Simple Structural Motifs Research Articles

CC+ : A searchable database of validated coiled coils in PDB structures and AlphaFold2 models.

α-Helical coiled coils are common tertiary and quaternary elements of protein structure. In coiled coils, two or more α helices wrap around each other to form bundles. This apparently simple structural motif can generate many architectures and topologies. Coiled coil-forming sequences can be predicted from heptad repeats of hydrophobic and polar residues, hpphppp, although this is not always reliable. Alternatively, coiled-coil structures can be identified using the program SOCKET, which finds knobs-into-holes (KIH) packing between side chains of neighboring helices. SOCKET also classifies coiled-coil architecture and topology, thus allowing sequence-to-structure relationships to be garnered. In 2009, we used SOCKET to create a relational database of coiled-coil structures, CC+ , from the RCSB Protein Data Bank (PDB). Here, we report an update of CC+ following an update of SOCKET (to Socket2) and the recent explosion of structural data and the success of AlphaFold2 in predicting protein structures from genome sequences. With the most-stringent SOCKET parameters, CC+ contains ≈12,000 coiled-coil assemblies from experimentally determined structures, and ≈120,000 potential coiled-coil structures within single-chain models predicted by AlphaFold2 across 48 proteomes. CC+ allows these and other less-stringently defined coiled coils to be searched at various levels of structure, sequence, and side-chain interactions. The identified coiled coils can be viewed directly from CC+ using the Socket2 application, and their associated data can be downloaded for further analyses. CC+ is available freely at http://coiledcoils.chm.bris.ac.uk/CCPlus/Home.html. It will be updated automatically. We envisage that CC+ could be used to understand coiled-coil assemblies and their sequence-to-structure relationships, and to aid protein design and engineering.

An Encodable Scaffold for Sequence-Specific Recognition of Duplex RNA.

RNA, unlike DNA, folds into a multitude of secondary and tertiary structures. This structural diversity has impeded the development of ligands that can sequence-specifically target this biomolecule. We sought to develop ligands for double-stranded RNA (dsRNA) segments, which are ubiquitous in RNA tertiary structure. The major groove of double-stranded DNA is sequence-specifically recognized by a range of dimeric helical transcription factors, including the basic leucine zippers (bZIP) and basic helix-loop-helix (bHLH) proteins; however, such simple structural motifs are not prevalent in RNA-binding proteins. We interrogated the high-resolution structures of DNA and RNA to identify requirements for a helix fork motif to occupy dsRNA major grooves akin to dsDNA. Our analysis suggested that the rigidity and angle of approach of dimeric helices in bZIP/bHLH motifs are not ideal for the binding of dsRNA major grooves. This investigation revealed that the replacement of the leucine zipper motifs in bHLH proteins with synthetic crosslinkers would allow recognition of dsRNA. We show that a model bHLH DNA-binding motif does not bind dsRNA but can be reengineered as an RNA ligand. Based on this hypothesis, we rationally designed a miniature synthetic crosslinked helix fork (CHF) as a generalizable proteomimetic scaffold for targeting dsRNA. We evaluated several CHF constructs against a set of RNA and DNA hairpins to probe the specificity of the designed construct. Our studies reveal a new class of proteomimetics as an encodable platform for sequence-specific recognition of dsRNA.

