correlated NMR spin relaxation to the zinc-finger protein CRP2(LIM2): evidence for collective motions in LIM domains

The solution structure of quail CRP2(LIM2) was significantly improved by using an increased number of NOE constraints obtained from a 13C,15N-labeled protein sample and by applying a recently developed triple-resonance cross-correlated relaxation experiment for the determination of the backbone dihedral angle psi. Additionally, the relative orientation of the 15N(i)-1HN(i) dipole and the 13CO(i) CSA tensor, which is related to both backbone angles phi and psi, was probed by nitrogen-carbonyl multiple-quantum relaxation and used as an additional constraint for the refinement of the local geometry of the metal-coordination sites in CRP2(LIM2). The backbone dynamics of residues located in the folded part of CRP2(LIM2) have been characterized by proton-detected 13C'(i-1)-15N(i) and 15N(i)-1HN(i) multiple-quantum relaxation, respectively. We show that regions having cross-correlated time modulation of backbone isotropic chemical shifts on the millisecond to microsecond time scale correlate with residues that are structurally altered in the mutant protein CRP2(LIM2)R122A (disruption of the CCHC zinc-finger stabilizing side-chain hydrogen bond) and that these residues are part of an extended hydrogen-bonding network connecting the two zinc-binding sites. This indicates the presence of long-range collective motions in the two zinc-binding subdomains. The conformational plasticity of the LIM domain may be of functional relevance for this important protein recognition motif.     

Mutational analysis and NMR spectroscopy of quail cysteine and glycine-rich protein CRP2 reveal an intrinsic segmental flexibility of LIM domains.

The LIM domain is a conserved cysteine and histidine-containing structural module of two tandemly arranged zinc fingers. It has been identified in single or multiple copies in a variety of regulatory proteins, either in combination with defined functional domains, like homeodomains, or alone, like in the CRP family of LIM proteins. Structural studies of CRP proteins have allowed a detailed evaluation of interactions in LIM-domains at the molecular level. The packing interactions in the hydrophobic core have been identified as a significant contribution to the LIM domain fold, whereas hydrogen bonding within each single zinc binding site stabilizes zinc finger geometry in a so-called "outer" or "indirect" coordination sphere. Here we report the solution structure of a point-mutant of the carboxyl-terminal LIM domain of quail cysteine and glycine-rich protein CRP2, CRP2(LIM2)R122A, and discuss the structural consequences of the disruption of the hydrogen bond formed between the guanidinium side-chain of Arg122 and the zinc-coordinating cysteine thiolate group in the CCHC rubredoxin-knuckle. The structural analysis revealed that the three-dimensional structure of the CCHC zinc binding site in CRP2(LIM2)R122A is adapted as a consequence of the modified hydrogen bonding pattern. Additionally, as a result of the conformational rearrangement of the zinc binding site, the packing interactions in the hydrophobic core region are altered, leading to a change in the relative orientation of the two zinc fingers with a concomitant change in the solvent accessibilities of hydrophobic residues located at the interface of the two modules. The backbone dynamics of residues located in the folded part of CRP2(LIM2)R122A have been characterized by proton-detected(15)N NMR spectroscopy. Analysis of the R2/R1ratios revealed a rotational correlation time of approximately 6.2 ns and tumbling with an axially symmetric diffusion tensor (D parallel/D perpendicular=1.43). The relaxation data were also analyzed using a reduced spectral density mapping approach. As in wild-type CRP2(LIM2), significant mobility on a picosecond/nanosecond time-scale was detected, and conformational exchange on a microsecond time-scale was identified for residues located in loop regions between secondary structure elements. In summary, the relative orientation of the two zinc binding sites and the accessibility of hydrophobic residues is not only determined by hydrophobic interactions, but can also be modified by the formation and/or breakage of hydrogen bonds. This may be important for the molecular interactions of an adaptor-type LIM domain protein in macromolecular complexes, particularly for the modulation of protein-protein interactions.     

