Dystrophin: More than just the sum of its parts

Dystrophin is one of a number of large cytoskeleton associated proteins that connect between various cytoskeletal elements and often are tethered to the membrane through other transmembrane protein complexes. These cytolinker proteins often provide structure and support to the cells where they are expressed, and mutations in genes encoding these proteins frequently gives rise to disease. Dystrophin is no exception in any of these respects, providing connections between a transmembrane complex known as the dystrophin–glycoprotein complex and the underlying cytoskeleton. The most established connection and possibly the most important is that to F-actin, but more recently evidence has been forthcoming of connections to membrane phospholipids, intermediate filaments and microtubules. Moreover it is becoming increasingly clear that the multiple spectrin-like repeats in the centre of the molecule, that had hitherto been thought to be largely redundant, harbour binding activities that have a significant impact on dystrophin functionality. This functionality is particularly apparent when assessed by the ability to rescue the dystrophic phenotype in mdx mice. This review will focus on the relatively neglected but functionally vital coiled-coil region of dystrophin, highlighting the structural relationships and interactions of the coiled-coil region and providing new insights into the functional role of this region.     

The Coiled-coil Structure Potential of the Laminin LCC Domain is Very Fragmented and Does Not Differentiate Between Natural and Non-detected Isoforms

Abstract

There are 15 known laminins, which differ in the isoforms of the three chains that assemble into the cross-shape molecules that are observed by electron microscopy. The amino acid sequences of the rod-like portion of the long arm have long been recognized as having a potential for coiled-coil structure formation; however, an experimental determination of its structure is hampered by the complexity of laminin, a multidomain, heterotrimeric, and glycosilated 800 kDa molecule. Here, we have investigated the coiled-coil structure potential of laminin to evaluate its distribution along the long arm, the presence of conserved patterns, and differences between natural and non-natural isoforms. With these aims, we have analysed the sequences of each laminin chain in the context of the three-chain assemblies to yield an overall score of coiled-coil potential for the 15 natural laminins and for the other 30 possible but non-detected ones. The potential has been calculated with two different existing methods to exclude algorithm specific biases and with different chain alignments to evaluate the dependency of the results on uncertainties in the specific alignment along the domain. The analysis shows that the distribution of the potential is discontinuous, highly fragmented along the arm, without a common pattern except for a higher potential at the C-terminus, and that natural and non-natural laminins cannot be distinguished based on their coiled-coil potential, indicating that other factors are responsible for the selection of chain assembly.     

Isolation and characterization of a novel v-SNARE family protein that interacts with a calcium-dependent protein kinase from the common ice plant, <Emphasis Type="Italic">Mesembryanthemum crystallinum</Emphasis>

McCPK1 (Mesembryanthemum crystallinum calcium-dependent protein kinase 1) mRNA expression is transiently salinity- and dehydration-stress responsive. The enzyme also undergoes dynamic subcellular localization changes in response to these same stresses. Using the yeast-two hybrid system, we have isolated and characterized a M. crystallinum CPK1 Adaptor Protein 2 (McCAP2). We show that McCPK1 interacts with the C-terminal, coiled-coil containing region of McCAP2 in the yeast two-hybrid system. This interaction was confirmed in vitro between the purified recombinant forms of each of the proteins and in vivo by coimmunoprecipitation experiments from plant extracts. McCAP2, however, was not a substrate for McCPK1. Computational threading analysis suggested that McCAP2 is a member of a novel family of proteins with unknown function also found in rice and Arabidopsis. These proteins contain coiled-coil spectrin repeat domains present in the syntaxin superfamily that participate in vesicular and protein trafficking. Consistent with the interaction data, subcellular localization and fractionation studies showed that McCAP2 colocalizes with McCPK1 to vesicular structures located on the actin cytoskeleton and within the endoplasmic reticulum in cells subjected to low humidity stress. McCAP2 also colocalizes with AtVTI1a, an Arabidopsis v-SNARE [vesicle-soluble N-ethyl maleimide-sensitive factor (NSF) attachment protein (SNAP) receptor] present in the trans-Golgi network (TGN) and prevacuolar compartments (PVCs). Both interaction and subcellular localization studies suggest that McCAP2 may possibly serve as an adaptor protein responsible for vesicle-mediated trafficking of McCPK1 to or from the plasma membrane along actin microfilaments of the cytoskeleton. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Dimerization of Neu/Erb2 transmembrane domain is controlled by membrane curvature

Secondary structures of the proto-oncogenic Neu/ErbB2 transmembrane segment and its mutant analogue have been determined in phospholipids. It is found that the mutated peptide possesses less helical character possibly due to the valine/glutamic acid point mutation. Embedding peptides in lipid systems whose topology can change from small (100-200 A) tumbling objects to bilayer discs of 450 A diameter leads to the finding that coiled-coil interactions are only observed in the presence of a bilayer membrane of low curvature, independent of mutation. This strongly suggests that any event that may change membrane topology can therefore perturb the dimerization/ologomerization and subsequent phosphorylation cascade leading to cell growth or cancer processes.     

