Ferredoxin-like Fold Research Articles

Cadherin-23, a protein essential for hearing, has an extracellular membrane-adjacent domain with a ferredoxin-like fold

Cadherin-23, a protein essential for hearing, has an extracellular membrane-adjacent domain with a ferredoxin-like fold

First 3-D structural evidence of a native-like intertwined dimer in the acylphosphatase family

Acylphosphatase (AcP, EC 3.6.1.7) is a small model protein conformed by a ferredoxin-like fold, profoundly studied to get insights into protein folding and aggregation processes. Numerous studies focused on the aggregation and/or amyloidogenic properties of AcPs suggest the importance of edge-β-strands in the process. In this work, we present the first crystallographic structure of Escherichia coli AcP (EcoAcP), showing notable differences with the only available NMR structure for this enzyme. EcoAcP is crystalised as an intertwined dimer formed by replacing a single C-terminal β-strand between two protomers, suggesting a flexible character of the C-terminal edge of EcoAcP. Despite numerous works where AcP from different sources have been used as a model system for protein aggregation, our domain-swapped EcoAcP structure is the first 3-D structural evidence of native-like aggregated species for any AcP reported to date, providing clues on molecular determinants unleashing aggregation.

Disrupting ACE2 Dimerization Mitigates the Infection by SARS-CoV-2 Pseudovirus

The coronavirus disease 2019 pandemic has caused over million death and 500 million reported cases globally. More effective antiviral medications are needed to curb the continued spread of this disease. The infection by SARS-CoV-2 virus is initiated via the interaction between the receptor binding domain (RBD) of the viral glycoprotein Spike (S protein) and the N-term peptidase domain of the angiotensin-converting enzyme 2 (ACE2) expressed on the host cell membrane. ACE2 forms a protein homodimer primarily through its ferredoxin-like fold domain (a.k.a., Neck-domain). We investigated whether the dimerization of ACE2 receptor plays a role in SARS-CoV-2 virus infection. We report here that the ACE2 receptor dimerization enhances the recognition of SARS-CoV-2 S protein. A 43-amino-acid peptide based on the N-term of Neck-domain could block the ACE2 dimerization and hence the interaction between RBD and ACE2 and mitigate the SARS-CoV-2 S protein pseudotyped virus/host cell interaction. Our study illustrated a new route to develop potential therapeutics for the prevention and treatment of SARS-CoV-2 viral infection.

Decoding an Amino Acid Sequence to Extract Information on Protein Folding.

Protein folding is a complicated phenomenon including various time scales (μs to several s), and various structural indices are required to analyze it. The methodologies used to study this phenomenon also have a wide variety and employ various experimental and computational techniques. Thus, a simple speculation does not serve to understand the folding mechanism of a protein. In the present review, we discuss the recent studies conducted by the author and their colleagues to decode amino acid sequences to obtain information on protein folding. We investigate globin-like proteins, ferredoxin-like fold proteins, IgG-like beta-sandwich fold proteins, lysozyme-like fold proteins and β-trefoil-like fold proteins. Our techniques are based on statistics relating to the inter-residue average distance, and our studies performed so far indicate that the information obtained from these analyses includes data on the protein folding mechanism. The relationships between our results and the actual protein folding phenomena are also discussed.

Crystal structure of a human MUC16 SEA domain reveals insight into the nature of the CA125 tumor marker

MUC16 is a membrane bound glycoprotein involved in the progression and metastasis of pancreatic and ovarian cancer. The protein is shed into the serum and the resulting cancer antigen 125 (CA125) can be detected by immunoassays. The CA125 epitope is used for monitoring ovarian cancer treatment progression, and has emerged as a potential target for antibody mediated immunotherapy. The extracellular tandem repeat domain of the protein is composed of repeating segments of heavily glycosylated sequence intermixed with homologous SEA (Sperm protein, Enterokinase and Agrin) domains. Here we report the purification and the first X-ray structure of a human MUC16 SEA domain. The structure was solved by molecular replacement using a Rosetta generated structure as a search model. The SEA domain reacted with three different MUC16 therapeutic antibodies, confirming that the CA125 epitope is localized to the SEA domain. The structure revealed a canonical ferredoxin-like fold, and contained a conserved disulfide bond. Analysis of the relative solvent accessibility of side chains within the SEA domain clarified the assignment of N-linked and O-linked glycosylation sites within the domain. A model of the glycosylated SEA domain revealed two major accessible faces, which likely represent the binding sites of CA125 specific antibodies. The results presented here will serve to accelerate future work to understand the functional role of MUC16 SEA domains and antibody recognition of the CA125 epitope.

