Coiled coils meet the chaperone world

Coiled coils are versatile structural modules that engage in a variety of cellular activities. Recent studies illuminate their role as substrate-binding elements in the chaperone cofactor prefoldin and in the AAA+ ATPases involved in protein (un)folding processes. The use of coiled coils to mediate the binding of non-native proteins represents a novel strategy in chaperone design and a new function for coiled coils.     

Facilitated release of substrate protein from prefoldin by chaperonin

Prefoldin is a chaperone that captures a protein-folding intermediate and transfers it to the group II chaperonin for correct folding. However, kinetics of interactions between prefoldin and substrate proteins have not been investigated. In this study, dissociation constants and dissociation rate constants of unfolded proteins with prefoldin were firstly measured using fluorescence microscopy. Our results suggest that binding and release of prefoldin from hyperthermophilic archaea with substrate proteins were in a dynamic equilibrium. Interestingly, the release of substrate proteins from prefoldin was facilitated when chaperonin was present, supporting a handoff mechanism of substrate proteins from prefoldin to the chaperonin.     

Functional characterization of recombinant prefoldin complexes from a hyperthermophilic archaeon, Thermococcus sp. strain KS-1

Prefoldin is a heterohexameric molecular chaperone complex that is found in the eukaryotic cytosol and also in archaea. It captures a nonnative protein and subsequently delivers it to a group II chaperonin for proper folding. Archaeal prefoldin is a heterocomplex containing two α subunits and four β subunits with the structure of a double β-barrel assembly, with six long coiled coils protruding from it like a jellyfish with six tentacles. We have studied the protein folding mechanism of group II chaperonin using those of Thermococcus sp. strain KS-1 (T. KS-1) because they exhibit high protein folding activity in vitro. We have also demonstrated functional cooperation between T. KS-1 chaperonins and prefoldin from Pyrococcus horikoshii OT3. Recent genome analysis has shown that Thermococcus kodakaraensis KOD1 contains two pairs of prefoldin subunit genes, correlating with the existence of two different chaperonin subunits. In this study, we characterized four different recombinant prefoldin complexes composed of two pairs of prefoldin subunits (α1, α2, β1, and β2) from T. KS-1. All of them (α1-β1, α2-β1, α1-β2, and α2-β2) exist as α₂β₄ heterohexamers and can protect several proteins from forming aggregates with different activities. We have also compared the collaborative activity between the prefoldin complexes and the cognate chaperonins. Prefoldin complexes containing the β1 subunit interacted with the chaperonins more strongly than those with the β2 subunit. The results suggest that Thermococcus spp. express different prefoldins for different substrates or conditions as chaperonins.     

蛋白质结构中卷曲螺旋的研究进展

卷曲螺旋 (coiledcoil)是存在于多种天然蛋白质中的结构模式 .近年来 ,通过对天然蛋白质中卷曲螺旋结构以及根据已有知识设计合成的卷曲螺旋结构的研究 ,已基本掌握了这类结构模式的特点 ,并将特异的卷曲螺旋结构应用于生化分析、工业、医药卫生等领域 .本文主要从天然蛋白质中卷曲螺旋的主要存在形式及其生物学功能、卷曲螺旋的主要结构特点、影响卷曲螺旋稳定性和结构特异性的因素、卷曲螺旋结构设计及其应用以及今后卷曲螺旋研究的主要发展方向等几个方面对近年来卷曲螺旋结构的研究进展情况进行了综述 .     

Location of disorder in coiled coil proteins is influenced by its biological role and subcellular localization: a GO-based study on human proteome

