The Pentameric Ligand-Gated Ion Channel Family: A New Member of the Voltage Gated Ion Channel Superfamily?

In this report we present seven lines of bioinformatic evidence supporting the conclusion that the Pentameric Ligand-gated Ion Channel (pLIC) Family is a member of the Voltage-gated Ion Channel (VIC) Superfamily. In our approach, we used the Transporter Classification Database (TCDB) as a reference and applied a series of bioinformatic methods to search for similarities between the pLIC family and members of the VIC superfamily. These include: (1) sequence similarity, (2) compatibility of topology and hydropathy profiles, (3) shared domains, (4) conserved motifs, (5) similarity of Hidden Markov Model profiles between families, (6) common 3D structural folds, and (7) clustering analysis of all families. Furthermore, sequence and structural comparisons as well as the identification of a 3-TMS repeat unit in the VIC superfamily suggests that the sixth transmembrane segment evolved into a re-entrant loop. This evidence suggests that the voltage-sensor domain and the channel domain have a common origin. The classification of the pLIC family within the VIC superfamily sheds light onto the topological origins of this family and its evolution, which will facilitate experimental verification and further research into this superfamily by the scientific community.

Ligandability assessment of the C-terminal Rel-homology domain of NFAT1.

Transcription factors are generally considered challenging, if not "undruggable", targets but they promise new therapeutic options due to their fundamental involvement in many diseases. In this study, we aim to assess the ligandability of the C-terminal Rel-homology domain of nuclear factor of activated T cells 1 (NFAT1), a TF implicated in T-cell regulation. Using a combination of experimental and computational approaches, we demonstrate that small molecule fragments can indeed bind to this protein domain. The newly identified binder is the first small molecule binder to NFAT1 validated with biophysical methods and an elucidated binding mode by X-ray crystallography. The reported eutomer/distomer pair provides a strong basis for potential exploration of higher potency binders on the path toward degrader or glue modalities.

Validation of an indirect ELISA assay for assessment of autoantibodies against full-length TRIM21 and its individual domains.

Anti-SSA-autoantibodies are common in patients with rheumatologic disease, especially Sjögren's syndrome, systemic lupus erythematosus and rheumatoid arthritis. They consist of both autoantibodies towards Ro60 and Ro52, the latter also known as TRIM21. TRIM21 is an intracellular protein consisting of four domains; PRY/SPRY, Coiled-Coil, B-box and RING. The aim of this study was to establish an indirect ELISA detecting autoantibodies towards both the full-length TRIM21 protein and its four domains. We expressed the five constructs, created, and validated indirect ELISA protocols for each target using plasma from anti-SSA positive patients and healthy controls. Our findings were validated to the clinically used standards. We measured significantly higher levels of autoantibodies towards our full-length TRIM21, and the PRY/SPRY, Coiled-Coil and RING domains in patients compared to healthy controls. No significant difference in the level of autoantibodies were detected against the B-box domain. Our setups had a signal to noise ratio in the range of 30 to 184, and an OD between 2 and 3. Readings did not decline using NaCl of 500 mM as wash, affirming the high binding affinity of the autoantibodies measured. Our protocols allow us to further study the different autoantibodies of anti-SSA positive patients. This creates the possibility to stratify our patients into subgroups regarding autoantibody profile and specific pheno- or endotype.

An Assessment of Quaternary Structure Functionality in Homomer Protein Complexes.

