Secondary Structure Elements Research Articles

Crystal structure of human peptidylarginine deiminase type VI (PAD6) provides insights into its inactivity.

Human peptidylarginine deiminase isoform VI (PAD6), which is predominantly limited to cytoplasmic lattices in the mammalian oocytes in ovarian tissue, is essential for female fertility. It belongs to the peptidylarginine deiminase (PAD) enzyme family that catalyzes the conversion of arginine residues to citrulline in proteins. In contrast to other members of the family, recombinant PAD6 was previously found to be catalytically inactive. We sought to provide structural insight into the human homologue to shed light on this observation. We report here the first crystal structure of PAD6, determined at 1.7 Å resolution. PAD6 follows the same domain organization as other structurally known PAD isoenzymes. Further structural analysis and size-exclusion chromatography show that PAD6 behaves as a homodimer similar to PAD4. Differential scanning fluorimetry suggests that PAD6 does not coordinate Ca2+ which agrees with acidic residues found to coordinate Ca2+ in other PAD homologs not being conserved in PAD6. The crystal structure of PAD6 shows similarities with the inactive state of apo PAD2, in which the active site conformation is unsuitable for catalytic citrullination. The putative active site of PAD6 adopts a non-productive conformation that would not allow protein-substrate binding due to steric hindrance with rigid secondary structure elements. This observation is further supported by the lack of activity on the histone H3 and cytokeratin 5 substrates. These findings suggest a different mechanism for enzymatic activation compared with other PADs; alternatively, PAD6 may exert a non-enzymatic function in the cytoplasmic lattice of oocytes and early embryos.

A unique approach for protein secondary structure comparison under TOPS representation

To unravel the intricate connection between protein function and protein structure, it is imperative to comprehensively evaluate protein secondary structure similarity from various perspectives. While numerous techniques have been suggested for comparing protein secondary structure elements (SSE), there continues to be a substantial need for finding alternative ways of comparing the same. In this paper, Topology of Protein Structure (TOPS) representations of protein secondary structures are considered to offer a new alignment-free method for evaluating similarities/dissimilarities of protein secondary structures. Initially, a two-dimensional numerical representation of the SSE is created, associating each point with a mass reflecting its frequency of occurrence. Then the means of coordinate values are determined by averaging weighted sums, and these mean values are subsequently used to calculate moments-of-inertia. Next, a four-component descriptor is generated out of the eigenvalues of the matrix and the mean values of the represented coordinates. Thereafter, Manhattan distance measure is used to obtain the distance matrix. This is finally applied to obtain the phylogenetic trees under the use of NJ method. SSE considered in the proposed method comprises 36-elements from the Chew-Kedem database giving five different taxa: globin, alpha-beta, tim-barrel, beta, and alpha. Phylogenetic trees were created for these SSE through the application of various methods: Clustal-Omega, LZ-Complexity, SED, TOPS + and TOC, to facilitate comparative analysis. Phylogenetic tree of the proposed method outperformed results of the previous methods when applied to the same SSE. Therefore, the method effectively constructs phylogenetic tree for analyzing protein secondary structure comparison. Communicated by Ramaswamy H. Sarma

Large-scale structure-informed multiple sequence alignment of proteins with SIMSApiper.

SIMSApiper is a Nextflow pipeline that creates reliable, structure-informed MSAs of thousands of protein sequences faster than standard structure-based alignment methods. Structural information can be provided by the user or collected by the pipeline from online resources. Parallelization with sequence identity-based subsets can be activated to significantly speed up the alignment process. Finally, the number of gaps in the final alignment can be reduced by leveraging the position of conserved secondary structure elements. The pipeline is implemented using Nextflow, Python3, and Bash. It is publicly available on github.com/Bio2Byte/simsapiper.