Intrinsic disorder in proteins has been explored to study lack of structure-function aspects of many proteins. The current study focuses on coiled coils which are often linked to intrinsic disorder. We present a sequence level analysis of human coiled coils to find out if this is universally true for all coiled coils. When annotated coiled-coil regions were collected from UniProt and investigated with disorder prediction tools namely-IUPred and DISpro, three patterns were commonly observed-disordered coiled coils (DisCCs), ordered coiled coils (OCCs) and the last one having a disordered region outside the coiled-coil region (DOCCs). Differential enrichment in the gene ontology was seen in these three categories. We found that OCCs are enriched in structural components of the extracellular space including the fibrinogen complex and laminin complex. On the contrary, DisCCs were found to be exclusively over-represented in proteins involved in actin filament, lamellipodium, cell junction, macromolecule complexes, ciliary rootlet and nucleolus. DOCCs are found to be associated with many regulatory and adaptor functions including positive regulation of calcium ion transport via store-operated calcium channel activity, cytoskeletal adaptor activity etc. Other than the GO-based analysis, sequence level analysis showed that disordered coiled-coil regions bear a high proportion of low-complexity regions as compared to ordered coiled coils. The former also has a higher probability of forming a dimer as compared to the ordered counterpart. Our study shows that the in silico approach of mapping of disorder in or around coiled coils in other biological systems or organisms can be applied to understand and rationalize the mode of action of these dynamic motifs.     

The Evolution and Structure Prediction of Coiled Coils across All Genomes

Coiled coils are α-helical interactions found in many natural proteins. Various sequence-based coiled-coil predictors are available, but key issues remain: oligomeric state and protein-protein interface prediction and extension to all genomes. We present SpiriCoil (http://supfam.org/SUPERFAMILY/spiricoil), which is based on a novel approach to the coiled-coil prediction problem for coiled coils that fall into known superfamilies: hundreds of hidden Markov models representing coiled-coil-containing domain families. Using whole domains gives the advantage that sequences flanking the coiled coils help. SpiriCoil performs at least as well as existing methods at detecting coiled coils and significantly advances the state of the art for oligomer state prediction. SpiriCoil has been run on over 16 million sequences, including all completely sequenced genomes (more than 1200), and a resulting Web interface supplies data downloads, alignments, scores, oligomeric state classifications, three-dimensional homology models and visualisation. This has allowed, for the first time, a genomewide analysis of coiled-coil evolution. We found that coiled coils have arisen independently de novo well over a hundred times, and these are observed in 16 different oligomeric states. Coiled coils in almost all oligomeric states were present in the last universal common ancestor of life. The vast majority of occasions that individual coiled coils have arisen de novo were before the last universal common ancestor of life; we do, however, observe scattered instances throughout subsequent evolutionary history, mostly in the formation of the eukaryote superkingdom. Coiled coils do not change their oligomeric state over evolution and did not evolve from the rearrangement of existing helices in proteins; coiled coils were forged in unison with the fold of the whole protein.     

Crystal structure of Skp, a prefoldin-like chaperone that protects soluble and membrane proteins from aggregation

The Seventeen Kilodalton Protein (Skp) is a trimeric periplasmic chaperone that assists outer membrane proteins in their folding and insertion into membranes. Here we report the crystal structure of Skp from E. coli. The structure of the Skp trimer resembles a jellyfish with alpha-helical tentacles protruding from a beta barrel body defining a central cavity. The architecture of Skp is unexpectedly similar to that of Prefoldin/GimC, a cytosolic chaperone present in eukaria and archea, that binds unfolded substrates in its central cavity. The ability of Skp to prevent the aggregation of model substrates in vitro is independent of ATP. Skp can interact directly with membrane lipids and lipopolysaccharide (LPS). These interactions are needed for efficient Skp-assisted folding of membrane proteins. We have identified a putative LPS binding site on the outer surface of Skp and propose a model for unfolded substrate binding.     

Subunit 1 of the prefoldin chaperone complex is required for lymphocyte development and function

Prefoldin is a hexameric chaperone that facilitates posttranslational folding of actins and other cytoskeletal proteins by the Tcp1-containing ring complex chaperonin, TriC. The present study characterized mice with a null mutation in Pfdn1, which encodes the first subunit of the Prefoldin complex. Pfdn1-deficient mice displayed phenotypes characteristic of defects in cytoskeletal function, including manifestations of ciliary dyskinesia, neuronal loss, and defects in B and T cell development and function. B and T cell maturation was markedly impaired at relatively early stages, namely at the transitions from pre-pro-B to pre-B cells in the bone marrow and from CD4-CD8- double-negative to CD4+CD8+ double-positive T cells in the thymus. In addition, mature B and T lymphocytes displayed cell activation defects upon Ag receptor cross-linking accompanied by impaired Ag receptor capping in B cells. These phenotypes illustrate the importance of cytoskeletal function in immune cell development and activation.     