Emergence of structure through protein–protein interactions and pH changes in dually predicted coiled-coil and disordered regions of centrosomal proteins

Human centrosomal proteins show a significant, 3.5 fold, bias to be both unstructured and coiled-coils with respect to generic human proteins, based on results from state of the art bioinformatics tools. We hypothesize that this bias means that these proteins adopt an ensemble of disordered and partially helical conformations, with the latter becoming stabilized when these proteins form complexes. Characterization of the structural properties of 13 peptides from 10 different centrosomal proteins ranging in size from 20 to 61 residues by biophysical methods led us to confirm our hypothesis in most cases. Interestingly, the secondary structure adopted by most of these peptides becomes stabilized at acidic pH and it is concentration dependent. For two of them, PIK3R1^453–513 and BRCA1^1253–1273, we observed not only the stabilization of helical structure through self-association, but also the presence of β-structures linked to the formation of high molecular weight oligomers. These oligomers are the predominant forms detected by CD, but unobservable by liquid state NMR. BRCA1^1397–1424 and MAP3K11^396–441 populate helical structures that can also self-associate at pH 3 through oligomeric species. Four peptides, derived from three proteins, namely CCNA2^103–123, BRCA1^1253–1273, BRCA1^1397–1424 and PIK3R1^453–513, can form intermolecular associations that are concomitant with alpha or beta structure stabilization. The self-phosphorylation previously described for the kinase NEK2 did not lead to any stabilization in the peptide's structure of NEK2^303–333, NEK2^341–361, and NEK2^410–430. Based on these results, obtained from a series of peptides derived from a significant number of different centrosomal proteins, we propose that conformational polymorphism, modulated by intermolecular interactions is a general property of centrosomal proteins.     

Structure-function analysis of GNIP, the glycogenin-interacting protein

Glycogenin is a self-glucosylating protein that initiates glycogen biosynthesis. We recently identified a family of proteins, GNIPs, that interact with glycogenin and stimulate its self-glucosylating activity [J. Biol. Chem. 277 (2002) 19331]. The GNIP gene (also called TRIM7) encodes at least four distinct isoforms of GNIP, three of which (GNIP1, GNIP2, and GNIP3) have in common a COOH-terminal B30.2 domain and predicted coiled-coil regions. Based on Western blot analysis, the GNIP1 protein is widely distributed in tissues. From analysis of a series of deletion mutants of GNIP2 using the yeast two-hybrid system, the B30.2 domain was found to be responsible for the interaction with glycogenin. A truncated form of recombinant GNIP2, lacking the NH₂-terminal coiled-coil region, was cross-linked to glycogenin by glutaraldehyde treatment, supporting the idea that the B30.2 domain was sufficient for the interaction. In the course of this study, GNIP2 was also found to interact with itself, via the coiled-coil domain. Heterologous interactions between GNIP1 and GNIP2 were also detected. Since glycogenin is also a dimer, higher order multimeric complexes between glycogenin and GNIPs would be possible.     

Crystal structure of the NEMO ubiquitin-binding domain in complex with Lys 63-linked di-ubiquitin

NEMO is essential for activation of the NF-κB signaling pathway, which is regulated by ubiquitination of proteins. The C-terminal leucine zipper of NEMO and its adjacent coiled-coil region (CC2-LZ) reportedly bind to linear ubiquitin chains with 1 μM affinity and to Lys 63-linked chains with 100 μM affinity. Here we report the crystal structure of the CC2-LZ region of mouse NEMO in complex with Lys 63-linked di-ubiquitin (K63-Ub₂) at 2.7 Å resolution. The ubiquitin-binding region consists of a 130 Å-long helix and forms a parallel coiled-coil dimer. The Ile 44-centered hydrophobic patch of ubiquitin is recognized in the middle of the NEMO ubiquitin-binding region. NEMO interacts with each K63-Ub₂via a single ubiquitin-binding site, consistent with low affinity binding with K63-Ub₂.

Structured summary

MINT-7262681: NEMO (uniprotkb:O88522) binds (MI:0407) to Ubiquitin (uniprotkb:P62991) by pull down (MI:0096)MINT-7262667: Ubiquitin (uniprotkb:P62991) and NEMO (uniprotkb:O88522) bind (MI:0407) by X-ray crystallography (MI:0114)     

The thermostability of an α-helical coiled-coil protein and its potential use in sensor applications

Coiled-coil proteins are assemblies of two to four α-helices that pack together in a parallel or anti-parallel fashion. Coiled-coil structures can confer a variety of functional capabilities, which include enabling proteins, such as myosin, to function in the contractile apparatus of muscle and non-muscle cells. The TlpA protein encoded by the virulence plasmid of Salmonella is an α-helical protein that forms an elongated coiled-coil homodimer. A number of studies have clearly established the role of TlpA as a temperature-sensing gene regulator, however the potential use of a TlpA in a thermo-sensor application outside of the organism has not been exploited. In this paper, we demonstrate that TlpA has several characteristics that are common with α-helical coiled-coils and its thermal folding and unfolding is reversible and rapid. TlpA is extremely sensitive to changes in temperature. We have also compared the heat-stability of TlpA with other structurally similar proteins. Using a folding reporter, in which TlpA is expressed as a C-terminal fusion with green fluorescent protein (GFP), we were able to use fluorescence as an indicator of folding and unfolding of the fusion protein. Our results on the rapid conformational changes inherent in TlpA support the previous findings and we present here preliminary data on the use of a GFP-TlpA fusion protein as temperature sensor.