Unveiling molecular details behind improved activity at neutral to alkaline pH of an engineered DyP-type peroxidase

DyP-type peroxidases (DyPs) are microbial enzymes that catalyze the oxidation of a wide range of substrates, including synthetic dyes, lignin-derived compounds, and metals, such as Mn2+ and Fe2+, and have enormous biotechnological potential in biorefineries. However, many questions on the molecular basis of enzyme function and stability remain unanswered. In this work, high-resolution structures of PpDyP wild-type and two engineered variants (6E10 and 29E4) generated by directed evolution were obtained. The X-ray crystal structures revealed the typical ferredoxin-like folds, with three heme access pathways, two tunnels, and one cavity, limited by three long loops including catalytic residues. Variant 6E10 displays significantly increased loops’ flexibility that favors function over stability: despite the considerably higher catalytic efficiency, this variant shows poorer protein stability compared to wild-type and 29E4 variants. Constant-pH MD simulations revealed a more positively charged microenvironment near the heme pocket of variant 6E10, particularly in the neutral to alkaline pH range. This microenvironment affects enzyme activity by modulating the pKa of essential residues in the heme vicinity and should account for variant 6E10 improved activity at pH 7–8 compared to the wild-type and 29E4 that show optimal enzymatic activity close to pH 4. Our findings shed light on the structure–function relationships of DyPs at the molecular level, including their pH-dependent conformational plasticity. These are essential for understanding and engineering the catalytic properties of DyPs for future biotechnological applications.

Structure and metal-binding properties of PA4063, a novel player in periplasmic zinc trafficking by Pseudomonas aeruginosa.

The capability to obtain essential nutrients in hostile environments is a critical skill for pathogens. Under zinc-deficient conditions, Pseudomonas aeruginosa expresses a pool of metal homeostasis control systems that is complex compared with other Gram-negative bacteria and has only been partially characterized. Here, the structure and zinc-binding properties of the protein PA4063, the first component of the PA4063-PA4066 operon, are described. PA4063 has no homologs in other organisms and is characterized by the presence of two histidine-rich sequences. ITC titration detected two zinc-binding sites with micromolar affinity. Crystallographic characterization, performed both with and without zinc, revealed an α/β-sandwich structure that can be classified as a noncanonical ferredoxin-like fold since it differs in size and topology. The histidine-rich stretches located at the N-terminus and between β3 and β4 are disordered in the apo structure, but a few residues become structured in the presence of zinc, contributing to coordination in one of the two sites. The ability to bind two zinc ions at relatively low affinity, the absence of catalytic cavities and the presence of two histidine-rich loops are properties and structural features which suggest that PA4063 might play a role as a periplasmic zinc chaperone or as a concentration sensor useful for optimizing the response of the pathogen to zinc deficiency.

The Status of Edge Strands in Ferredoxin-Like Fold

There is an opinion in professional literature that edge-strands in β-sheet are critical to the processes of amyloid transformation. Propagation of fibrillar forms mainly takes place on the basis of β-sheet type interactions. In many proteins, the edge strands represent only a partially matched form to the β-sheet. Therefore, the edge-strand takes slightly distorted forms. The assessment of the level of arrangement can be carried out based on studying the secondary structure as well as the structure of the hydrophobic core. For this purpose, a fuzzy oil drop model was used to determine the contribution of each fragment with a specific secondary structure to the construction of the system being the effect of a certain synergy, which results in the construction of a hydrophobic core. Studying the participation of β-sheets edge fragments in the hydrophobic core construction is the subject of the current analysis. Statuses of these edge fragments in β-sheets in ferredoxin-like folds are treated as factors that disturb the symmetry of the system.

SARS-CoV2 infectivity is potentially modulated by host redox status

The current coronavirus disease (COVID-19) outbreak caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV2) has emerged as a threat to global social and economic systems. Disparity in the infection of SARS-CoV2 among host population and species is an established fact without any clear explanation. To initiate infection, viral S-protein binds to the Angiotensin-Converting Enzyme 2 (ACE2) receptor of the host cell. Our analysis of retrieved amino acid sequences deposited in data bases shows that S-proteins and ACE2 are rich in cysteine (Cys) residues, many of which are conserved in various SARS-related coronaviruses and participate in intra-molecular disulfide bonds. High-resolution protein structures of S-proteins and ACE2 receptors highlighted the probability that two of these disulfide bonds are potentially redox-active, facilitating the primal interaction between the receptor and the spike protein. Presence of redox-active disulfides in the interacting parts of S-protein, ACE2, and a ferredoxin-like fold domain in ACE2, strongly indicate the role of redox in COVID-19 pathogenesis and severity. Resistant animals lack a redox-active disulfide (Cys133-Cys141) in ACE2 sequences, further strengthening the redox hypothesis for infectivity. ACE2 is a known regulator of oxidative stress. Augmentation of cellular oxidation with aging and illness is the most likely explanation of increased vulnerability of the elderly and persons with underlying health conditions to COVID-19.