It has been recently suggested that a significant fraction of homomer protein-protein interfaces evolve neutrally, without contributing to function, due to a hydrophobic bias in missense mutations. However, the fraction of such gratuitous complexes is currently unknown. Here, we quantified the fraction of homodimers where multimerization is unlikely to contribute to their biochemical function. We show that: 1) ligand binding-site structure predicts whether a homomer is functional or not; the vast majority of homodimers with multichain binding-sites (MBS) are likely to be functional, while in homodimers with single-chain binding-sites (SBS) and small to medium interfaces, quaternary structure is unlikely to be functional in a significant fraction-35%, even up to 42%-of complexes; 2) the hydrophobicity of interfaces changes little with the strength of selection, and the amino acid composition of interfaces is shaped by the "hydrophobic ratchet" in both types, but they are not in a strict equilibrium with mutations; particularly cysteines are much more abundant in mutations than in interfaces or surfaces; 3) in MBS homomers, the interfaces are conserved, while in a high fraction of SBS homomers, the interface is not more conserved than the solvent-accessible surface; and 4) MBS homomer interfaces coevolve more strongly with ligand binding sites than the interfaces of SBS homomers, and MBS complexes have higher capacity to transfer information from ligands across the interfaces than SBS homomers, explaining the enrichment of allostery in the former.

High-accuracy protein model quality assessment using attention graph neural networks.

Great improvement has been brought to protein tertiary structure prediction through deep learning. It is important but very challenging to accurately rank and score decoy structures predicted by different models. CASP14 results show that existing quality assessment (QA) approaches lag behind the development of protein structure prediction methods, where almost all existing QA models degrade in accuracy when the target is a decoy of high quality. How to give an accurate assessment to high-accuracy decoys is particularly useful with the available of accurate structure prediction methods. Here we propose a fast and effective single-model QA method, QATEN, which can evaluate decoys only by their topological characteristics and atomic types. Our model uses graph neural networks and attention mechanisms to evaluate global and amino acid level scores, and uses specific loss functions to constrain the network to focus more on high-precision decoys and protein domains. On the CASP14 evaluation decoys, QATEN performs better than other QA models under all correlation coefficients when targeting average LDDT. QATEN shows promising performance when considering only high-accuracy decoys. Compared to the embedded evaluation modules of predicted ${C}_{\alpha^{-}} RMSD$ (pRMSD) in RosettaFold and predicted LDDT (pLDDT) in AlphaFold2, QATEN is complementary and capable of achieving better evaluation on some decoy structures generated by AlphaFold2 and RosettaFold. These results suggest that the new QATEN approach can be used as a reliable independent assessment algorithm for high-accuracy protein structure decoys.

Complexes++: Efficient and versatile coarse-grained simulations of protein complexes and their dense solutions.

The interior of living cells is densely filled with proteins and their complexes, which perform multitudes of biological functions. We use coarse-grained simulations to reach the system sizes and time scales needed to study protein complexes and their dense solutions and to interpret experiments. To take full advantage of coarse-graining, the models have to be efficiently implemented in simulation engines that are easy to use, modify, and extend. Here, we introduce the Complexes++ simulation software to simulate a residue-level coarse-grained model for proteins and their complexes, applying a Markov chain Monte Carlo engine to sample configurations. We designed a parallelization scheme for the energy evaluation capable of simulating both dilute and dense systems efficiently. Additionally, we designed the software toolbox pycomplexes to easily set up complex topologies of multi-protein complexes and their solutions in different thermodynamic ensembles and in replica-exchange simulations, to grow flexible polypeptide structures connecting ordered protein domains, and to automatically visualize structural ensembles. Complexes++ simulations can easily be modified and they can be used for efficient explorations of different simulation systems and settings. Thus, the Complexes++ software is well suited for the integration of experimental data and for method development.

Markov State Modeling Analysis Captures Changes in the Temperature-Sensitive N-Terminal and ß-Turn Regions of the p53 DNA-Binding Domain.