Role of topological constraints in the all-or-none transition of a globular protein model: theory of the helix-coil transition in doubly crosslinked, coiled coils

Employing a recently developed statistical mechanical theory, the alpha-helix-to-random-coil transition in two-chain, coiled coils is shown to possess many of the essential qualitative features of the equilibrium folding process in globular proteins. The role of short vs. long range interactions in stabilizing the native structure is examined. We demonstrate in doubly crosslinked coiled coils how, due to the role of loop entropy, an intrinsically continuous conformational transition evolves into one well approximated by an all-or-none transition. Thus the present work points out the crucial role played by loop entropy in the conformational transition in coiled coils in particular and perhaps in globular proteins in general.     

Varied effects of Pyrococcus furiosus prefoldin and P. furiosus chaperonin on the refolding reactions of substrate proteins

Prefoldin is a molecular chaperone found in the archaeal and eukaryotic cytosol. Prefoldin can stabilize tentatively nascent polypeptide chains or non-native forms of mainly cytoskeletal proteins, which are subsequently delivered to group II chaperonin to accomplish their precise folding. However, the detailed mechanism is not well known, especially with regard to endogenous substrate proteins. Here, we report the effects of Pyrococcus furiosus prefoldin (PfuPFD) on the refolding reactions of Pyrococcus furiosus citrate synthase (PfuCS) and Aequorea enhanced green fluorescence protein (GFPuv) in the presence or absence of Pyrococcus furiosus chaperonin (PfuCPN). We confirmed that both PfuPFD and PfuCPN interacted with PfuCS and GFPuv refolding intermediates. However, the interactions between chaperone and substrate were different for each case, as was the final effect on the refolding reaction. Effects on the refolding reaction varied from passive effects such as ATP-dependent binding and release (PfuCPN towards GFPuv) and binding which leads to folding arrest (PfuPFD towards GFPuv), to active effects such as net increase in thermal stability (PfuCPN towards PfuCS) to an active improvement in refolding yield (PfuPFD towards PfuCS). We postulate that differences in molecular interactions between substrate and chaperone lead to these differences in chaperoning effects.     

Coiled‐coils: The long and short of it

Coiled‐coils are found in proteins throughout all three kingdoms of life. Coiled‐coil domains of some proteins are almost invariant in sequence and length, betraying a structural and functional role for amino acids along the entire length of the coiled‐coil. Other coiled‐coils are divergent in sequence, but conserved in length, thereby functioning as molecular spacers. In this capacity, coiled‐coil proteins influence the architecture of organelles such as centrioles and the Golgi, as well as permit the tethering of transport vesicles. Specialized coiled‐coils, such as those found in motor proteins, are capable of propagating conformational changes along their length that regulate cargo binding and motor processivity. Coiled‐coil domains have also been identified in enzymes, where they function as molecular rulers, positioning catalytic activities at fixed distances. Finally, while coiled‐coils have been extensively discussed for their potential to nucleate and scaffold large macromolecular complexes, structural evidence to substantiate this claim is relatively scarce.     

Divergent substrate-binding mechanisms reveal an evolutionary specialization of eukaryotic prefoldin compared to its archaeal counterpart

Prefoldin (PFD) is a molecular chaperone that stabilizes and then delivers unfolded proteins to a chaperonin for facilitated folding. The PFD hexamer has undergone an evolutionary change in subunit composition, from two PFDalpha and four PFDbeta subunits in archaea to six different subunits (two alpha-like and four beta-like subunits) in eukaryotes. Here, we show by electron microscopy that PFD from the archaeum Pyrococcus horikoshii (PhPFD) selectively uses an increasing number of subunits to interact with nonnative protein substrates of larger sizes. PhPFD stabilizes unfolded proteins by interacting with the distal regions of the chaperone tentacles, a mechanism different from that of eukaryotic PFD, which encapsulates its substrate inside the cavity. This suggests that although the fundamental functions of archaeal and eukaryal PFD are conserved, their mechanism of substrate interaction have diverged, potentially reflecting a narrower range of substrates stabilized by the eukaryotic PFD.     

