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

Abstract

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.

A Cysteine-rich LIM-only Protein Mediates Regulation of Smooth Muscle-specific Gene Expression by cGMP-dependent Protein Kinase

“…The deletion mutant CRP)ZF3/4 was inactive, suggesting PKG binding to CRP4 was required. The effect of CRP4 on SRF DNA binding is similar to (55,56). Based on the data of Chang et al (26) for CRP2/smLIM, we propose that SRF and GATA6 bind to ZF1 and ZF4 of CRP4, respectively.…”

“…CRP4 is homologous to CRP1 and CRP2/smLIM, and their high degree of sequence similarity suggests similar functions. Solution structures of CRP1 and -2 demonstrate that the proteins' two LIM domains are independent folding units bridged by a flexible linker region, suggesting an adaptor or linker role for the proteins (55,56). Similar to CRP4, CRP1, and CRP2/smLIM co-operate with SRF and GATA factors to transactivate SM-specific genes; CRP2 exists in a trimeric complex, with SRF and GATA4 (or -6) docking to the N-terminal and C-terminal LIM domains, respectively (26).…”

The human and mouse orthologous LIM-only proteins respectively encoded in chromosome 6 and 17 show a different expression pattern

“…Three-dimensional solution structures of CRP1 [26] and CRP2 [27] have shown that the two LIM domains are independent modules which do not interact directly and are coupled by a flexible linker in both molecules. The CRPs have the capacity to interact with specific proteins, as these CRPs have two LIM domains, it is postulated that they act as adapters bridging two distinct proteins.…”

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

“…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 Glu 11 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 Lys 30 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 Glu 11 HN-Lys 30 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.…”