An Encodable Scaffold for Sequence‐Specific Recognition of Duplex RNA

AbstractRNA, unlike DNA, folds into a multitude of secondary and tertiary structures. This structural diversity has impeded the development of ligands that can sequence‐specifically target this biomolecule. We sought to develop ligands for double‐stranded RNA (dsRNA) segments, which are ubiquitous in RNA tertiary structure. The major groove of double‐stranded DNA is sequence‐specifically recognized by a range of dimeric helical transcription factors, including the basic leucine zippers (bZIP) and basic helix‐loop‐helix (bHLH) proteins; however, such simple structural motifs are not prevalent in RNA‐binding proteins. We interrogated the high‐resolution structures of DNA and RNA to identify requirements for a helix fork motif to occupy dsRNA major grooves akin to dsDNA. Our analysis suggested that the rigidity and angle of approach of dimeric helices in bZIP/bHLH motifs are not ideal for the binding of dsRNA major grooves. This investigation revealed that the replacement of the leucine zipper motifs in bHLH proteins with synthetic crosslinkers would allow recognition of dsRNA. We show that a model bHLH DNA‐binding motif does not bind dsRNA but can be reengineered as an RNA ligand. Based on this hypothesis, we rationally designed a miniature synthetic crosslinked helix fork (CHF) as a generalizable proteomimetic scaffold for targeting dsRNA. We evaluated several CHF constructs against a set of RNA and DNA hairpins to probe the specificity of the designed construct. Our studies reveal a new class of proteomimetics as an encodable platform for sequence‐specific recognition of dsRNA.

Secondary Structure Formation in Hybrid Synthetic/Peptide Polymers: Insights from Molecular Dynamics Simulations

AbstractProteins and peptides exhibit an immense variety of structures, which are generally classified according to simple structural motifs (mainly α helices and β sheets). Considerable efforts have been invested in understanding the relationship between chemical structure (primary structure) of peptides and their spatial motifs (secondary structure). However, little is known about the possibility to interfere intentionally in these structural driving forces, for example, by inserting (short) artificial polymer chains in the peptide backbone. Structure formation on such hybrid synthetic/biochemical polymers is still an emerging field of research. Here, molecular dynamics simulations are used to illustrate the influence of inserted polyethylene segments on the secondary structure of several peptide homopolymers. A loss of structure of ≈50% when the peptide chain length drops to ten amino acids and a practically complete absence for even shorter peptide segments.

Stimuli responsive hierarchical assembly of supramolecular conductive fibers from coiled-coil building blocks.

Proteins and peptides provide inspiration for synthetic stimuli-responsive nanomaterials. In this work, the assembly mechanism of a de novo designed peptide that forms α-helical coiled-coil hexamer fibers was probed through point mutations of the amino acid sequence and varying assembly solution pH. Buffer pH affects multi-scale aspects of supramolecular assembly, including peptide monomer secondary structure and interactions between α-helical coiled-coil hexamer building blocks. These higher order assembly interfaces are formed by electrostatic networks between solvent-exposed polarizable side chains, as observed by cryo-electron microscopy. One of the resulting hierarchical structures was a novel assembly of α-helical cross coiled-coils that exhibits electrical conductivity. Here, we show that pH-gated assembly can be achieved with only one amino acid mutation. This work opens materials design for more stimuli-responsive and functional materials with simple pH-gating structural motifs.

Interpretable Deep-Learning Unveils Structure-Property Relationships in Polybenzenoid Hydrocarbons.

In this work, interpretable deep learning was used to identify structure-property relationships governing the HOMO-LUMO gap and the relative stability of polybenzenoid hydrocarbons (PBHs) using a ring-based graph representation. This representation was combined with a subunit-based perception of PBHs, allowing chemical insights to be presented in terms of intuitive and simple structural motifs. The resulting insights agree with conventional organic chemistry knowledge and electronic structure-based analyses and also reveal new behaviors and identify influential structural motifs. In particular, we evaluated and compared the effects of linear, angular, and branching motifs on these two molecular properties and explored the role of dispersion in mitigating the torsional strain inherent in nonplanar PBHs. Hence, the observed regularities and the proposed analysis contribute to a deeper understanding of the behavior of PBHs and form the foundation for design strategies for new functional PBHs.

An evolutionary history of the CoA-binding protein Nat/Ivy.