Packing and hydrophobicity effects on protein folding and stability: effects of beta-branched amino acids, valine and isoleucine, on the formation and stability of two-stranded alpha-helical coiled coils/leucine zippers.

The aim of this study was to examine the differences between hydrophobicity and packing effects in specifying the three-dimensional structure and stability of proteins when mutating hydrophobes in the hydrophobic core. In DNA-binding proteins (leucine zippers), Leu residues are conserved at positions "d," and beta-branched amino acids, Ile and Val, often occur at positions "a" in the hydrophobic core. In order to discern what effect this selective distribution of hydrophobes has on the formation and stability of two-stranded alpha-helical coiled coils/leucine zippers, three Val or three Ile residues were simultaneously substituted for Leu at either positions "a" (9, 16, and 23) or "d" (12, 19, and 26) in both chains of a model coiled coil. The stability of the resulting coiled coils was monitored by CD in the presence of Gdn.HCl. The results of the mutations of Ile to Val at either positions "a" or "d" in the reduced or oxidized coiled coils showed a significant hydrophobic effect with the additional methylene group in Ile stabilizing the coiled coil (delta delta G values range from 0.45 to 0.88 kcal/mol/mutation). The results of mutations of Leu to Ile or Val at positions "a" in the reduced or oxidized coiled coils showed a significant packing effect in stabilizing the coiled coil (delta delta G values range from 0.59 to 1.03 kcal/mol/mutation). Our results also indicate the subtle control hydrophobic packing can have not only on protein stability but on the conformation adopted by the amphipathic alpha-helices. These structural findings correlate with the observation that in DNA-binding proteins, the conserved Leu residues at positions "d" are generally less tolerant of amino acid substitutions than the hydrophobic residues at positions "a."     

Structure and molecular dynamics simulation of archaeal prefoldin: the molecular mechanism for binding and recognition of nonnative substrate proteins

Prefoldin (PFD) is a heterohexameric molecular chaperone complex in the eukaryotic cytosol and archaea with a jellyfish-like structure containing six long coiled-coil tentacles. PFDs capture protein folding intermediates or unfolded polypeptides and transfer them to group II chaperonins for facilitated folding. Although detailed studies on the mechanisms for interaction with unfolded proteins or cooperation with chaperonins of archaeal PFD have been performed, it is still unclear how PFD captures the unfolded protein. In this study, we determined the X-ray structure of Pyrococcus horikoshii OT3 PFD (PhPFD) at 3.0 Å resolution and examined the molecular mechanism for binding and recognition of nonnative substrate proteins by molecular dynamics (MD) simulation and mutation analyses. PhPFD has a jellyfish-like structure with six long coiled-coil tentacles and a large central cavity. Each subunit has a hydrophobic groove at the distal region where an unfolded substrate protein is bound. During MD simulation at 330 K, each coiled coil was highly flexible, enabling it to widen its central cavity and capture various nonnative proteins. Docking MD simulation of PhPFD with unfolded insulin showed that the β subunit is essentially involved in substrate binding and that the α subunit modulates the shape and width of the central cavity. Analyses of mutant PhPFDs with amino acid replacement of the hydrophobic residues of the β subunit in the hydrophobic groove have shown that βIle107 has a critical role in forming the hydrophobic groove.     

Sequence divergence of coiled coils--structural rods, myosin filament packing, and the extraordinary conservation of cohesins