The transcription factor p53 is one of the most widely studied cancer proteins. Its temperature-sensitive nature suggests reduction in functionality at physiological temperatures. Temperature-induced conformational variations and their impact on its functional ability still remain unexplored. A total of 20.8 µs molecular dynamics simulations of wildtype p53 in the apo and the DNA-bound states have been performed at 300 K and 310 K. Further, Markov State Modeling (MSM) analyses were performed, considering Cα-Cα distances as reaction coordinates. Filtering of these distances based on correlation with the time-independent components (tICs) resulted in 16 and 32 distances for apo and DNA-bound systems, respectively. Individual MSM analyses using these filtered distances were performed for both p53 systems. These Cα-Cα residue pairs belonged to the N-terminal, S6/7 ß-turn, loop L2, loop L3, and hydrophobic core residues. At physiological temperatures, apo-p53 exhibits exposure of its hydrophobic core, where the temperature-sensitive hotspot residues were also located. This exposure was the result of the S6/7 ß-turn and N-terminal moving apart. In the DNA-bound p53 system, loop L1 attains an open conformation at physiological temperatures, which weakens the DNA binding. It is already known that p53 mutants that lack DNA binding also tend to show similar conformational variations. The S6/7 ß-turn along with the already known functionally important loop L2 may pose as regions to be targeted to overcome the loss in binding of temperature-sensitive wildtype p53. Rescue strategies directed toward these temperature-sensitive regions may be useful to recuperate its strong binding at physiological temperatures.

Towards a generic prototyping approach for therapeutically-relevant peptides and proteins in a cell-free translation system.

Advances in peptide and protein therapeutics increased the need for rapid and cost-effective polypeptide prototyping. While in vitro translation systems are well suited for fast and multiplexed polypeptide prototyping, they suffer from misfolding, aggregation and disulfide-bond scrambling of the translated products. Here we propose that efficient folding of in vitro produced disulfide-rich peptides and proteins can be achieved if performed in an aggregation-free and thermodynamically controlled folding environment. To this end, we modify an E. coli-based in vitro translation system to allow co-translational capture of translated products by affinity matrix. This process reduces protein aggregation and enables productive oxidative folding and recycling of misfolded states under thermodynamic control. In this study we show that the developed approach is likely to be generally applicable for prototyping of a wide variety of disulfide-constrained peptides, macrocyclic peptides with non-native bonds and antibody fragments in amounts sufficient for interaction analysis and biological activity assessment.

Computational design of a neutralizing antibody with picomolar binding affinity for all concerning SARS-CoV-2 variants.

Coronavirus disease 2019, caused by SARS-CoV-2, remains an on-going pandemic, partly due to the emergence of variant viruses that can "break-through" the protection of the current vaccines and neutralizing antibodies (nAbs), highlighting the needs for broadly nAbs and next-generation vaccines. We report an antibody that exhibits breadth and potency in binding the receptor-binding domain (RBD) of the virus spike glycoprotein across SARS coronaviruses. Initially, a lead antibody was computationally discovered and crystallographically validated that binds to a highly conserved surface of the RBD of wild-type SARS-CoV-2. Subsequently, through experimental affinity enhancement and computational affinity maturation, it was further developed to bind the RBD of all concerning SARS-CoV-2 variants, SARS-CoV-1 and pangolin coronavirus with pico-molar binding affinities, consistently exhibited strong neutralization activity against wild-type SARS-CoV-2 and the Alpha and Delta variants. These results identify a vulnerable target site on coronaviruses for development of pan-sarbecovirus nAbs and vaccines.

Assessment of Scavenger Receptor Cysteine-Rich Domain Binding to Bacteria.