Remote loop evolution reveals a complex biological function for chitinase enzymes beyond the active site

Loops are small secondary structural elements that play a crucial role in the emergence of new enzyme functions. However, the evolutionary molecular mechanisms how proteins acquire these loop elements and obtain new function is poorly understood. To address this question, we study glycoside hydrolase family 19 (GH19) chitinase—an essential enzyme family for pathogen degradation in plants. By revealing the evolutionary history and loops appearance of GH19 chitinase, we discover that one loop which is remote from the catalytic site, is necessary to acquire the new antifungal activity. We demonstrate that this remote loop directly accesses the fungal cell wall, and surprisingly, it needs to adopt a defined structure supported by long-range intramolecular interactions to perform its function. Our findings prove that nature applies this strategy at the molecular level to achieve a complex biological function while maintaining the original activity in the catalytic pocket, suggesting an alternative way to design new enzyme function.

The Inhibition of Gag-Pol Expression by the Restriction Factor Shiftless Is Dispensable for the Restriction of HIV-1 Infection.

The interferon-induced host cell protein Shiftless (SFL) inhibits -1 programmed ribosomal frameshifting (-1PRF) required for the expression of HIV-1 Gal-Pol and the formation of infectious HIV-1 particles. However, the specific regions in SFL required for antiviral activity and the mechanism by which SFL inhibits -1PRF remain unclear. Employing alanine scanning mutagenesis, we found that basic amino acids in the predicted zinc ribbon motif of SFL are essential for the suppression of Gag-Pol expression but dispensable for anti-HIV-1 activity. We have shown that SFL inhibits the expression of the murine leukemia virus (MLV) Gag-Pol polyprotein and the formation of infectious MLV particles, although Gag-Pol expression of MLV is independent of -1PRF but requires readthrough of a stop codon. These findings indicate that SFL might inhibit HIV-1 infection by more than one mechanism and that SFL might target programmed translational readthrough as well as -1PRF signals, both of which are regulated by mRNA secondary structure elements.

Backbone chemical shift and secondary structure assignments for mouse siderocalin

The lipocalin protein family is a structurally conserved group of proteins with a variety of biological functions defined by their ability to bind small molecule ligands and interact with partner proteins. One member of this family is siderocalin, a protein found in mammals. Its role is discussed in inflammatory processes, iron trafficking, protection against bacterial infections and oxidative stress, cell migration, induction of apoptosis, and cancer. Though it seems to be involved in numerous essential pathways, the exact mechanisms are often not fully understood. The NMR backbone assignments for the human siderocalin and its rat ortholog have been published before. In this work we describe the backbone NMR assignments of siderocalin for another important model organism, the mouse - data that might become important for structure-based drug discovery. Secondary structure elements were predicted based on the assigned backbone chemical shifts using TALOS-N and CSI 3.0, revealing a high content of beta strands and one prominent alpha helical region. Our findings correlate well with the known crystal structure and the overall conserved fold of the lipocalin family.

Emergence of two distinct spatial folds in a pair of plant virus proteins encoded by nested genes

Virus genomes may encode overlapping or nested open reading frames that increase their coding capacity. It is not known whether the constraints on spatial structures of the two encoded proteins limit evolvability of nested genes. We examine the evolution of a pair of proteins, p22 and p19, encoded by nested genes in plant viruses from the genus Tombusvirus. The known structure of p19, a suppressor of RNA silencing, belongs to the RAGNYA fold from the alpha+beta class. The structure of p22, the cell-to-cell movement protein from the 30K family widespread in plant viruses, is predicted with the AlphaFold approach, suggesting a single jelly-roll fold core from the all-beta class, structurally similar to capsid proteins from plant and animal viruses. The nucleotide and codon preferences impose modest constraints on the types of secondary structures encoded in the alternative reading frames, nonetheless allowing for compact, well-ordered folds from different structural classes in two similarly-sized nested proteins. Tombusvirus p22 emerged through radiation of the widespread 30K family, which evolved by duplication of a virus capsid protein early in the evolution of plant viruses, whereas lineage-specific p19 may have emerged by stepwise increase in the length of the overprinted gene and incremental acquisition of functionally active secondary structure elements by the protein product. This evolution of p19 towards the RAGNYA fold represents one of the first documented examples of protein structure convergence in naturally occurring proteins.

Structural and physical basis for the elasticity of elastin.