Nat/Ivy is a diverse and ubiquitous CoA-binding evolutionary lineage that catalyzes acyltransferase reactions, primarily converting thioesters into amides. At the heart of the Nat/Ivy fold is a phosphate-binding loop that bears a striking resemblance to that of P-loop NTPases-both are extended, glycine-rich loops situated between a β-strand and an α-helix. Nat/Ivy, therefore, represents an intriguing intersection between thioester chemistry, a putative primitive energy currency, and an ancient mode of phospho-ligand binding. Current evidence suggests that Nat/Ivy emerged independently of other cofactor-utilizing enzymes, and that the observed structural similarity-particularly of the cofactor binding site-is the product of shared constraints instead of shared ancestry. The reliance of Nat/Ivy on a β-α-β motif for CoA-binding highlights the extent to which this simple structural motif may have been a fundamental evolutionary "nucleus" around which modern cofactor-binding domains condensed, as has been suggested for HUP domains, Rossmanns, and P-loop NTPases. Finally, by dissecting the patterns of conserved interactions between Nat/Ivy families and CoA, the coevolution of the enzyme and the cofactor was analyzed. As with the Rossmann, it appears that the pyrophosphate moiety at the center of the cofactor predates the enzyme, suggesting that Nat/Ivy emerged sometime after the metabolite dephospho-CoA.

Evolution of cooperation with asymmetric social interactions

How cooperation emerges in human societies is both an evolutionary enigma and a practical problem with tangible implications for societal health. Population structure has long been recognized as a catalyst for cooperation because local interactions facilitate reciprocity. Analysis of population structure typically assumes bidirectional social interactions. But human social interactions are often unidirectional-where one individual has the opportunity to contribute altruistically to another, but not conversely-as the result of organizational hierarchies, social stratification, popularity effects, and endogenous mechanisms of network growth. Here we expand the theory of cooperation in structured populations to account for both uni- and bidirectional social interactions. Even though unidirectional interactions remove the opportunity for reciprocity, we find that cooperation can nonetheless be favored in directed social networks and that cooperation is provably maximized for networks with an intermediate proportion of unidirectional interactions, as observed in many empirical settings. We also identify two simple structural motifs that allow efficient modification of interaction directions to promote cooperation by orders of magnitude. We discuss how our results relate to the concepts of generalized and indirect reciprocity.

Prediction of Spin Density, Baird-Antiaromaticity, and Singlet-Triplet Energy Gap in Triplet-State Polybenzenoid Systems from Simple Structural Motifs.

Triplet-state aromaticity has been recently proposed as a strategy for designing functional organic electronic compounds, many of which are polycyclic aromatic systems. However, in many cases, the aromatic nature of the triplet state cannot be easily predicted. Moreover, it is often unclear how specific structural manipulations affect the electronic properties of the excited-state compounds. Herein, the relationship between the structure of polybenzenoid hydrocarbons (PBHs) and their spin-density distribution and aromatic character in the first triplet excited state is studied. Although a direct link is not immediately visible, classifying the PBHs according to their annulation sequence reveals regularities. Based on these, a set of guidelines is defined to qualitatively predict the location of spin and paratropicity and the singlet-triplet energy gap in larger PBHs, using only their smaller tri- and tetracyclic components, and subsequently tested on larger systems.

Cover Feature: Prediction of Spin Density, Baird‐Antiaromaticity, and Singlet–Triplet Energy Gap in Triplet‐State Polybenzenoid Systems from Simple Structural Motifs (Chem. Eur. J. 23/2021)

Structure, sequence, spin: The structures of polybenzenoid hydrocarbons are analyzed in terms of the sequence of tricyclic annulations, revealing regularities in the location of spin density and Baird-antiaromaticity. The two properties localize onto specific tri- and tetracyclic substructures according to a hierarchy: linear, zig-zag, curved. The same preferences are reflected in the relative energy of the T1 systems and in the singlet-triplet energy gaps. More information can be found in the Full Paper by R. Gershoni-Poranne et al. on page 6923.

The Associative Memory, Water Mediated, Structure and Energy Model (AWSEM)-Amylometer: Predicting Amyloid Propensity and Fibril Topology Using an Optimized Folding Landscape Model.