Interaction of hCLIM1, an enigma family protein, with alpha-actinin 2

Enigma proteins are proteins that possess a PDZ domain at the amino terminal and one to three LIM domains at the carboxyl terminal. They are cytoplasmic proteins that are involved with the cytoskeleton and signal transduction pathway. By virtue of the two protein interacting domains, they are capable of protein-protein interactions. Here we report a study on a human Enigma protein hCLIM1, in particular. Our study describes the interaction of the human 36 kDa carboxyl terminal LIM domain protein (hCLIM1), the human homologue of CLP36 in rat, with alpha-actinin 2, the skeletal muscle isoform of alpha-actinin. hCLIM1 protein was shown to interact with alpha-actinin 2 by yeast two-hybrid screening and immunochemical analyses. Yeast two-hybrid analyses also demonstrated that the LIM domain of hCLIM1 binds to the EF-hand region of alpha-actinin 2, defining a new mode of LIM domain interactions. Immunofluorescent study demonstrates that hCLIM1 colocalizes with alpha-actinin at the Z-disks in human myocardium. Taken together, our experimental results suggest that hCLIM1is a novel cytoskeletal protein and may act as an adapter that brings other proteins to the cytoskeleton.     

Solution structure of the chicken cysteine-rich protein, CRP1, a double-LIM protein implicated in muscle differentiation

The mechanism by which the contractile machinery of muscle is assembled and maintained is not well-understood. Members of the cysteine-rich protein (CRP) family have been implicated in these processes. Three vertebrate CRPs (CRP1-3) that exhibit developmentally regulated muscle-specific expression have been identified. All three proteins are associated with the actin cytoskeleton, and one has been shown to be required for striated muscle structure and function. The vertebrate CRPs identified to date display a similar molecular architecture; each protein is comprised of two tandemly arrayed LIM domains, protein-binding motifs found in a number of proteins with roles in cell differentiation. Each LIM domain coordinates two Zn(II) ions that are bound independently in CCHC (C=Cys, H=His) and CCCC modules. Here we describe the solution structure of chicken CRP1 determined by homonuclear and 1H-15N heteronuclear magnetic resonance spectroscopy. Comparison of the structures of the two LIM domains of CRP1 reveals a high degree of similarity in their tertiary folds. In addition, the two component LIM domains represent two completely independent folding units and exhibit no apparent interactions with each other. The structural independence and spatial separation of the two LIM domains of CRP1 are compatible with an adapter or linker role for the protein.     

Cysteine-rich protein 1 (CRP1) regulates actin filament bundling

Background  

Cysteine-rich protein 1 (CRP1) is a LIM domain containing protein localized to the nucleus and the actin cytoskeleton. CRP1 has been demonstrated to bind the actin-bundling protein α-actinin and proposed to modulate the actin cytoskeleton; however, specific regulatory mechanisms have not been identified.     

Cypher, a striated muscle-restricted PDZ and LIM domain-containing protein, binds to alpha-actinin-2 and protein kinase C.

We have cloned and characterized a novel striated muscle-restricted protein (Cypher) that has two mRNA splice variants, designated Cypher1 and Cypher2. Both proteins contain an amino-terminal PDZ domain. Cypher1, but not Cypher2, contains three carboxyl-terminal LIM domains and an amino acid repeat sequence that exhibits homology to a repeat sequence found in the largest subunit of RNA polymerase II. cypher1 and cypher2 mRNAs exhibited identical expression patterns. Both are exclusively expressed in cardiac and striated muscle in embryonic and adult stages. By biochemical assays, we have demonstrated that Cypher1 and Cypher2 bind to alpha-actinin-2 via their PDZ domains. This interaction has been further confirmed by immunohistochemical studies that demonstrated co-localization of Cypher and alpha-actinin at the Z-lines of cardiac muscle. We have also found that Cypher1 binds to protein kinase C through its LIM domains. Phosphorylation of Cypher by protein kinase C has demonstrated the functional significance of this interaction. Together, our data suggest that Cypher1 may function as an adaptor in striated muscle to couple protein kinase C-mediated signaling, via its LIM domains, to the cytoskeleton (alpha-actinin-2) through its PDZ domain.     