Elastin function is to endow vertebrate tissues with elasticity so that they can adapt to local mechanical constraints. The hydrophobicity and insolubility of the mature elastin polymer have hampered studies of its molecular organisation and structure-elasticity relationships. Nevertheless, a growing number of studies from a broad range of disciplines have provided invaluable insights, and several structural models of elastin have been proposed. However, many questions remain regarding how the primary sequence of elastin (and the soluble precursor tropoelastin) governs the molecular structure, its organisation into a polymeric network, and the mechanical properties of the resulting material. The elasticity of elastin is known to be largely entropic in origin, a property that is understood to arise from both its disordered molecular structure and its hydrophobic character. Despite a high degree of hydrophobicity, elastin does not form compact, water-excluding domains and remains highly disordered. However, elastin contains both stable and labile secondary structure elements. Current models of elastin structure and function are drawn from data collected on tropoelastin and on elastin-like peptides (ELPs) but at the tissue level, elasticity is only achieved after polymerisation of the mature elastin. In tissues, the reticulation of tropoelastin chains in water defines the polymer elastin that bears elasticity. Similarly, ELPs require polymerisation to become elastic. There is considerable interest in elastin especially in the biomaterials and cosmetic fields where ELPs are widely used. This review aims to provide an up-to-date survey of/perspective on current knowledge about the interplay between elastin structure, solvation, and entropic elasticity.

Inhibitory potential of recombinant and native peanut Bowman-Birk inhibitor against proteases of human blood coagulation pathway

Plant proteinase/protease inhibitors (PIs) have therapeutic potential besides their role in regulating endogenous protease activity in several physiological processes. They exert their function by inhibiting the catalytic activity of specific proteases. The use of PIs for inhibiting coagulation factors and slowing down the blood coagulation process has emerged as a critical intervention point for developing antithrombotic agents. The present study has demonstrated the characterization of bacterially expressed recombinant peanut Bowman-Birk inhibitor (rPnBBI) and evaluation of its anticoagulant potential compared to native PnBBI (isoinhibitors pool) purified from peanuts. The full-length BBI gene was cloned, and rPnBBI (7.53 kDa) expressed in the soluble fraction of Escherichia coli BL21 (DE3) strain was isolated by passing through the trypsin affinity column. The purified rPnBBI with β-sheets as major secondary structural elements exhibited inhibitory activity towards bovine trypsin and chymotrypsin at diverse pH and temperatures. Both, rPnBBI and PnBBI showed anticoagulant effects by prolonging the activated partial thromboplastin time (aPTT) and inhibiting the activity of proteases/factors (plasma kallikrein, FXIIa, FXIa, FIXa, and FXa) involved in the blood coagulation pathway. Besides, rPnBBI and PnBBI showed a similar inhibitory tendency towards FXIIa, FXIa, and FIXa, while rPnBBI exhibited a higher inhibition potential than PnBBI towards FXa. Contrarily, PnBBI showed higher inhibitory potential than rPnBBI on plasma kallikrein. Altogether, the bioactive BBI molecules from the natural food source peanuts showed significant anticoagulant potential, which could be exploited in the prophylaxis of thromboembolic disorders associated with various human diseases.

Analysis of the inter-domain orientation of tandem RRM domains with diverse linkers: connecting experimental with AlphaFold2 predicted models.

The RNA recognition motif (RRM) is the most prevalent RNA binding domain in eukaryotes and is involved in most RNA metabolism processes. Single RRM domains have a limited RNA specificity and affinity and tend to be accompanied by other RNA binding domains, frequently additional RRMs that contribute to an avidity effect. Within multi-RRM proteins, the most common arrangement are tandem RRMs, with two domains connected by a variable linker. Despite their prevalence, little is known about the features that lead to specific arrangements, and especially the role of the connecting linker. In this work, we present a novel and robust way to investigate the relative domain orientation in multi-domain proteins using inter-domain vectors referenced to a stable secondary structure element. We apply this method to tandem RRM domains and cluster experimental tandem RRM structures according to their inter-domain and linker-domain contacts, and report how this correlates with their orientation. By extending our analysis to AlphaFold2 predicted structures, with particular attention to the inter-domain predicted aligned error, we identify new orientations not reported experimentally. Our analysis provides novel insights across a range of tandem RRM orientations that may help for the design of proteins with a specific RNA binding mode.