The scavenger receptor cysteine-rich SRCR domain is an ancient protein domain found in SR-A and SR-I scavenger receptors, which is characterized by a conserved arrangement of cysteines (Martinez et al., Pharmacol Rev 63(4):967-1000, 2011; Sarrias et al., Crit Rev Immunol 24(1):1-37, 2004; Telfer and Baldwin, Cell Immunol 296(1):76-86, 2015; PrabhuDas et al., J Immunol, 2017. 198(10):3775-3789). SRCR domains are divided into group A and group B SRCR domains by virtue of how many cysteines they contain and the resulting disulfide bonding pattern. Group B SRCR domains, found in WC1, CD163, CD5, CD6, Spα and DMBT1, are approximately 100-110 amino acids long and contain 6-8 cysteines predicted to form 3-4 disulfide bonds. The crystal structure of a CD5 group B SRCR domain predicts a fold of two beta-sheets and an alpha helix (Rodamilans et al., J Biol Chem 282(17):12669-12677, 2007; Wang et al., Mol Immunol 48:801-809, 2011). SRCR domains bind to many different types of chemical compounds found on cells, viruses, and microbes and are usually found in multiples in the extracellular domains of transmembrane proteins or in secreted proteins. Small amino acid differences between these SRCR domains lead to significant differences in binding affinity. In addition, SRCR domain genes contain allelic polymorphisms and can be extensively duplicated. Thus, single and duplicated SRCR domain protein gene loci encode a large tunable binding potential. Binding to pathogen-associated molecular patterns (PAMPs) combined with signaling potential predicts an important role for these molecules in the immune response. WC1 SRCR domains bind to the spirochetes Leptospira and Borrelia (Hsu et al., J Immunol 194(5):2280-2288, 2015). CD6 (Sarrias et al., Proc Natl Acad Sci U S A 104(28):11724-11729, 2007), Spα (Sarrias et al., J Biol Chem 280(42):35391-35398, 2005), CD163A (Fabriek et al., Blood 113(4):887-892, 2009) and DMBT1 (Madsen et al., Eur J Immunol 33(8):2327-2336, 2003) bind to Gram-positive and Gram-negative bacteria; CD5 binds to yeast (Vera et al., Proc Natl Acad Sci U S A 106(5):1506-1511, 2009). Identified ligands include lipoteichoic acid, lipopolysaccharide, poly-phosphorylated, and -sulfated compounds such as dextran sulfate sodium, leucine-rich repeat proteins, and fungal mannose (Sarrias et al., Proc Natl Acad Sci U S A 104(28):11724-11729, 2007; Sarrias et al., J Biol Chem 280(42):35391-35398, 2005; Fabriek et al., Blood 113(4):887-892, 2009; Vera et al., Proc Natl Acad Sci U S A 106(5):1506-1511, 2009; End et al., Eur J Immunol 39(3):833-842, 2009; Loimaranta et al., J Biol Chem 284(28):18614-18623, 2009). A conserved linear binding motif (VEVLXXXXW) in an external loop in the SRCR domain has been identified in CD163A and DMBT1 and can be used as a peptide that aggregates bacteria (Fabriek et al., Blood 113(4):887-892, 2009; Bikker et al., J Biol Chem 279(46):47699-47703, 2004; Leito et al., Biol Chem 389(9):1193-1200, 2008). In contrast, WC1 binding to bacteria is mediated by a noncontinuous motif in the native protein, and mutation of the VEVLXXXXW motif has no effect upon bacterial binding (Hsu et al., J Immunol 194(5):2280-2288, 2015). Thus, bacterial binding studies with WC1 SRCR domains must be done with native, correctly disulfide bonded, protein, ideally posttranslationally modified in mammalian cells.WC1 is found in the genomes of most mammals, reptiles, and birds and is expressed exclusively on γδ T cells in ruminants. The 13 bovine WC1 genes encode up to 11 extracellular SRCR domains, organized in the SRCR domain pattern of a1-[b2-c3-d4-e5-d6]-[b7-c8-d9-e10-d'11], where the alphabet designations indicate homology between genes and across species (Chen et al., BMC Genet 13:86, 2012; Herzig et al., BMC Evol Biol 10:181, 2010; Herzig and Baldwin, BMC Genomics 10:191, 2009). Some of the signaling co-receptor WC1 molecules are required for the γδ T cell response to Leptospira (Wang et al., Mol Immunol 48:801-809, 2011; Rogers et al., J Immunol 174(6):3386-3393, 2005; Wang et al., Eur J Immunol 39(1):254-266, 2009). The WC1 expressed on responsive γδ T cells is correlated with its direct binding to Leptospira via some of its SRCR domains (Hsu et al., J Immunol 194(5):2280-2288, 2015). Because WC1+ γδ T cells share a restriction in their γδ TCRs and WC1 has TCR co-receptor activity, we hypothesize that WC1 co-ligation with the TCR plays the determining role in the activation of WC1+ γδ T cells by pathogens. Classification of the binding of WC1 SRCR domains, their ligands, and their role in the interaction of 𝛾δ T cells with pathogens relevant to the host will allow these cells to be recruited in next-generation vaccines to pathogens that have significant negative economic and health impact.