Crystal structure of the Csm5 subunit of the type III-A CRISPR-Cas system

The Csm complex eliminates foreign RNA and DNA in the microbial defense CRISPR-Cas system. Csm5, one of the five subunits in the complex, facilitates crRNA maturation and target RNA binding in the type III system. However, the exact functional mechanism of Csm5 has remained elusive. Here, we report the crystal structure of the apo form of the Csm5 subunit at a resolution of 2.6 Å. Structural comparison of amino acids in the complex bound to RNA exhibits notable conformational changes in the crRNA and the target RNA binding sites. Shifts in the β-hairpin motif (β5-β6), α13 helix (resides 352-383), and G-rich loop (residues 335-337) in the C-terminal domain indicate an induced movement by crRNA binding. The positively charged residues (Lys 92, Arg 95 and Lys 96) located in the β-α4 loop of the target RNA interface show high conformational flexibility, while three-helix bundles (α1-α3) of the N-domain involved in Csm2 binding exhibit a rotational shift. The altered architecture of the Csm5 subunit demonstrates remarkable versatility of the ferredoxin-like fold in the RNA binding protein and provides a structural basis for the mechanism for crRNA and target RNA binding in the type III-A Crispr-Cas system.

Structure and functional implications of WYL domain-containing bacterial DNA damage response regulator PafBC

In mycobacteria, transcriptional activator PafBC is responsible for upregulating the majority of genes induced by DNA damage. Understanding the mechanism of PafBC activation is impeded by a lack of structural information on this transcription factor that contains a widespread, but poorly understood WYL domain frequently encountered in bacterial transcription factors. Here, we determine the crystal structure of Arthrobacter aurescens PafBC. The protein consists of two modules, each harboring an N-terminal helix-turn-helix DNA-binding domain followed by a central WYL and a C-terminal extension (WCX) domain. The WYL domains exhibit Sm-folds, while the WCX domains adopt ferredoxin-like folds, both characteristic for RNA-binding proteins. Our results suggest a mechanism of regulation in which WYL domain-containing transcription factors may be activated by binding RNA or other nucleic acid molecules. Using an in vivo mutational screen in Mycobacterium smegmatis, we identify potential co-activator binding sites on PafBC.

Structural characterization and heterologous expression of a new cyt gene cloned from Bacillus thuringiensis.

Bacillus thuringiensis (Bt) strains produce Cry (crystal) and Cyt (cytolytic) proteins belonging to the group of bacterial toxins known as pore-forming toxins (PFTs), which interact with midgut cells of target insects to create pores, disruption of ion homeostasis and eventual death. PFTs have synergistic insecticidal activities and have been used as biopesticides against agriculturally important insects. Identification of new Cyt proteins is important because of their specific toxicity towards hemipteran pests, against which the Cry proteins are not effective. We have structurally characterized a cyt (cyt1007) gene from an Indian Bt isolate SK-1007. The presence of a "Bacillus thuringiensis toxin" domain and maximum identity of 36% with Cyt2Ca in the deduced amino acid sequence indicated Cyt1007 protein to be a new member of Cyt family. Three dimensional (3D) modeling (PMDB ID: PM0081490) revealed that it adopts a typical ferredoxin-like fold, and is composed of a single domain of α/β architecture, in which a single β sheet is surrounded by two α helical layers. The putative lipid binding site and probable mode of action of Cyt1007 protein were predicted through comparative analysis with other Cyt toxins and their distant homologs Evf (Erwinia virulence factor) and VVA2 (Volvatoxin A2). Heterologous expression of cyt1007 gene as a 25kDa protein in Escherichia coli was achieved at high levels in both soluble and insoluble fractions. Affinity chromatography-based purification yielded 83.6% purified Cyt1007 protein, which can be used for downstream applications for the investigation of its toxicity. Graphical abstract Steps in the structural characterization and heterologous expression of a new cyt gene cloned from Bacillus thuringiensis.

