高等植物的LRR蛋白:结构与功能

ＬＲＲ结构存在于细胞定位和功能上各不相同的多种蛋白质中,与蛋白质之间的相互作用和细胞内的信号传递过程密切相关。植物中含ＬＲＲ的蛋白主要有类受体蛋白激酶、抗病基因编码的蛋白和多聚半乳糖醛酸酶抑制蛋白等,它们分别在细胞的生长发育、抗病反应等过程中发挥着重要作用,其相似的ＬＲＲ结构为从分子水平上研究这些蛋白的作用机制提供了结构基础。     

The spectrin repeat: a structural platform for cytoskeletal protein assemblies

Spectrin repeats are three-helix bundle structures which occur in a large number of diverse proteins, either as single copies or in tandem arrangements of multiple repeats. They can serve structural purposes, by coordination of cytoskeletal interactions with high spatial precision, as well as a 'switchboard' for interactions with multiple proteins with a more regulatory role. We describe the structure of the alpha-actinin spectrin repeats as a prototypical example, their assembly in a defined antiparallel dimer, and the interactions of spectrin repeats with multiple other proteins. The alpha-actinin rod domain shares several features common to other spectrin repeats. (1) The rod domain forms a rigid connection between two actin-binding domains positioned at the two ends of the alpha-actinin dimer. The exact distance and rigidity are important, for example, for organizing the muscle Z-line and maintaining its architecture during muscle contraction. (2) The spectrin repeats of alpha-actinin have evolved to make tight antiparallel homodimer contacts. (3) The spectrin repeats are important interaction sites for multiple structural and signalling proteins. The interactions of spectrin repeats are, however, diverse and defy any simple classification of their preferred interaction sites, which is possible for other domains (e.g. src-homology domains 3 or 2). Nevertheless, the binding properties of the repeats perform important roles in the biology of the proteins where they are found, and lead to the assembly of complex, multiprotein structures involved both in cytoskeletal architecture as well as in forming large signal transduction complexes.     

Conservation of inter-residue interactions and prediction of folding rates of domain repeats

Domains are the main structural and functional units of larger proteins. They tend to be contiguous in primary structure and can fold and function independently. It has been observed that 10–20% of all encoded proteins contain duplicated domains and the average pairwise sequence identity between them is usually low. In the present study, we have analyzed the structural similarity between domain repeats of proteins with known structures available in the Protein Data Bank using structure-based inter-residue interaction measures such as the number of long-range contacts, surrounding hydrophobicity, and pairwise interaction energy. We used RADAR program for detecting the repeats in a protein sequence which were further validated using Pfam domain assignments. The sequence identity between the repeats in domains ranges from 20 to 40% and their secondary structural elements are well conserved. The number of long-range contacts, surrounding hydrophobicity calculations and pairwise interaction energy of the domain repeats clearly reveal the conservation of 3-D structure environment in the repeats of domains. The proportions of mainchain–mainchain hydrogen bonds and hydrophobic interactions are also highly conserved between the repeats. The present study has suggested that the computation of these structure-based parameters will give better clues about the tertiary environment of the repeats in domains. The folding rates of individual domains in the repeats predicted using the long-range order parameter indicate that the predicted folding rates correlate well with most of the experimentally observed folding rates for the analyzed independently folded domains.     

Structural characterization of the tumor suppressor p16, an ankyrin-like repeat protein.

The p16 protein has been identified as a tumor suppressor that functions by inhibiting the cyclin-dependent kinases CDK4 and CDK6. Deletions or point mutations in the p16 gene have been found in a number cancers, emphasizing its importance in regulating cell cycle progression. Inhibition by p16 occurs through protein-protein interactions with its targets. This is not surprising, since p16 is thought to contain ankyrin-like repeats, motifs implicated in protein-protein interactions. Our goal was to identify structural characteristics of p16 not only as an important step towards understanding CDK4 inhibition but also to explore the role of ankyrin repeats in the p16 structure, as no detailed structure of any protein containing these motifs has been reported. We have expressed, refolded, and purified p16 from E. coli and have shown it to be functionally active by specific binding to CDK4. Analytical ultracentrifugation has shown that p16 weakly self-associates to form dimers with a Kd = 270 microM. The CD spectrum indicates that the protein is composed of 33% alpha-helix, 22% beta-sheet, 19% beta-turn, and 27% other (which includes aromatic and random coil contributions). Further CD experiments suggest that p16 exhibits low structural stability with a delta G of -2.3 kcal/mol. This weak stability is a consequence of a highly dynamic structure as measured by ANS-binding, NMR hydrogen-deuterium exchange, and fluorescence. It is possible that a well-defined tertiary structure is imparted upon the binding of p16 to CDK4.     