Hotspots for mutations in the SARS-CoV-2 spike glycoprotein: a correspondence analysis.

Spike glycoprotein (Sgp) is liable for binding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to the host receptors. Since Sgp is the main target for vaccine and drug designing, elucidating its mutation pattern could help in this regard. This study is aimed at investigating the correspondence of specific residues to the SgpSARS-CoV-2 functionality by explorative interpretation of sequence alignments. Centrality analysis of the Sgp dissects the importance of these residues in the interaction network of the RBD-ACE2 (receptor-binding domain) complex and furin cleavage site. Correspondence of RBD to threonine500 and asparagine501 and furin cleavage site to glutamine675, glutamine677, threonine678, and alanine684 was observed; all residues are exactly located at the interaction interfaces. The harmonious location of residues dictates the RBD binding property and the flexibility, hydrophobicity, and accessibility of the furin cleavage site. These species-specific residues can be assumed as real targets of evolution, while other substitutions tend to support them. Moreover, all these residues are parts of experimentally identified epitopes. Therefore, their substitution may affect vaccine efficacy. Higher rate of RBD maintenance than furin cleavage site was predicted. The accumulation of substitutions reinforces the probability of the multi-host circulation of the virus and emphasizes the enduring evolutionary events.

Genetic and Bioinformatic Strategies to Improve Diagnosis in Three Inherited Bleeding Disorders in Bogotá, Colombia.

Inherited bleeding disorders (IBDs) are the most frequent congenital diseases in the Colombian population; three of them are hemophilia A (HA), hemophilia B (HB), and von Willebrand Disease (VWD). Currently, diagnosis relies on multiple clinical laboratory assays to assign a phenotype. Due to the lack of accessibility to these tests, patients can receive an incomplete diagnosis. In these cases, genetic studies reinforce the clinical diagnosis. The present study characterized the molecular genetic basis of 11 HA, three HB, and five VWD patients by sequencing the F8, F9, or the VWF gene. Twelve variations were found in HA patients, four in HB patients, and 19 in WVD patients. From these variations a total of 25 novel variations were found. Disease-causing variations were used as positive controls for validation of the high-resolution melting (HRM) variant-scanning technique. This approach is a low-cost genetic diagnostic method proposed to be incorporated in developing countries. For the data analysis, we developed an accessible open-source code in Python that improves HRM data analysis with better sensitivity of 95% and without bias when using different HRM equipment and software. Analysis of amplicons with a length greater than 300 bp can be performed by implementing an analysis by denaturation domains.

An Assessment of Serological Assays for SARS-CoV-2 as Surrogates for Authentic Virus Neutralization.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019 and has since caused a global pandemic resulting in millions of cases and deaths. Diagnostic tools and serological assays are critical for controlling the outbreak, especially assays designed to quantitate neutralizing antibody levels, considered the best correlate of protection. As vaccines become increasingly available, it is important to identify reliable methods for measuring neutralizing antibody responses that correlate with authentic virus neutralization but can be performed outside biosafety level 3 (BSL3) laboratories. While many neutralizing assays using pseudotyped virus have been developed, there have been few studies comparing the different assays to each other as surrogates for authentic virus neutralization. Here, we characterized three enzyme-linked immunosorbent assays (ELISAs) and three pseudotyped vesicular stomatitis virus (VSV) neutralization assays and assessed their concordance with authentic virus neutralization. The most accurate assays for predicting authentic virus neutralization were luciferase- and secreted embryonic alkaline phosphatase (SEAP)-expressing pseudotyped virus neutralizations, followed by green fluorescent protein (GFP)-expressing pseudotyped virus neutralization, and then the ELISAs. IMPORTANCE The ongoing COVID-19 pandemic is caused by infection with severe acute respiratory syndrome virus 2 (SARS-CoV-2). Prior infection or vaccination can be detected by the presence of antibodies in the blood. Antibodies in the blood are also considered to be protective against future infections from the same virus. The "gold standard" assay for detecting protective antibodies against SARS-CoV-2 is neutralization of authentic SARS-CoV-2 virus. However, this assay can only be performed under highly restrictive biocontainment conditions. We therefore characterized six antibody-detecting assays for their correlation with authentic virus neutralization. The significance of our research is in outlining the advantages and disadvantages of the different assays and identifying the optimal surrogate assay for authentic virus neutralization. This will allow for more accurate assessments of protective immunity against SARS-CoV-2 following infection and vaccination.