Biosynthesis of the nickel-pincer nucleotide cofactor of lactate racemase requires a CTP-dependent cyclometallase

Bacterial lactate racemase is a nickel-dependent enzyme that contains a cofactor, nickel pyridinium-3,5-bisthiocarboxylic acid mononucleotide, hereafter named nickel-pincer nucleotide (NPN). The LarC enzyme from the bacterium Lactobacillus plantarum participates in NPN biosynthesis by inserting nickel ion into pyridinium-3,5-bisthiocarboxylic acid mononucleotide. This reaction, known in organometallic chemistry as a cyclometalation, is characterized by the formation of new metal-carbon and metal-sulfur σ bonds. LarC is therefore the first cyclometallase identified in nature, but the molecular mechanism of LarC-catalyzed cyclometalation is unknown. Here, we show that LarC activity requires Mn2+-dependent CTP hydrolysis. The crystal structure of the C-terminal domain of LarC at 1.85 Å resolution revealed a hexameric ferredoxin-like fold and an unprecedented CTP-binding pocket. The loss-of-function of LarC variants with alanine variants of acidic residues leads us to propose a carboxylate-assisted mechanism for nickel insertion. This work also demonstrates the in vitro synthesis and purification of the NPN cofactor, opening new opportunities for the study of this intriguing cofactor and of NPN-utilizing enzymes.

Seeing the light with BLUF proteins.

First described about 15years ago, BLUF (Blue Light Using Flavin) domains are light-triggered switches that control enzyme activity or gene expression in response to blue light, remaining activated for seconds or even minutes after stimulation. The conserved, ferredoxin-like fold holds a flavin chromophore that captures the light and somehow triggers downstream events. BLUF proteins are found in both prokaryotes and eukaryotes and have a variety of architectures and oligomeric forms, but the BLUF domain itself seems to have a well-preserved structure and mechanism that have been the focus of intense study for a number of years. Crystallographic and NMR structures of BLUF domains have been solved, but the conflicting models have led to considerable debate about the atomic details of photo-activation. Advanced spectroscopic and computational methods have been used to analyse the early events after photon absorption, but these too have led to widely differing conclusions. New structural models are improving our understanding of the details of the mechanism and may lead to novel tailor-made tools for optogenetics.

Crystal structure and biochemical features of dye-decolorizing peroxidase YfeX from Escherichia coli O157 Asp143 and Arg232 play divergent roles toward different substrates

YfeX from Escherichia coli O157 is a bacterial dye-decolorizing peroxidase that represents both dye-decoloring activity and typical peroxidase activity. We reported the crystal structure of YfeX bound to heme at 2.09 Å resolution. The YfeX monomer resembles a ferredoxin-like fold and contains two domains. The three conserved residues surrounding the heme group are His215, Asp143 and Arg232. His215 functions as the proximal axial ligand of the heme iron atom. Biochemical data show that the catalytic significance of the conserved Asp143 and Arg232 depends on the substrate types and that YfeX may adopt various catalytic mechanisms toward divergent substrates. In addition, it is observed that an access tunnel spans from the protein molecular surface to the heme distal region, it serves as the passageway for the entrance and binding of the H2O2.

An atypical segment swap in the DN and DC domains of the Acr_tran family resistance-nodulation-cell division pump.

Domain/segment swapping is an exchange of equivalent secondary structure element(s) among two or more protein domains resulting in the reconstitution of the original fold while simultaneously causing oligomerization. Here we report an example of the outer membrane factor docking region of the Acr_tran family (PF00873) resistance-nodulation-cell division pump, in which a swapped, misfolded state, of the ferredoxin-like fold of the DN and DC domains, effectuates oligomerization. The atypical segment swap and the associated displacement of a region of the ferredoxin-like fold leads to a topology that is distinct from the original fold. To our knowledge, such segment swaps and associated fold change are rare. This exemplifies the role of functional constraints including oligomerization that determine the interplay between sequence and the three-dimensional structure of proteins.

Compact Structure Patterns in Proteins

Globular proteins typically fold into tightly packed arrays of regular secondary structures. We developed a model to approximate the compact parallel and antiparallel arrangement of α-helices and β-strands, enumerated all possible topologies formed by up to five secondary structural elements (SSEs), searched for their occurrence in spatial structures of proteins, and documented their frequencies of occurrence in the PDB. The enumeration model grows larger super-secondary structure patterns (SSPs) by combining pairs of smaller patterns, a process that approximates a potential path of protein fold evolution. The most prevalent SSPs are typically present in superfolds such as the Rossmann-like fold, the ferredoxin-like fold, and the Greek key motif, whereas the less frequent SSPs often possess uncommon structure features such as split β-sheets, left-handed connections, and crossing loops. This complete SSP enumeration model, for the first time, allows us to investigate which theoretically possible SSPs are not observed in available protein structures. All SSPs with up to four SSEs occurred in proteins. However, among the SSPs with five SSEs, approximately 20% (218) are absent from existing folds. Of these unobserved SSPs, 80% contain two or more uncommon structure features. To facilitate future efforts in protein structure classification, engineering, and design, we provide the resulting patterns and their frequency of occurrence in proteins at: http://prodata.swmed.edu/ssps/.