The amino acid sequences of the long, anti-parallel coiled coils of the cohesin subunits SMC1 and SMC3 are almost totally conserved in mammals. To understand this exceptional conservation more broadly, we analyzed amino acid sequence variation for several groups of coiled-coil proteins. Some long coiled coils, including giantin, NuMA, and Ndc80p/Nuf2p diverge approximately 20% from humans to rodents, suggesting they function as spacer rods, whose sequence divergence is constrained only by the need to maintain the coiled-coil structure. Other coiled coils such as skeletal muscle myosin, intermediate filaments, and the lamins diverge only 1-3%. We suggest that this sequence divergence is constrained by the extensive packing contacts over the entire surface of the coiled-coil. The coiled coils of SMC5/6 and SMC2/4 (condensin) are slightly more constrained than the presumed spacer rods, diverging 10-15%. Conversely, the coiled coils of SMC1/3 (cohesin) diverge only 0.0-1.0%. This extreme constraint suggests that the entire surface of the coiled coil is intimately involved in the mechanism of sister chromatid cohesion. Direct binding of the coiled coils to chromatin, or perhaps the need to avoid such binding, are two possible mechanisms. Finally, analysis of the heptad repeat shows that the a and d positions are more constrained in spacer rods, and the bcefg positions more constrained in skeletal muscle myosin.     

Unraveling double stranded alpha-helical coiled coils: an x-ray diffraction study on hard alpha-keratin fibers

Transformations of proteins secondary and tertiary structures are generally studied in globular proteins in solution. In fibrous proteins, such as hard alpha-keratin, that contain long and well-defined double stranded alpha-helical coiled coil domains, such study can be directly done on the native fibrous tissue. In order to assess the structural behavior of the coiled coil domains under an axial mechanical stress, wide angle x-ray scattering and small angle x-ray scattering experiments have been carried out on stretched horse hair fibers at relative humidity around 30%. Our observations of the three major axial spacings as a function of the applied macroscopic strain have shown two rates. Up to 4% macroscopic strain the coiled coils were slightly distorted but retained their overall conformation. Above 4% the proportion of coiled coil domains progressively decreased. The main and new result of our study is the observation of the transition from alpha-helical coiled coils to disordered chains instead of the alpha-helical coiled coil to beta-sheet transition that occurs in wet fibers.     

A filamentous molecular chaperone of the prefoldin family from the deep-sea hyperthermophile Methanocaldococcus jannaschii

Prefoldin is a molecular chaperone found in the domains eukarya and archaea that acts in conjunction with Group II chaperonin to correctly fold other nascent proteins. Previously, our group identified a putative single subunit of prefoldin, gamma PFD, that was up-regulated in response to heat stress in the hyperthermophilic archaeon Methanocaldococcus jannaschii. In order to characterize this protein, we subcloned and expressed it and the other two prefoldin subunits from M. jannaschii, alpha and beta PFD, into Eschericia coli and characterized the proteins. Whereas alpha and beta PFD readily assembled into the expected hexamer, gamma PFD would not assemble with either protein. Instead, gamma PFD forms long filaments of defined dimensions measuring 8.5 nm x 1.7-3.5 nm and lengths exceeding 1 microm. Filamentous gamma PFD acts as a molecular chaperone through in vitro assays, in a manner comparable to PFD. A possible molecular model for filament assembly is discussed.     

Phylogenetic occurrence of coiled coil proteins: Implications for tissue structure in metazoa via a coiled coil tissue matrix

We examined GenBank sequence files with a heptad repeat analysis program to assess the phylogenetic occurrence of coiled coil proteins, how heptad repeat domains are organized within them, and what structural/functional categories they comprise. Of 102,007 proteins analyzed, 5.95% (6,074) contained coiled coil domains; 1.26% (1,289) contained “extended” (> 75 amino acid) domains. While the frequency of proteins containing coiled coils was surprisingly constant among all biota, extended coiled coil proteins were fourfold more frequent in the animal kingdom and may reflect early events in the divergence of plants and animals. Structure/function categories of extended coils also revealed phylogenetic differences. In pathogens and parasites, many extended coiled coil proteins are external and bind host proteins. In animals, the majority of extended coiled coil proteins were identified as constituents of two protein categories: 1) myosins and motors; or 2) components of the nuclear matrix-intermediate filament scaffold. This scaffold, produced by sequential extraction of epithelial monolayers in situ, contains only 1–2% of the cell mass while accurately retaining morphological features of living epithelium and is greatly enriched in proteins with extensive, interrupted coiled coil forming domains. The increased occurrence of this type of protein in Metazoa compared with plants or protists leads us to hypothesize a tissue-wide matrix of coiled coil interactions underlying metazoan differentiated cell and tissue structure.