Ankyrin: structure, properties, and functions

Recent data on characteristics of the structure, functions, and main properties of ankyrins (proteins that are linkers between the spectrin-based cytoskeleton and integral membrane proteins) are summarized. The interactions of ankyrins with band-3 protein, P-type ATPases, ion channels, receptors, and protein kinase C are considered. The structure of ankyrin repeats that are often contained in other proteins (which are not classified with the ankyrin family) and ensure protein-protein interactions as well as interactions between proteins and nucleic acids is described in details. The mechanisms of regulation of the ability of ankyrins to interact with other proteins (alternative splicing and post-translational modification, including phosphorylation) are also considered.     

A dominant negative mutant of 2-5A-dependent RNase suppresses antiproliferative and antiviral effects of interferon.

2-5A-dependent RNase is the terminal factor in the interferon-regulated 2-5A system thought to function in both the molecular mechanism of interferon action and in the general control of RNA stability. However, direct evidence for specific functions of 2-5A-dependent RNase has been generally lacking. Therefore, we developed a strategy to block the 2-5A system using a truncated form of 2-5A-dependent RNase which retains 2-5A binding activity while lacking RNase activity. When the truncated RNase was stably expressed to high levels in murine cells, it prevented specific rRNA cleavage in response to 2-5A transfection and the cells were unresponsive to the antiviral activity of interferon alpha/beta for encephalomyocarditis virus. Remarkably, cells expressing the truncated RNase were also resistant to the antiproliferative activity of interferon. The truncated RNase is a dominant negative mutant that binds 2-5A and that may interfere with normal protein-protein interactions through nine ankyrin-like repeats.     

CLIPR-59, a new trans-Golgi/TGN cytoplasmic linker protein belonging to the CLIP-170 family

The microtubule cytoskeleton plays a fundamental role in cell organization and membrane traffic in higher eukaryotes. It is well established that molecular motors are involved in membrane-microtubule interactions, but it has also been proposed that nonmotor microtubule-binding (MTB) proteins known as CLIPs (cytoplasmic linker proteins) have basic roles in these processes. We report here the characterization of CLIPR-59, a CLIP-170-related protein localized to the trans-most part of the Golgi apparatus. CLIPR-59 contains an acidic region followed by three ankyrin-like repeats and two CLIP-170-related MTB motifs. We show that the 60-amino acid-long carboxy-terminal domain of CLIPR-59 is necessary and sufficient to achieve Golgi targeting, which represents the first identification of a membrane targeting domain in a CLIP-170-related protein. The MTB domain of CLIPR-59 is functional because it localizes to microtubules when expressed as a fragment in HeLa cells. However, our results suggest that this domain is normally inhibited by the presence of adjacent domains, because neither full-length CLIPR-59 nor a CLIPR-59 mutant missing its membrane-targeting region localize to microtubules. Consistent with this observation, overexpression of CLIPR-59 does not affect the microtubule network. However, CLIPR-59 overexpression strongly perturbs early/recycling endosome-TGN dynamics, implicating CLIPR-59 in the regulation of this pathway.     