Structure and dynamics of the human muscle LIM protein

The family of cysteine rich proteins (CRP) comprises three closely homologous members that have been reported to interact with α-actinin. Muscular LIM protein (MLP/CRP3), the skeletal muscle variant, was originally discovered as a positive regulator of myogenesis and is suggested to be part of the stretch sensor of the myofibril through its interaction with telethonin (T-Cap). We determined the structure of both LIM domains of human MLP by nuclear magnetic resonance spectroscopy. We confirm by ¹⁵N relaxation measurements that both LIM domains act as independent units and that the adjacent linker regions are fully flexible. With the published structures of CRP1 and CRP2, the complete family has now been structurally characterized.     

LIM-only protein FHL3 interacts with CDC25B2 phosphatase

LIM domain proteins are important regulators of the growth, determination, and differentiation of cells. In this report, FHL3 (human four-and-a-half LIM-only protein 3) is shown to interact with human phosphatase CDC25B, a cell cycle regulator involved in the control of G2/M. We found that this interaction was specific to the CDC25B2 isoform. Deletion and point mutation studies indicated that the second LIM domain of FHL3 was essential for this interaction. FRET experiments in C2C12 cells showed that, although both proteins were colocated in the cytoplasm and the nucleus, they interacted only in the nucleus. Finally, we showed that FHL3 binding impaired neither CDC25B2 phosphatase activity nor its localization. Further work is now needed to elucidate the consequences of this interaction on myoblast fate decision and cycle control.     

Cysteine-rich LIM-only proteins CRP1 and CRP2 are potent smooth muscle differentiation cofactors

Cysteine-rich LIM-only proteins, CRP1 and CRP2, expressed during cardiovascular development act as bridging molecules that associate with serum response factor and GATA proteins. SRF-CRP-GATA complexes strongly activated smooth muscle gene targets. CRP2 was found in the nucleus during early stages of coronary smooth muscle differentiation from proepicardial cells. A dominant-negative CRP2 mutant blocked proepicardial cells from differentiating into smooth muscle cells. Together with SRF and GATA proteins, CRP1 and CRP2 converted pluripotent 10T1/2 fibroblasts into smooth muscle cells, while muscle LIM protein CRP3 inhibited the conversion. Thus, LIM-only proteins of the CRP family play important roles in organizing multiprotein complexes, both in the cytoplasm, where they participate in cytoskeletal remodeling, and in the nucleus, where they strongly facilitate smooth muscle differentiation.     

Targeted disruption of the mouse <Emphasis Type="Italic">Csrp2</Emphasis>gene encoding the cysteine- and glycine-rich LIM domain protein CRP2 result in subtle alteration of cardiac ultrastructure

Background  

The cysteine and glycine rich protein 2 (CRP2) encoded by the Csrp2 gene is a LIM domain protein expressed in the vascular system, particularly in smooth muscle cells. It exhibits a bimodal subcellular distribution, accumulating at actin-based filaments in the cytosol and in the nucleus. In order to analyze the function of CRP2 in vivo, we disrupted the Csrp2 gene in mice and analysed the resulting phenotype.     

ZRP-1, a zyxin-related protein, interacts with the second PDZ domain of the cytosolic protein tyrosine phosphatase hPTP1E.

Protein-protein interactions play an important role in the specificity of cellular signaling cascades. By using the yeast two-hybrid system, a specific interaction was identified between the second PDZ domain of the cytosolic protein tyrosine phosphatase hPTP1E and a novel protein, which was termed ZRP-1 to indicate its sequence similarity to the Zyxin protein family. The mRNA encoding this protein is distributed widely in human tissues and contains an open reading frame of 1428 base pairs, predicting a polypeptide of 476 amino acid residues. The deduced protein displays a proline-rich amino-terminal region and three double zinc finger LIM domains at its carboxyl terminus. The specific interaction of this novel protein with the second PDZ domain of hPTP1E was demonstrated both in vitro, using bacterially expressed proteins, and in vivo, by co-immunoprecipitation studies. Deletion analysis indicated that an intact carboxyl terminus is required for its interaction with the second PDZ domain of hPTP1E in the yeast two-hybrid system and suggested that other sequences, including the LIM domains, also participate in the interaction. The genomic organization of the ZRP-1 coding sequence is identical to that of the lipoma preferred partner gene, another Zyxin-related protein, suggesting that the two genes have evolved from a recent gene duplication event.     