Amyloids are fibrillar protein aggregates with simple repeated structural motifs in their cores, usually β-strands but sometimes α-helices. Identifying the amyloid-prone regions within protein sequences is important both for understanding the mechanisms of amyloid-associated diseases and for understanding functional amyloids. Based on the crystal structures of seven cross-β amyloidogenic peptides with different topologies and one recently solved cross-α fiber structure, we have developed a computational approach for identifying amyloidogenic segments in protein sequences using the Associative memory, Water mediated, Structure and Energy Model (AWSEM). The AWSEM-Amylometer performs favorably in comparison with other predictors in predicting aggregation-prone sequences in multiple data sets. The method also predicts well the specific topologies (the relative arrangement of β-strands in the core) of the amyloid fibrils. An important advantage of the AWSEM-Amylometer over other existing methods is its direct connection with an efficient, optimized protein folding simulation model, AWSEM. This connection allows one to combine efficient and accurate search of protein sequences for amyloidogenic segments with the detailed study of the thermodynamic and kinetic roles that these segments play in folding and aggregation in the context of the entire protein sequence. We present new simulation results that highlight the free energy landscapes of peptides that can take on multiple fibril topologies. We also demonstrate how the Amylometer methodology can be straightforwardly extended to the study of functional amyloids that have the recently discovered cross-α fibril architecture.

Biphasic Supramolecular Self-Assembly of Ferric Ions and Tannic Acid across Interfaces for Nanofilm Formation.

Cell nanoencapsulation provides a chemical tool for the isolation and protection of living cells from harmful, and often lethal, external environments. Although several strategies are available to form nanometric films, most methods heavily rely on time-consuming multistep processes and are not biocompatible. Here, the interfacial supramolecular self-assembly and film formation of ferric ions (FeIII ) and tannic acid (TA) in biphasic systems is reported, where FeIII and TA come into contact each other and self-assemble across the interface of two immiscible phases. The interfacial nanofilm formation developed is simple, fast, and cytocompatible. Its versatility is demonstrated with various biphasic systems: hollow microcapsules, encasing microbial or mammalian cells, that are generated at the water-oil interface in a microfluidic device; a cytoprotective FeIII -TA shell that forms on the surface of the alginate microbead, which then entraps probiotic Lactobacillus acidophilus; and a pericellular FeIII -TA shell that forms on individual Saccharomyces cerevisiae. This biphasic interfacial reaction system provides a simple but versatile structural motif in materials science, as well as advancing chemical manipulability of living cells.

Irreversible Cysteine-Selective Protein Labeling Employing Modular Electrophilic Tetrafluoroethylation Reagents.

Fluoroalkylation reagents based on hypervalent iodine are widely used to transfer fluoroalkyl moieties to various nucleophiles. However, the transferred groups have so far been limited to simple structural motifs. We herein report a reagent featuring a secondary amine that can be converted to amide, sulfonamide, and tertiary amine derivatives in one step. The resulting reagents bear manifold functional groups, many of which would not be compatible with the original synthetic pathway. Exploiting this structural versatility and the known high reactivity toward thiols, the new-generation reagents were used in bioconjugation with an artificial retro-aldolase, containing an exposed cysteine and a reactive catalytic lysine. Whereas commercial reagents based on maleimide and iodoacetamide labeled both sites, the iodanes exclusively modified the cysteine residue. The study thus demonstrates that modular fluoroalkylation reagents can be used as tools for cysteine-selective bioconjugation.

Key Structural Motifs To Predict the Cage Topology in Endohedral Metallofullerenes

We show that the relative isomer stability of fullerene anions is essentially governed by a few simple structural motifs, requiring only the connectivity information between atoms. Relative energies of a large number of isomers of fullerene anions, C(2n)(q) (2n = 68-104; q = -2, -4, -6), can be satisfactorily reproduced by merely counting the numbers of seven kinds of hexagon-based motifs. The dependence of stability on these motifs varies with the charge state, which reflects the fact that the isomeric form of the carbon cage in endohedral metallofullerenes (EMFs) often differs from that in neutral empty fullerenes. The chemical origin of the stabilization differences between motifs is discussed on the basis of electronic and strain effects as well as aromaticity. On the basis of this simple model, the extraordinary abundance of the icosahedral C80 cage in EMFs can be easily understood. We also provide an explanation for why the well-known isolated pentagon rule is often violated in smaller EMFs. Finally, simple topological indices are proposed for quantitatively predicting the relative stability of fullerene anions, allowing a rapid determination of suitable hosting cages in EMFs by just counting three simple structural motifs.