Sequential unfolding of ankyrin repeats in tumor suppressor p16

The ANK repeat is a ubiquitous 33-residue motif that adopts a beta hairpin helix-loop-helix fold. Multiple tandem repeats stack in a linear manner to produce an elongated structure that is stabilized predominantly by short-range interactions between residues close in sequence. The tumor suppressor p16(INK4) consists of four repeats and represents the minimal ANK folding unit. We found from Phi value analysis that p16 unfolded sequentially. The two N-terminal ANK repeats, which are distorted from the canonical ANK structure in all INK4 proteins and which are important for functional specificity, were mainly unstructured in the rate-limiting transition state for folding/unfolding, while the two C-terminal repeats were fully formed. A sequential unfolding mechanism could have implications for the cellular fate of wild-type and cancer-associated mutant p16 proteins.     

Structural domains required for channel function of the mouse transient receptor potential protein homologue TRP1beta

Transient receptor potential proteins (TRP) are supposed to participate in the formation of store-operated Ca(2+) influx channels by co-assembly. However, little is known which domains facilitate the interaction of subunits. Contribution of the N-terminal coiled-coil domain and ankyrin-like repeats and the putative pore region of the mouse TRP1beta (mTRP1beta) variant to the formation of functional cation channels were analyzed following overexpression in HEK293 (human embryonic kidney) cells. MTRP1beta expressing cells exhibited enhanced Ca(2+) influx and enhanced whole-cell membrane currents compared to mTRP1beta deletion mutants. Using a yeast two-hybrid assay only the coiled-coil domain facilitated homodimerization of the N-terminus. These results suggest that the N-terminus of mTRP1beta is required for structural organization thus forming functional channels.     

Evolution and function of CAG/polyglutamine repeats in protein-protein interaction networks

Expanded runs of consecutive trinucleotide CAG repeats encoding polyglutamine (polyQ) stretches are observed in the genes of a large number of patients with different genetic diseases such as Huntington's and several Ataxias. Protein aggregation, which is a key feature of most of these diseases, is thought to be triggered by these expanded polyQ sequences in disease-related proteins. However, polyQ tracts are a normal feature of many human proteins, suggesting that they have an important cellular function. To clarify the potential function of polyQ repeats in biological systems, we systematically analyzed available information stored in sequence and protein interaction databases. By integrating genomic, phylogenetic, protein interaction network and functional information, we obtained evidence that polyQ tracts in proteins stabilize protein interactions. This happens most likely through structural changes whereby the polyQ sequence extends a neighboring coiled-coil region to facilitate its interaction with a coiled-coil region in another protein. Alteration of this important biological function due to polyQ expansion results in gain of abnormal interactions, leading to pathological effects like protein aggregation. Our analyses suggest that research on polyQ proteins should shift focus from expanded polyQ proteins into the characterization of the influence of the wild-type polyQ on protein interactions.     

Common structural constituents confer I kappa B activity to NF-kappa B p105 and I kappa B/MAD-3.

The vertebrate NF-kappa B/c-rel inhibitors MAD-3/I kappa B alpha, I kappa B gamma/pdI and bcl-3 all share a conserved ankyrin repeat domain (ARD) consisting of six complete repeats, a short acidic motif and/or an incomplete seventh repeat. We present here a detailed analysis of the domain in p105/pdI and MAD-3/I kappa B involved in inhibition of DNA binding and in protein interaction with rel factors. We demonstrate that in both cases an acidic region and six ankyrin-like repeats are sufficient and required for protein interaction with the rel factors. However, for p105/pdI to achieve the high affinity needed to suppress DNA binding, an incomplete seventh repeat is required in addition. Both pdI and MAD-3 associate with rel proteins by forming heterotrimeric complexes, as shown by native gel analysis and by cross-linking. Furthermore, we demonstrate that deletion of only three amino acids in the first repeat converts the subunit specificity of the p105 ARD into that of MAD-3/I kappa B. We conclude that functionally the ARD in these molecules has a modular structure, with different subregions determining the specificity for the NF-kappa B subunits p50 and p65.     