On the origin of optical rotation changes during the κ-carrageenan disorder-to-order transition

It is well established that solutions of both polymeric and oligomeric κ-carrageenan exhibit a clear change in optical rotation (OR), in concert with gel-formation for polymeric samples, as the solution is cooled in the presence of certain ions. The canonical interpretation – that this OR change reflects a ‘coil-to-helix transition’ in single chains – has seemed unambiguous; the solution- or ‘disordered’-state structure has ubiquitously been assumed to be a ‘random coil’, and the helical nature of carrageenan in the solid-state was settled in the 1970s. However, recent work has found that κ-carrageenan contains substantial helical secondary structure elements in the disordered-state, raising doubts over the validity of this interpretation. To investigate the origins of the OR, density-functional theory calculations were conducted using atomic models of κ-carrageenan oligomers. Changes were found to occur in the predicted OR owing purely to dimerization of chains, and - together with the additional effects of slight changes in conformation that occur when separated helical chains form double-helices - the predicted OR changes are qualitatively consistent with experimental results. These findings contribute to a growing body of evidence that the carrageenan ‘disorder-to-order’ transition is a cooperative process, and have further implications for the interpretation of OR changes demonstrated by macromolecules in general.

Folded Alpha Helical Putative New Proteins from Apilactobacillus kunkeei

The emergence of new proteins is a central question in biology. Most tertiary protein folds known to date appear to have an ancient origin, but it is clear from bioinformatic analyses that new proteins continuously emerge in all organismal groups. However, there is a paucity of experimental data on new proteins regarding their structure and biophysical properties. We performed a detailed phylogenetic analysis and identified 48 putative open reading frames in the honeybee-associated bacterium Apilactobacillus kunkeei for which no or few homologs could be identified in closely-related species, suggesting that they could be relatively new on an evolutionary time scale and represent recently evolved proteins. Using circular dichroism-, fluorescence- and nuclear magnetic resonance (NMR) spectroscopy we investigated six of these proteins and show that they are not intrinsically disordered, but populate alpha-helical dominated folded states with relatively low thermodynamic stability (0–3 kcal/mol). The NMR and biophysical data demonstrate that small new proteins readily adopt simple folded conformations suggesting that more complex tertiary structures can be continuously re-invented during evolution by fusion of such simple secondary structure elements. These findings have implications for the general view on protein evolution, where de novo emergence of folded proteins may be a common event.

NMR and Single-Molecule FRET Insights into Fast Protein Motions and Their Relation to Function.

Proteins often undergo large-scale conformational transitions, in which secondary and tertiary structure elements (loops, helices, and domains) change their structures or their positions with respect to each other. Simple considerations suggest that such dynamics should be relatively fast, but the functional cycles of many proteins are often relatively slow. Sophisticated experimental methods are starting to tackle this dichotomy and shed light on the contribution of large-scale conformational dynamics to protein function. In this review, we focus on the contribution of single-molecule Förster resonance energy transfer and nuclear magnetic resonance (NMR) spectroscopies to the study of conformational dynamics. We briefly describe the state of the art in each of these techniques and then point out their similarities and differences, as well as the relative strengths and weaknesses of each. Several case studies, in which the connection between fast conformational dynamics and slower function has been demonstrated, are then introduced and discussed. These examples include both enzymes and large protein machines, some of which have been studied by both NMR and fluorescence spectroscopies.

Structural basis of DNA recognition of the Campylobacter jejuni CosR regulator.

Campylobacter jejuni has emerged as an antibiotic-resistant threat worldwide. CosR is an essential regulator for this bacterium and is important for Campylobacter adaptation to various stresses. Here, we describe the structural basis of CosR binding to target DNA as determined by cryo-electron microscopy and X-ray crystallography. Since CosR is a potential target for intervention, our studies may facilitate the development of novel therapeutics to combat C. jejuni infection.