Designing Covalently Linked Heterodimeric Four-Helix Bundles.

De novo design has proven a powerful methodology for understanding protein folding and function, and for mimicking or even bettering the properties of natural proteins. Extensive progress has been made in the design of helical bundles, simple structural motifs that can be nowadays designed with a high degree of precision. Among helical bundles, the four-helix bundle is widespread in nature, and is involved in numerous and fundamental processes. Representative examples are the carboxylate bridged diiron proteins, which perform a variety of different functions, ranging from reversible dioxygen binding to catalysis of dioxygen-dependent reactions, including epoxidation, desaturation, monohydroxylation, and radical formation. The "Due Ferri" (two-irons; DF) family of proteins is the result of a de novo design approach, aimed to reproduce in minimal four-helix bundle models the properties of the more complex natural diiron proteins, and to address how the amino acid sequence modulates their functions. The results so far obtained point out that asymmetric metal environments are essential to reprogram functions, and to achieve the specificity and selectivity of the natural enzymes. Here, we describe a design method that allows constructing asymmetric four-helix bundles through the covalent heterodimerization of two different α-helical harpins. In particular, starting from the homodimeric DF3 structure, we developed a protocol for covalently linking the two α2 monomers by using the Cu(I) catalyzed azide-alkyne cycloaddition. The protocol was then generalized, in order to include the construction of several linkers, in different protein positions. Our method is fast, low cost, and in principle can be applied to any couple of peptides/proteins we desire to link.

Simple structure, complex function

The four-helix bundle is a simple structural motif, widespread in nature, that is involved in numerous and fundamental processes. This portfolio is now expanded by the report of a four-helix bundle protein able to store copper for particulate methane monooxygenase, an enzyme that catalyzes methane oxidation.

Evolving Wonder-RNAs in a Test Tube

RNA is a captivating polymer: it is chemically simple yet functionally diverse. RNA has been sought out by Mother Nature to take on many important biological functions, from genetic information encoding and transmission, to enzymatic catalysis and molecular recognition, to regulation of gene expression. Along the way, Nature has produced so many interesting RNA species, and, perhaps out of necessity, given them different sizes. At one end of the spectrum are microRNAs made of merely *22 nucleotides, and at the other end is the human 28S ribosomal RNA composed of 5,070 nucleotides! Yet, most functional RNA molecules, including many ribozymes and riboswitches, are middle-ofthe-roaders having 50–200 nucleotides. As far as the complexity of the structure and function is concerned, the size of the RNA does matter. The most fitting example is the ribosome, which is designed to execute an essential and demanding chemical transformation in a highly organized manner. Nature has adequately dealt with this challenge by creating one of most spectacular macromolecular assemblies in cells. Among several dozen macromolecules used to construct the ribosome are two super-sized RNAs that contain a few thousand nucleotides. One of Nature’s most intriguing but unsolved mysteries is the evolution process behind the selection of such long functional RNA molecules. It has been postulated that the modern size of ribosomal RNAs is a consequence of amalgamation of smaller fragments (Clark 1987; Gray and Schnare 1996) and sequence comparison studies have suggested that some ribosomal RNA domains are indeed older than others (Gray and Schnare 1996; Bokov and Steinberg 2009). Evidently, Nature has found a way to patiently build a complex function through progressive, domain-by-domain construction of ‘‘wonder-RNAs’’. A distinct trait of Homo sapiens is to learn and apply. Based on the principle of Darwinian evolution, in vitro selection technique was devised in 1990 (Ellington and Szostak 1990; Tuerk and Gold 1990), and has since been used to create numerous functional RNA (or single-stranded DNA) molecules. Key to the success of each in vitro selection experiment is the simultaneous exploration of a vast sequence space covered by a random library containing as many as 10 RNA siblings. Although many fascinating functional nucleic acids have been derived by this approach, large and complex wonder-RNAs are few and far between (Bartel and Szostak 1993). This may simply be an inevitable outcome of two inherent drawbacks associated with the random library approach: the inefficient sequence space coverage and the tyranny of simple structural motifs. A standard DNA synthesis produces *0.1 mg of DNA (*10 molecules), just enough to cover the entire sequence space of a 25-nucleotide sequence (4 & 10). When the random domain increases to 50 nucleotides (still smaller than a tRNA), the sequence space rises to *10 (4 & 10), translating to *35,000 t of DNA! Thus, the sequence space coverage for a long RNA is categorically infinitesimal. Even if we assume there are complex RNAs in such a library, the chance of finding them is further diminished due to the overabundance of simple structural motifs in the library (Schlosser and Li 2005). Computational analysis has shown that random libraries are primarily populated with simple structural motifs like hairpin loops and three-way junctions and have extremely few more intricate structural motifs often found in large biological RNAs (Gevertz et al. 2005). Y. Li (&) Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4K1, Canada e-mail: liying@mcmaster.ca

