Structure of the molecular chaperone prefoldin: unique interaction of multiple coiled coil tentacles with unfolded proteins

Prefoldin (GimC) is a hexameric molecular chaperone complex built from two related classes of subunits and present in all eukaryotes and archaea. Prefoldin interacts with nascent polypeptide chains and, in vitro, can functionally substitute for the Hsp70 chaperone system in stabilizing non-native proteins for subsequent folding in the central cavity of a chaperonin. Here, we present the crystal structure and characterization of the prefoldin hexamer from the archaeum Methanobacterium thermoautotrophicum. Prefoldin has the appearance of a jellyfish: its body consists of a double beta barrel assembly with six long tentacle-like coiled coils protruding from it. The distal regions of the coiled coils expose hydrophobic patches and are required for multivalent binding of nonnative proteins.     

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 .     

MultiCoil: A program for predicting two-and three-stranded coiled coils

A new multidimensional scoring approach for identifying and distinguishing trimeric and dimeric coiled coils is implemented in the MultiCoil program. The program extends the two-stranded coiled-coil prediction program PairCoil to the identification of three-stranded coiled coils. The computations are based upon data gathered from a three-stranded coiled-coil database comprising 6,319 amino acid residues, as well as from the previously constructed two-stranded coiled-coil database. In addition to identifying coiled coils not predicted by the two-stranded database programs, MultiCoil accurately classifies the oligomerization states of known dimeric and trimeric coiled coils. Analysis of the MultiCoil scores provides insight into structural features of coiled coils, and yields estimates that 0.9% of all protein residues form three-stranded coiled coils and that 1.5% form two-stranded coiled coils. The MultiCoil program is available at http://theory.Ics.mit.edu/multicoil.     

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.     

Statistical analysis of intrahelical ionic interactions in alpha-helices and coiled coils

There are many controversies concerning whether ionic interactions in alpha-helices and coiled coils actually contribute to the stabilisation and formation of these structures. Here we used a statistical approach to probe this question. We extracted unique alpha-helical and coiled coil structures from the protein database and analysed the ionic interactions between positively and negatively charged residues. The ionic interactions were categorized according to the type, spacing and order of the residues involved. Separate datasets were produced depending on the number of alpha-helices in the coiled coils and the mutual orientation of the helices. We compared the frequency of residue configurations able to form ionic interactions with their probability to form the interaction. We found a correlation between the two variables in alpha-helices, antiparallel two-stranded coiled coils and parallel two-stranded coiled coils. This indicates that some ionic interactions are indeed important for the formation and stabilisation of alpha-helices and coiled coils. We concluded that the configurations, which have simultaneously a large probability to form the ionic interaction and a frequent occurrence, are those, which have the most stabilising effect. These are the 4RE, 3ER and 4ER interactions.     

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.     

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.     

Exploring alternate states and oligomerization preferences of coiled‐coils by de novo structure modeling

Homomeric coiled‐coils can self‐assemble into a wide range of structural states with different helix topologies and oligomeric states. In this study, we have combined de novo structure modeling with stability calculations to simultaneously predict structure and oligomeric states of homomeric coiled‐coils. For dimers an asymmetric modeling protocol was developed. Modeling without symmetry constraints showed that backbone asymmetry is important for the formation of parallel dimeric coiled‐coils. Collectively, our results demonstrate that high‐resolution structure of coiled‐coils, as well as parallel and antiparallel orientations of dimers and tetramers, can be accurately predicted from sequence. De novo modeling was also used to generate models of competing oligomeric states, which were used to compare stabilities and thus predict the native stoichiometry from sequence. In a benchmark set of 33 coiled‐coil sequences, forming dimers to pentamers, up to 70% of the oligomeric states could be correctly predicted. The calculations demonstrated that the free energy of helix folding could be an important factor for determining stability and oligomeric state of homomeric coiled‐coils. The computational methods developed here should be broadly applicable to studies of sequence‐structure relationships in coiled‐coils and the design of higher order assemblies with improved oligomerization specificity. Proteins 2015; 83:235–247. © 2014 Wiley Periodicals, Inc.     

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.     

Fifty years of coiled-coils and alpha-helical bundles: a close relationship between sequence and structure

alpha-Helical coiled coils are remarkable for the diversity of related conformations that they adopt in both fibrous and globular proteins, and for the range of functions that they exhibit. The coiled coils are based on a heptad (7-residue), hendecad (11-residue) or a related quasi-repeat of apolar residues in the sequences of the alpha-helical regions involved. Most of these, however, display one or more sequence discontinuities known as stutters or stammers. The resulting coiled coils vary in length, in the number of chains participating, in the relative polarity of the contributing alpha-helical regions (parallel or antiparallel), and in the pitch length and handedness of the supercoil (left- or right-handed). Functionally, the concept that a coiled coil can act only as a static rod is no longer valid, and the range of roles that these structures have now been shown to exhibit has expanded rapidly in recent years. An important development has been the recognition that the delightful simplicity that exists between sequence and structure, and between structure and function, allows coiled coils with specialized features to be designed de novo.     

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.     

FTIR-Spectroscopy of multistranded coiled coil proteins.