Recent advances in nanomaterials-based electrochemical immunosensors and aptasensors for HER2 assessment in breast cancer.

Human epidermal growth factor receptor 2 (HER2) is one of the key molecular targets in breast cancer pathogenesis. Overexpression and/or amplification of HER2 in approximately 15-20% of breast cancer patients is associated with high mortality and poor prognosis. Accumulating evidence shows that accurate and sensitive detection of HER2 improves the survival outcomes for HER2-positive breast cancer patients from targeted therapies. The current methods of clinical determination of HER2 expression levels are based on slide-based assays that rely on invasively collected primary tumours. Alternatively, ELISA-based detection of the shredded HER2 extracellular domain (HER2-ECD) of has been suggested as a surrogate method for monitoring disease progress and treatment response in breast cancer patients. In the past decade, biosensors have emerged as an alternative modality for the detection of circulating HER2-ECD in human serum samples. In particular, electrochemical biosensors based on nanomaterials and antibodies and aptamers have been increasingly developed as promising tools for rapid, sensitive, and cost-effective detection of HER2-ECD. These biosensors harness the high affinity and specificity of antibodies and aptamers, and unique conductive properties, biocompatibility, large surface area, and chemical stability of nanomaterials for selective and sensitive assessment of the HER2. This review provides an overview of the recent advances in the application of nanomaterials-based immunosensors and aptasensors for detection of circulating HER2-ECD. In particular, various electrochemical techniques, detection approaches, and nanomaterials are discussed. Further, analytical figures of merit of various HER2 immunosensors and aptasensors are compared. Finally, possible challenges and potential opportunities for biosensor-based detection of HER2-ECD are discussed.

TIAMMAt: Leveraging Biodiversity to Revise Protein Domain Models, Evidence from Innate Immunity.

Sequence annotation is fundamental for studying the evolution of protein families, particularly when working with nonmodel species. Given the rapid, ever-increasing number of species receiving high-quality genome sequencing, accurate domain modeling that is representative of species diversity is crucial for understanding protein family sequence evolution and their inferred function(s). Here, we describe a bioinformatic tool called Taxon-Informed Adjustment of Markov Model Attributes (TIAMMAt) which revises domain profile hidden Markov models (HMMs) by incorporating homologous domain sequences from underrepresented and nonmodel species. Using innate immunity pathways as a case study, we show that revising profile HMM parameters to directly account for variation in homologs among underrepresented species provides valuable insight into the evolution of protein families. Following adjustment by TIAMMAt, domain profile HMMs exhibit changes in their per-site amino acid state emission probabilities and insertion/deletion probabilities while maintaining the overall structure of the consensus sequence. Our results show that domain revision can heavily impact evolutionary interpretations for some families (i.e., NLR's NACHT domain), whereas impact on other domains (e.g., rel homology domain and interferon regulatory factor domains) is minimal due to high levels of sequence conservation across the sampled phylogenetic depth (i.e., Metazoa). Importantly, TIAMMAt revises target domain models to reflect homologous sequence variation using the taxonomic distribution under consideration by the user. TIAMMAt's flexibility to revise any subset of the Pfam database using a user-defined taxonomic pool will make it a valuable tool for future protein evolution studies, particularly when incorporating (or focusing) on nonmodel species.

Assessment of humoral and cellular immunity induced by the BNT162b2 SARS-CoV-2 vaccine in healthcare workers, elderly people, and immunosuppressed patients with autoimmune disease.