Interaction of the heart-specific LIM domain protein, FHL2, with DNA-binding nuclear protein, hNP220

Using a yeast two-hybrid library screen, we have identified that the heart specific FHL2 protein, four-and-a-half LIM protein 2, interacted with human DNA-binding nuclear protein, hNP220. Domain studies by the yeast two-hybrid interaction assay revealed that the second LIM domain together with the third and the fourth LIM domains of FHL2 were responsible to the binding with hNP220. Using green fluorescent protein (GFP)-FHL2 and blue fluorescent protein (BFP)-hNP220 fusion proteins co-expressed in the same cell, we demonstrated a direct interaction between FHL2 and hNP220 in individual nucleus by two-fusion Fluorescence Resonance Energy Transfer (FRET) assay. Besides, Western blot analysis using affinity-purified anti-FHL2 antipeptide antibodies confirmed a 32-kDa protein of FHL2 in heart only. Virtually no expression of FHL2 protein was detected in brain, liver, lung, kidney, testis, skeletal muscle, and spleen. Moreover, the expression of FHL2 protein was also detectable in the human diseased heart tissues. Our results imply that FHL2 protein can shuttle between cytoplasm and nucleus and may act as a molecular adapter to form a multicomplex with hNP220 in the nucleus, thus we speculate that FHL2 may be particularly important for heart muscle differentiation and the maintenance of the heart phenotype.     

T2BP, a novel TRAF2 binding protein, can activate NF-kappaB and AP-1 without TNF stimulation.

TRAF2 is a key molecule involved in TNF signaling, which is crucial for the regulation of inflammatory processes. We have identified a novel TRAF2 binding protein, designated as T2BP (TRAF2 binding protein), by a mammalian two-hybrid screening approach. T2BP is a relatively small protein of 184 amino acids, which includes a forkhead-associated domain, the phosphopeptide binding motif. The interaction domain search showed that the TRAF domain in TRAF2 is required for the binding to T2BP whereas almost the entire protein in T2BP binds to TRAF2. The interaction was further confirmed by co-immunoprecipitation. Expression profiling for T2BP and TRAF2 revealed an ubiquitous expression in adult mouse tissues. Overexpression of T2BP in HEK293 cells activated NF-kappaB and AP-1 in a dose dependent manner as well as seen in the TNF-treated control cells. Our results suggest that T2BP is involved in the TNF-mediated signaling by its interaction with TRAF2.     

Novel interaction partners of the CD2BP2-GYF domain

The GYF domain of CD2BP2 serves as an adapter that recognizes proline-rich sequences in intracellular proteins. Although the T cell adhesion molecule CD2 and the core splicing protein SmB/B' were previously shown to interact with CD2BP2-GYF, we are now using a general approach to identify putative GYF domain target sites within the human proteome. The phage display-derived recognition motif for CD2BP2-GYF is PPG(W/F/Y/M/L). SPOT analysis confirmed that the GYF domain interacts with peptides from human proteins containing the consensus site. Epitope mapping by NMR spectroscopy performed for several peptides revealed a conserved binding surface. A direct interaction of the CD2BP2-GYF domain with the novel protein interaction partners PI31 and NPWBP was verified by yeast two-hybrid analysis.     

