Mutational analysis of the metal sites in an LIM domain

Michelsen, James W.; Sewell, A.K.; Louis, Heather A.; Olsen, Jay I.; Davis, Darrell R.; Winge, Dennis R.; Beckerle, Mary C.

doi:10.1016/s0021-9258(19)78098-3

Cited by 62 publications

(11 citation statements)

References 28 publications

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“…Briefly, the engineered expression vectors were introduced into Escherichia coli BL21(DE3) cells, and the cells were induced to produce the protein of interest by exposure to 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (Sigma). Proteins were isolated from the bacterial cell lysates essentially as described previously (1,14,16,32). For CRP1 and LIM2, bacteria were lysed in 10 mM KCl, 10 mM potassium phosphate (pH 7.2), and 10 mM dithiothreitol, and soluble proteins were dialyzed versus column buffer (10 mM potassium phosphate (pH 7.2), 10 mM KCl, 0.01% 2-mercaptoethanol) prior to cation-exchange chromatography on CM-52 (Whatman); proteins were eluted from the column with a linear KCl (0-250 mM) gradient in column buffer.…”

Section: Methodsmentioning

confidence: 99%

Solution Structure of the Chicken Cysteine-Rich Protein, CRP1, a Double-LIM Protein Implicated in Muscle Differentiation^,

et al. 1999

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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.

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Section: Methodsmentioning

confidence: 99%

Solution Structure of the Chicken Cysteine-Rich Protein, CRP1, a Double-LIM Protein Implicated in Muscle Differentiation^,

et al. 1999

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“…The CRP1 column was prepared from bacterially expressed avian smooth muscle CRP1. We have shown previously that bacterially expressed CRP1 exhibits a native structure (Michelsen et al, 1993(Michelsen et al, , 1994 and retains the ability to bind zyxin (Schmeichel and Beckerle, 1994). When a 27-34% ammonium sulfate precipitate from the avian smooth muscle extract (Fig.…”

Section: Recovery Of ␣ -Actinin From a Crp1-affinity Columnmentioning

confidence: 96%

CRP1, a LIM Domain Protein Implicated in Muscle Differentiation, Interacts with α-Actinin

Pomiès

Louis

Beckerle

1997

The Journal of Cell Biology

129

139

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Members of the cysteine-rich protein (CRP) family are LIM domain proteins that have been implicated in muscle differentiation. One strategy for defining the mechanism by which CRPs potentiate myogenesis is to characterize the repertoire of CRP binding partners. In order to identify proteins that interact with CRP1, a prominent protein in fibroblasts and smooth muscle cells, we subjected an avian smooth muscle extract to affinity chromatography on a CRP1 column. A 100-kD protein bound to the CRP1 column and could be eluted with a high salt buffer; Western immunoblot analysis confirmed that the 100-kD protein is α-actinin. We have shown that the CRP1–α-actinin interaction is direct, specific, and saturable in both solution and solid-phase binding assays. The K d for the CRP1–α-actinin interaction is 1.8 ± 0.3 μM. The results of the in vitro protein binding studies are supported by double-label indirect immunofluorescence experiments that demonstrate a colocalization of CRP1 and α-actinin along the actin stress fibers of CEF and smooth muscle cells. Moreover, we have shown that α-actinin coimmunoprecipitates with CRP1 from a detergent extract of smooth muscle cells. By in vitro domain mapping studies, we have determined that CRP1 associates with the 27-kD actin–binding domain of α-actinin. In reciprocal mapping studies, we showed that α-actinin interacts with CRP1-LIM1, a deletion fragment that contains the NH2-terminal 107 amino acids (aa) of CRP1. To determine whether the α-actinin binding domain of CRP1 would localize to the actin cytoskeleton in living cells, expression constructs encoding epitope-tagged full-length CRP1, CRP1-LIM1(aa 1-107), or CRP1-LIM2 (aa 108-192) were microinjected into cells. By indirect immunofluorescence, we have determined that full-length CRP1 and CRP1-LIM1 localize along the actin stress fibers whereas CRP1-LIM2 fails to associate with the cytoskeleton. Collectively these data demonstrate that the NH2-terminal part of CRP1 that contains the α-actinin–binding site is sufficient to localize CRP1 to the actin cytoskeleton. The association of CRP1 with α-actinin may be critical for its role in muscle differentiation.

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“…The LIM motif is defined by a cysteine-rich consensus sequence, CX2CXI6_ 23HX2CX2CX2CXI6.2tCX2_3(C,H,D) (Freyd et al, 1990;Karlsson et al, 1990;Sadler et al, 1992). Together the conserved Cys, His, and Asp residues coordinate two zinc atoms per LIM domain, giving rise to a double zinc finger (Michelsen et al, 1993(Michelsen et al, , 1994Kosa et al, 1994). The LIM domain has been shown to mediate specific protein-protein interactions and, in this way, may regulate protein activity and localization (Feuerstein et al, 1994;Schmeichel and Beckerle, 1994;Valge-Archer et al, 1994;Wu and Gill, 1994).…”

mentioning

confidence: 99%

Two muscle-specific LIM proteins in Drosophila.

Stronach

Siegrist

Beckerle

1996

The Journal of Cell Biology

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Mutational analysis of the metal sites in an LIM domain

Cited by 62 publications

References 28 publications

Solution Structure of the Chicken Cysteine-Rich Protein, CRP1, a Double-LIM Protein Implicated in Muscle Differentiation^,

Solution Structure of the Chicken Cysteine-Rich Protein, CRP1, a Double-LIM Protein Implicated in Muscle Differentiation^,

CRP1, a LIM Domain Protein Implicated in Muscle Differentiation, Interacts with α-Actinin

Two muscle-specific LIM proteins in Drosophila.

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Mutational analysis of the metal sites in an LIM domain

Cited by 62 publications

References 28 publications

Solution Structure of the Chicken Cysteine-Rich Protein, CRP1, a Double-LIM Protein Implicated in Muscle Differentiation,

Solution Structure of the Chicken Cysteine-Rich Protein, CRP1, a Double-LIM Protein Implicated in Muscle Differentiation,

CRP1, a LIM Domain Protein Implicated in Muscle Differentiation, Interacts with α-Actinin

Two muscle-specific LIM proteins in Drosophila.

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Product

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Solution Structure of the Chicken Cysteine-Rich Protein, CRP1, a Double-LIM Protein Implicated in Muscle Differentiation^,

Solution Structure of the Chicken Cysteine-Rich Protein, CRP1, a Double-LIM Protein Implicated in Muscle Differentiation^,