The first ankyrin-like repeat is the minimum indispensable key structure for functional assembly of homo- and heteromeric TRPC4/TRPC5 channels

The closely related TRPC4 and TRPC5 proteins, members of the canonical transient receptor potential (TRPC) family, assemble into either homo- or heterotetrameric, non-selective cation-channels. To elucidate domains that mediate channel complex formation, we evaluated dominant negative effects of N- or C-terminal TRPC4/5 fragments on respective currents of full-length proteins overexpressed in HEK293 cells with whole-cell electrophysiology. Confocal F?rster Resonance Energy Transfer (FRET) measurements enabled to probe the interaction potential of these CFP/YFP-labelled fragments in vivo. Only N-terminal fragments that included the first ankyrin-like repeat potently down-regulated TRPC4/TRPC5 currents, while fragments including either the second ankyrin-like repeat and the coiled-coil domain or the C-terminus remained ineffective. Total internal reflection fluorescence (TIRF) microscopy data suggested that the dominant negative N-terminal fragments led to a predominantly intracellular localisation of coexpressed TRPC5 proteins. FRET measurements clearly revealed that only fragments including the first ankyrin-like repeat were able to multimerise. Moreover a TRPC5 mutant that lacked the first ankyrin-like repeat was unable to homo-multimerise, failed to interact with wild-type TRPC5 and resulted in non-functional channels.     

Structural analysis of the plakin domain of bullous pemphigoid antigen1 (BPAG1) suggests that plakins are members of the spectrin superfamily

Bullous pemphigoid antigen 1 (BPAG1) is a member of the plakin family of proteins. The plakins are multi-domain proteins that have been shown to interact with microtubules, actin filaments and intermediate filaments, as well as proteins found in cellular junctions. These interactions are mediated through different domains on the plakins. The interactions between plakins and components of specialized cell junctions such as desmosomes and hemidesmosomes are mediated through the so-called plakin domain, which is a common feature of the plakins. We report the crystal structure of a stable fragment from BPAG1, residues 226-448, defined by limited proteolysis of the whole plakin domain. The structure, determined by single-wavelength anomalous diffraction phasing from a selenomethionine-substituted crystal at 3.0 A resolution, reveals a tandem pair of triple helical bundles closely related to spectrin repeats. Based on this structure and analysis of sequence conservation, we propose that the architecture of plakin domains is defined by two pairs of spectrin repeats interrupted by a putative Src-Homology 3 (SH3) domain.     

The ANK repeat: a ubiquitous motif involved in macromolecular recognition

Many proteins rely on stable, noncovalent interactions with other macromolecules to perform their function. The identification of a repeated sequence motif, the ANK repeat, in diverse proteins whose common function involves binding to other proteins indicates one way nature may achieve a wide range of protein-protein interactions. In this article, we describe evidence that these ANK repeats are involved in the specific recognition of proteins and possibly DNA, and present a model for the folding of the motif.     

Expansion of protein domain repeats

Many proteins, especially in eukaryotes, contain tandem repeats of several domains from the same family. These repeats have a variety of binding properties and are involved in protein–protein interactions as well as binding to other ligands such as DNA and RNA. The rapid expansion of protein domain repeats is assumed to have evolved through internal tandem duplications. However, the exact mechanisms behind these tandem duplications are not well-understood. Here, we have studied the evolution, function, protein structure, gene structure, and phylogenetic distribution of domain repeats. For this purpose we have assigned Pfam-A domain families to 24 proteomes with more sensitive domain assignments in the repeat regions. These assignments confirmed previous findings that eukaryotes, and in particular vertebrates, contain a much higher fraction of proteins with repeats compared with prokaryotes. The internal sequence similarity in each protein revealed that the domain repeats are often expanded through duplications of several domains at a time, while the duplication of one domain is less common. Many of the repeats appear to have been duplicated in the middle of the repeat region. This is in strong contrast to the evolution of other proteins that mainly works through additions of single domains at either terminus. Further, we found that some domain families show distinct duplication patterns, e.g., nebulin domains have mainly been expanded with a unit of seven domains at a time, while duplications of other domain families involve varying numbers of domains. Finally, no common mechanism for the expansion of all repeats could be detected. We found that the duplication patterns show no dependence on the size of the domains. Further, repeat expansion in some families can possibly be explained by shuffling of exons. However, exon shuffling could not have created all repeats.