Evolutionary relationships between heme-binding ferredoxin α + β barrels.

BackgroundThe α + β barrel superfamily of the ferredoxin-like fold consists of a functionally diverse group of evolutionarily related proteins. The barrel architecture of these proteins is formed by either homo-/hetero-dimerization or duplication and fusion of ferredoxin-like domains. Several members of this superfamily bind heme in order to carry out their functions.ResultsWe analyze the heme-binding sites in these proteins as well as their barrel topologies. Our comparative structural analysis of these heme-binding barrels reveals two distinct modes of packing of the ferredoxin-like domains to constitute the α + β barrel, which is typified by the Type-1/IsdG-like and Type-2/OxdA-like proteins, respectively. We examine the heme-binding pockets and explore the versatility of the α + β barrels ability to accommodate heme or heme-related moieties, such as siroheme, in at least three different sites, namely, the mode seen in IsdG/OxdA, Cld/DyP/EfeB/HemQ and siroheme decarboxylase barrels.ConclusionsOur study offers insights into the plausible evolutionary relationships between the two distinct barrel packing topologies and relate the observed heme-binding sites to these topologies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-1033-6) contains supplementary material, which is available to authorized users.

Crystal structures of CRISPR-associated Csx3 reveal a manganese-dependent deadenylation exoribonuclease

In prokaryotes, the CRISPR/Cas system is known to target and degrade invading phages and foreign genetic elements upon subsequent infection. However, the structure and function of many Cas proteins remain largely unknown, due to the high diversity of Cas proteins. Here we report 3 crystal structures of Archaeoglobus fulgidus Csx3 (AfCsx3) in free form, in complex with manganese ions and in complex with a single-stranded RNA (ssRNA) fragment, respectively. AfCsx3 harbors a ferredoxin-like fold and forms dimer both in the crystal and in solution. Our structure-based biochemical analysis demonstrates that the RNA binding sites and cleavage sites are located at 2 separate surfaces within the AfCsx3 dimer, suggesting a model to bind, tether and cleave the incoming RNA substrate. In addition, AfCsx3 displays robust 3′-deadenylase activity in the presence of manganese ions, which strongly suggests that AfCsx3 functions as a deadenylation exonuclease. Taken together, our results indicate that AfCsx3 is a Cas protein involved in RNA deadenylation and provide a framework for understanding the role of AfCsx3 in the Type III-B CRISPR/Cas system.

Structure and catalytic mechanism of the evolutionarily unique bacterial chalcone isomerase.

Flavonoids represent a large class of secondary metabolites produced by plants. These polyphenolic compounds are well known for their antioxidative abilities, are antimicrobial phytoalexins responsible for flower pigmentation to attract pollinators and, in addition to other properties, are also specific bacterial regulators governing the expression of Rhizobium genes involved in root nodulation (Firmin et al., 1986). The bacterial chalcone isomerase (CHI) from Eubacterium ramulus catalyses the first step in a flavanone-degradation pathway by ring opening of (2S)-naringenin to form naringenin chalcone. The structural biology and enzymology of plant CHIs have been well documented, whereas the existence of bacterial CHIs has only recently been elucidated. This first determination of the structure of a bacterial CHI provides detailed structural insights into the key step of the flavonoid-degradation pathway. The active site could be confirmed by co-crystallization with the substrate (2S)-naringenin. The stereochemistry of the proposed mechanism of the isomerase reaction was verified by specific (1)H/(2)H isotope exchange observed by (1)H NMR experiments and was further supported by mutagenesis studies. The active site is shielded by a flexible lid, the varying structure of which could be modelled in different states of the catalytic cycle using small-angle X-ray scattering data together with the crystallographic structures. Comparison of bacterial CHI with the plant enzyme from Medicago sativa reveals that they have unrelated folds, suggesting that the enzyme activity evolved convergently from different ancestor proteins. Despite the lack of any functional relationship, the tertiary structure of the bacterial CHI shows similarities to the ferredoxin-like fold of a chlorite dismutase and the stress-related protein SP1.