Structures closed into cycles in globular proteins

Different types of structures closed into cycles are widespread at all the levels of structural organization of proteins. β-Hairpins, triple-stranded β-sheets, and βαβ-units represent simple structural motifs closed into cycles by systems of hydrogen bonds. Secondary closing of these simple motifs into larger cycles by means of different superhelices, split β-hairpins, or SS-bridges results in formation of complex structural motifs such as abcd-units, φ-motifs, five- and seven-segment α/β-motifs, etc. At the level of tertiary structure many proteins and domains fold into structures closed into cylinders. Apparently, closing the motifs and domains into cycles and cylinders results in formation of more cooperative and stable structures as compared with open ones, and this may be the reason for high frequencies of occurrence of the motifs in proteins.

Structural motifs are closed into cycles in proteins

β-Hairpins, triple-strand β-sheets and βαβ-units represent simple structural motifs closed into cycles by systems of hydrogen bonds. Secondary closing of these simple motifs into large cycles by means of different superhelices, split β-hairpins or SS-bridges results in the formation of more complex structural motifs having unique overall folds and unique handedness such as abcd-units, φ -motifs, five- and seven-segment α/β-motifs. Apparently, the complex structural motifs are more cooperative and stable and this may be one of the main reasons of high frequencies of occurrence of the motifs in proteins.

Exploring the folding energy landscape of a series of designed consensus tetratricopeptide repeat proteins

Repeat proteins contain short, tandem arrays of simple structural motifs (20-40 aa). These stack together to form nonglobular structures that are stabilized by short-range interactions from residues close in primary sequence. Unlike globular proteins, they have few, if any, long-range nonlocal stabilizing interactions. One ubiquitous repeat is the tetratricopeptide motif (TPR), a 34-aa helix-turn-helix motif. In this article we describe the folding kinetics of a series of 7 designed TPR proteins that are assembled from arraying identical designed consensus repeats (CTPRan). These range from the smallest 2-repeat protein to a large 10-repeat protein (approximately 350 aa). In particular, we describe how the energy landscape changes with the addition of repeat units. The data reveal that although the CTPRa proteins have low local frustration, their highly symmetric, modular native structure is reflected in their multistate kinetics of unfolding and folding. Moreover, although the initial folding of all CTPRan proteins involves a nucleus with similar solvent accessibility, their subsequent folding to the native structure depends directly on repeat number. This corresponds to an increasingly complex landscape that culminates in CTPRa10 populating a misfolded, off-pathway intermediate. These results extend our current understanding of the malleable folding pathways of repeat proteins and highlight the consequences of adding identical repeats to the energy landscape.