Protein-protein interaction of FHL3 with FHL2 and visualization of their interaction by green fluorescent proteins (GFP) two-fusion fluorescence resonance energy transfer (FRET)

LIM domain proteins are found to be important regulators in cell growth, cell fate determination, cell differentiation and remodeling of the cell cytoskeleton. Human Four-and-a-half LIM-only protein 3 (FHL3) is a type of LIM-only protein that contains four tandemly repeated LIM motifs with an N-terminal single zinc finger (half LIM motif). FHL3 expresses predominantly in human skeletal muscle. In this report, FHL3 was shown to be a novel interacting partner of FHL2 using the yeast two-hybrid assay. Furthermore, site-directed mutagenesis of FHL3 indicated that the LIM2 of FHL3 is the essential LIM domain for interaction with FHL2. Green fluorescent protein (GFP) was used to tag FHL3 in order to study its distribution during myogenesis. Our result shows that FHL3 was localized in the focal adhesions and nucleus of the cells. FHL3 mainly stayed in the focal adhesion during myogenesis. Moreover, using site-directed mutagenesis, the LIM1 of FHL3 was identified as an essential LIM domain for its subcellular localization. Mutants of GFP have given rise to a novel technique, two-fusion fluorescence resonance energy transfer (FRET), in the determination of protein-protein interaction at particular subcellular locations of eukaryotic cells. To determine whether FHL2 and FHL3 can interact with one another and to locate the site of this interaction in a single intact mammalian cell, we fused FHL2 and FHL3 to different mutants of GFP and studied their interactions using FRET. BFP/GFP fusion constructs were cotransfected into muscle myoblast C2C12 to verify the colocalization and subcellular localization of FRET. We found that FHL2 and FHL3 were colocalized in the mitochondria of the C2C12 cells and FRET was observed by using an epi-fluorescent microscope equipped with an FRET specific filter set.     

Characterization of PINCH-2, a new focal adhesion protein that regulates the PINCH-1-ILK interaction,cell spreading,and migration

Integrin-linked kinase (ILK) is a multidomain protein that plays important roles at cell-extracellular matrix (ECM) adhesion sites. We describe here a new LIM-domain containing protein (termed as PINCH-2) that forms a complex with ILK. PINCH-2 is co-expressed with PINCH-1 (previously known as PINCH), another member of the PINCH protein family, in a variety of human cells. Immunofluorescent staining of cells with PINCH-2-specific antibodies show that PINCH-2 localizes to both cell-ECM contact sites and the nucleus. Deletion of the first LIM (LIM1) domain of PINCH-2 abolished the ability of PINCH-2 to form a complex with ILK. The ILK-binding defective LIM1-deletion mutant, unlike the wild type PINCH-2 or the ILK-binding competent LIM5-deletion mutant, was incapable of localizing to cell-ECM contact sites, suggesting that ILK binding is required for this process. Importantly, the PINCH-2-ILK and PINCH-1-ILK interactions are mutually exclusive. Overexpression of PINCH-2 significantly inhibited the PINCH-1-ILK interaction and reduced cell spreading and migration. These results identify a novel nuclear and focal adhesion protein that associates with ILK and reveals an important role of PINCH-2 in the regulation of the PINCH-1-ILK interaction, cell shape change, and migration.     

The zyxin-related protein TRIP6 interacts with PDZ motifs in the adaptor protein RIL and the protein tyrosine phosphatase PTP-BL

The small adaptor protein RIL consists of two segments, the C-terminal LIM and the N-terminal PDZ domain, which mediate multiple protein-protein interactions. The RIL LIM domain can interact with PDZ domains in the protein tyrosine phosphatase PTP-BL and with the PDZ domain of RIL itself. Here, we describe and characterise the interaction of the RIL PDZ domain with the zyxin-related protein TRIP6, a protein containing three C-terminal LIM domains. The second LIM domain in TRIP6 is sufficient for a strong interaction with RIL. A weaker interaction with the third LIM domain in TRIP6, including the proper C-terminus, is also evident. TRIP6 also interacts with the second out of five PDZ motifs in PTP-BL. For this interaction to occur both the third LIM domain and the proper C-terminus are necessary. RNA expression analysis revealed overlapping patterns of expression for TRIP6, RIL and PTP-BL, most notably in tissues of epithelial origin. Furthermore, in transfected epithelial cells TRIP6 can be co-precipitated with RIL and PTP-BL PDZ polypeptides, and a co-localisation of TRIP6 and RIL with Factin structures is evident. Taken together, PTP-BL, RIL and TRIP6 may function as components of multi-protein complexes at actin-based sub-cellular structures.