Coiled coils of different order were investigated using infrared (IR) spectroscopy. Recently, we demonstrated that dimeric coiled coils display unique vibrational spectra with at least three separable bands instead of only one band of a classical alpha-helix in the amide I region.This was attributed to a distortion of the helical structure by the supercoil bending, giving rise to bands that are not observed in the undistorted helix. Here, we investigated coiled coils forming trimers, tetramers, and pentamers. These higher order coiled coils, in general, possess larger superhelical pitches, resulting in a smaller helical distortion. We found that all coiled coils studied, including the native dimeric GCN4 leucine zipper and its variants leading to parallel trimers and tetramers as well as the rod portions of fibritin (parallel trimer), alpha-actinin (antiparallel spectrin type trimer), and COMP (parallel pentamer), displayed the typical three band pattern of the coiled coil amide I spectra. However, the separation of these three bands and their positional deviation from the classical alpha-helical band position was correlated to the extent of the helical distortion as reflected by the pitch values of the supercoils. The most pronounced spectral anomaly was found for the tropomyosin dimer with a reported helical pitch of 137 A, whereas the smallest spectral distortion was found for the pentameric COMP complex and the tetrameric leucine zipper mutant, both with a pitch of about 205 A.     

Generalized Crick equations for modeling noncanonical coiled coils

Crick envisaged the alpha-helical coiled coil to result from systematic bending of an alpha-helix such that every seventh residue was structurally equivalent, and he derived equations for the coordinates of the backbone atoms. Crick's predictions were vindicated experimentally and coiled-coil sequences were shown to have hydrophobic residues alternately spaced 3 and 4 residues apart. Nonetheless, in some coiled coils such canonical heptad repeats are interrupted by inserts of 3 or 4 residues generating decad and hendecad motifs. The supercoiling of the coiled coils varies with the sequence pattern, being left- or right-handed in purely heptad-based or hendecad-based motifs, respectively. To model coiled coils with a mixture of motifs, we describe how Crick's equations can be modified for cases where the pitch is not constant. Using the analogy of the bending of a beam, we took the tilt angle to change linearly with distance along the major helix and the pitch of a motif to be affected by neighboring motifs depending on the rigidity of the alpha-helical strands. We tested our approach by fitting the two-, three-, and four-stranded noncanonical coiled coils of GrpE, hemagglutinin, and tetrabrachion. The backbone atoms of the model and crystal structures agreed with root mean square deviations of <1.1 A.     

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."     

Computational assessment of folding energy landscapes in heterodimeric coiled coils

The coiled coil structural motif consists of alpha helices supercoiling around each other to form staggered knobs‐into‐holes packing. Such structures are deceptively simple, especially as they often can be described with parametric equations, but are known to exist in various conformations. Even the simplest systems, consisting of 2 monomers, can assemble into a wide range of states. They can form canonical as well as noncanonical coiled coils, be parallel or antiparallel, where helices associate with different degrees of shift, tilt, and rotation. Here, we investigate the energy landscape of heterodimeric coiled coils by carrying out de novo folding simulations starting from amino acid sequence. We folded a diverse set of 22 heterodimers and demonstrate that the approach is capable of identifying the atomic details in the experimental structure in the majority of cases. Our methodology also enables exploration of alternative states that can be accessible in solution beyond the experimentally determined structure. For many systems, we observe folding energy landscapes with multiple energy minima and several isoenergetic states. By comparing coiled coils from single domains and those extracted from larger proteins, we find that standalone coiled coils have deeper energy wells at the experimentally determined conformation. By folding the competing homodimeric states in addition to the heterodimers, we observe that the structural specificity towards the heteromeric state is often small. Taken together, our results demonstrate that de novo folding simulations can be a powerful tool to characterize structural specificity of coiled coils when coupled to assessment of energy landscapes.     

Conformational transition between four and five-stranded phenylalanine zippers determined by a local packing interaction

Alpha-helical coiled coils play a crucial role in mediating specific protein-protein interactions. However, the rules and mechanisms that govern helix-helix association in coiled coils remain incompletely understood. Here we have engineered a seven heptad "Phe-zipper" protein (Phe-14) with phenylalanine residues at all 14 hydrophobic a and d positions, and generated a further variant (Phe-14(M)) in which a single core Phe residue is substituted with Met. Phe-14 forms a discrete alpha-helical pentamer in aqueous solution, while Phe-14(M) folds into a tetrameric helical structure. X-ray crystal structures reveal that in both the tetramer and the pentamer the a and d side-chains interlock in a classical knobs-into-holes packing to produce parallel coiled-coil structures enclosing large tubular cavities. However, the presence of the Met residue in the apolar interface of the tetramer markedly alters its local coiled-coil conformation and superhelical geometry. Thus, short-range interactions involving the Met side-chain serve to preferentially select for tetramer formation, either by inhibiting a nucleation step essential for pentamer folding or by abrogating an intermediate required to form the pentamer. Although specific trigger sequences have not been clearly identified in dimeric coiled coils, higher-order coiled coils, as well as other oligomeric multi-protein complexes, may require such sequences to nucleate and direct their assembly.     

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.