Amino Acid Polymerization on Silica Surfaces.

The polymerization of unactivated amino acids (AAs) is an important topic because of its applications in various fields including industrial medicinal chemistry and prebiotic chemistry. Silica as a promoter for this reaction, is of great interest owing to its large abundance and low cost. The amide/peptide bond synthesis on silica has been largely demonstrated but suffers from a lack of knowledge regarding its reaction mechanism, the key parameters, and surface features that influence AA adsorption and reactivity, the selectivity of the reaction product, the role of water in the reaction, etc. The present review addresses these problems by summarizing experimental and modeling results from the literature and attempts to rationalize some apparent divergences in published results. After briefly presenting the main types of silica surface sites and other relevant macroscopic features, we discuss the different deposition procedures of AAs, whose importance is often neglected. We address the possible AA adsorption mechanisms including covalent grafting and H-bonding and show that they are highly dependent on silanol types and density. We then consider how the adsorption mechanisms determine the occurrence and outcome of AA condensation (formation of cyclic dimers or of long linear chains), and outline some recent results that suggest significant polymerization selectivity in systems containing several AAs, as well as the formation of specific elements of secondary structure in the growing polypeptide chains.

Biophysical characterization and insights into the oligomeric nature of CD2-associated protein.

Glomerular podocytes are specialized epithelial cells localized to the blood-urine interface of the kidney. Podocyte slit-diaphragm (SD), a size-and-charge-selective junction, is instrumental in blood ultrafiltration and the formation of protein-free urine. The SD consists of macromolecular complexes of several proteins, such as nephrin, podocin, and CD2-associated protein (CD2AP). CD2AP is an adapter protein and is considered to be crucial for the integrity of SD. Mutations in the SD proteins cause nephrotic syndrome (NS), characterized by proteinuria. SD proteins' structural features must be elucidated to understand the mechanism of proteinuria in NS. In this study, we expressed, purified, and biophysically characterized heterologously expressed human CD2AP. Codon-optimized human CD2AP was expressed in E. coli Rosetta cells. The recombinant protein was induced with 1 mM IPTG and purified by Ni-NTA affinity chromatography. Analytical size-exclusion chromatography, blue native-PAGE, circular dichroism, and fluorescence spectroscopy were performed to decipher the oligomeric nature, secondary structural content, and tertiary packing of CD2AP. Our analysis revealed that CD2AP adopts a predominantly disordered secondary structure despite exhibiting moderate tertiary packing, characterized by low helical and β-sheet content. CD2AP readily assembles into homo-oligomers, with octamers and tetramers constituting the primary population. Interestingly, the inherent flexibility of CD2AP's secondary structural elements appears resistant to thermal denaturation. Frameshift mutation (p.K579Efs*7) that leads to loss of the coiled-coil domain promotes aberrant oligomerization of CD2AP through SH3 domains. We successfully expressed full-length human CD2AP in a heterologous system, wherein the secondary structure of CD2AP is predominantly disordered. CD2AP can form higher-order oligomers, and the significance of these oligomers and the impact of mutations in the context of size-selective permeability of SD needs further investigation.

Essential dynamics of ubiquitin in water and in a natural deep eutectic solvent.

Natural deep eutectic solvents (NADESs) comprised of osmolytes are of interest as potential biomolecular (cryo)protectants. However, the way these solvents influence the structure and dynamics of biomolecules as well as the role of water remains poorly understood. We carried out principal component analysis of various secondary structure elements of ubiquitin in water and a betaine : glycerol : water (1 : 2 : ζ; ζ = 0, 1, 2, 5, 10, 20, 45) NADES, from molecular dynamics trajectories, to gain insight into the protein dynamics as it undergoes a transition from a highly viscous anhydrous to an aqueous environment. A crossover of the protein's essential dynamics at ζ ∼ 5, induced by solvent-shell coupled fluctuations, is observed, indicating that ubiquitin might (re)fold in the NADES upon water addition at ζ > ∼5. Further, in contrast to water, the anhydrous NADES preserves ubiquitin's essential modes at high temperatures explaining the protein's seemingly enhanced thermal stability.

The SKMT Algorithm: A method for assessing and comparing underlying protein entanglement.

We present fast and simple-to-implement measures of the entanglement of protein tertiary structures which are appropriate for highly flexible structure comparison. These are performed using the SKMT algorithm, a novel method of smoothing the Cα backbone to achieve a minimal complexity curve representation of the manner in which the protein's secondary structure elements fold to form its tertiary structure. Its subsequent complexity is characterised using measures based on the writhe and crossing number quantities heavily utilised in DNA topology studies, and which have shown promising results when applied to proteins recently. The SKMT smoothing is used to derive empirical bounds on a protein's entanglement relative to its number of secondary structure elements. We show that large scale helical geometries dominantly account for the maximum growth in entanglement of protein monomers, and further that this large scale helical geometry is present in a large array of proteins, consistent across a number of different protein structure types and sequences. We also show how these bounds can be used to constrain the search space of protein structure prediction from small angle x-ray scattering experiments, a method highly suited to determining the likely structure of proteins in solution where crystal structure or machine learning based predictions often fail to match experimental data. Finally we develop a structural comparison metric based on the SKMT smoothing which is used in one specific case to demonstrate significant structural similarity between Rossmann fold and TIM Barrel proteins, a link which is potentially significant as attempts to engineer the latter have in the past produced the former. We provide the SWRITHE interactive python notebook to calculate these metrics.

Glutamine-rich regions of the disordered CREB transactivation domain mediate dynamic intra- and intermolecular interactions.

The cyclic AMP response element (CRE) binding protein (CREB) is a transcription factor that contains a 280-residue N-terminal transactivation domain and a basic leucine zipper that mediates interaction with DNA. The transactivation domain comprises three subdomains, the glutamine-rich domains Q1 and Q2 and the kinase inducible activation domain (KID). NMR chemical shifts show that the isolated subdomains are intrinsically disordered but have a propensity to populate local elements of secondary structure. The Q1 and Q2 domains exhibit a propensity for formation of short β-hairpin motifs that function as binding sites for glutamine-rich sequences. These motifs mediate intramolecular interactions between the CREB Q1 and Q2 domains as well as intermolecular interactions with the glutamine-rich Q1 domain of the TATA-box binding protein associated factor 4 (TAF4) subunit of transcription factor IID (TFIID). Using small-angle X-ray scattering, NMR, and single-molecule Förster resonance energy transfer, we show that the Q1, Q2, and KID regions remain dynamically disordered in a full-length CREB transactivation domain (CREBTAD) construct. The CREBTAD polypeptide chain is largely extended although some compaction is evident in the KID and Q2 domains. Paramagnetic relaxation enhancement reveals transient long-range contacts both within and between the Q1 and Q2 domains while the intervening KID domain is largely devoid of intramolecular interactions. Phosphorylation results in expansion of the KID domain, presumably making it more accessible for binding the CBP/p300 transcriptional coactivators. Our study reveals the complex nature of the interactions within the intrinsically disordered transactivation domain of CREB and provides molecular-level insights into dynamic and transient interactions mediated by the glutamine-rich domains.

Location of S-nitrosylated cysteines in protein three-dimensional structures.

Although S-nitrosylation of cysteines is a common protein posttranslational modification, little is known about its three-dimensional structural features. This paper describes a systematic survey of the data available in the Protein Data Bank. Several interesting observations could be made. (1) As a result of radiation damage, S-nitrosylated cysteines (Snc) are frequently reduced, at least partially. (2) S-nitrosylation may be a protection against irreversible thiol oxidation; because the NO group of Snc is relatively accessible to the solvent, it may act as a cork to protect the sulfur atoms of cysteines from oxidation by molecular oxygen to sulfenic, sulfinic, and sulfonic acid; moreover, Snc are frequently found at the start or end of helices and strands and this might shield secondary structural elements from unfolding.