The development of vaccines to prevent SARS-CoV-2 infection has mainly relied on the induction of neutralizing antibodies (nAbs) to the Spike protein of SARS-CoV-2, but there is growing evidence that T cell immune response can contribute to protection as well. In this study, an anti-receptor binding domain (RBD) antibody assay and an INFγ-release assay (IGRA) were used to detect humoral and cellular responses to the Pfizer-BioNTech BNT162b2 vaccine in three separate cohorts of COVID-19-naïve patients: 108 healthcare workers (HCWs), 15 elderly people, and 5 autoimmune patients treated with immunosuppressive agents. After the second dose of vaccine, the mean values of anti-RBD antibodies (Abs) and INFγ were 123.33 U/mL (range 27.55-464) and 1513 mIU/mL (range 145-2500) in HCWs and 210.7 U/mL (range 3-500) and 1167 mIU/mL (range 83-2500) in elderly people. No correlations between age and immune status were observed. On the contrary, a weak but significant positive correlation was found between INFγ and anti-RBD Abs values (rho = 0.354, p = 0.003). As to the autoimmune cohort, anti-RBD Abs were not detected in the two patients with absent peripheral CD19+B cells, despite high INFγ levels being observed in all 5 patients after vaccination. Even though the clinical relevance of T cell response has not yet been established as a correlate of vaccine-induced protection, IGRA testing has showed optimal sensitivity and specificity to define vaccine responders, even in patients lacking a cognate antibody response to the vaccine.

Gram-negative outer-membrane proteins with multiple ß-barrel domains.

Outer-membrane beta barrels (OMBBs) are found in the outer membrane of gram-negative bacteria and eukaryotic organelles. OMBBs fold as antiparallel ß-sheets that close onto themselves, forming pores that traverse the membrane. Currently known structures include only one barrel, of 8 to 36 strands, per chain. The lack of multi-OMBB chains is surprising, as most OMBBs form oligomers, and some function only in this state. Using a combination of sensitive sequence comparison methods and coevolutionary analysis tools, we identify many proteins combining multiple beta barrels within a single chain; combinations that include eight-stranded barrels prevail. These multibarrels seem to be the result of independent, lineage-specific fusion and amplification events. The absence of multibarrels that are universally conserved in bacteria with an outer membrane, coupled with their frequent de novo genesis, suggests that their functions are not essential but rather beneficial in specific environments. Adjacent barrels of complementary function within the same chain may allow for functions beyond those of the individual barrels.

Antibacterial effects assessment on some livestock pathogens, thermal stability and proposing a probable reason for different levels of activity of thanatin.

There is a continuing need to prevent the increasing use of common antibiotic and find the replacement to combat the drug/antibiotic resistant bacteria such as antimicrobial peptides (AMPs) such as thanatin peptide. In this study, recombinant thanatin peptide was expressed in the HEK293 cell line. Then the antimicrobial properties of this peptide on some poultry and farm animal's pathogen strains were assessed. The thermal-stability of thanatin was predicted in various temperatures through in silico analysis. Afterwards, according to Minimum Inhibitory Concentration (MIC) results, Escherichia coli and Pseudomonas aeruginosa were chosen to test the hypothesis of LptA/LptD-thanatin interaction, computationally. Relative amino acid sequences and crystallography structures were retrieved and missed tertiary structures were predicted. The interaction of thanatin with LptA and LptD of Escherichia coli and Pseudomonas aeruginosa were analyzed subsequently. The antibacterial activity of thanatin peptide was evaluated between 6.25 and 100 µg/mL using minimum inhibitory concentration. Also, the amounts of minimum bactericidal concentrations (MBC) were between 12.5 and 200 µg/mL. The bioinformatics analysis followed by the in vitro assessment, demonstrated that thanatin would be thermally stable in the body temperature of poultry and farm animals. Thanatin could penetrate to the outer membrane domain of LptD in Escherichia coli and it could block the transition path of this protein while the entrance of LptD in Pseudomonas aeruginosa was blocked for thanatin by extra residues in comparison with Escherichia coli LptD. In addition, the quality of interaction, with regard to the number and distance of interactions which leads to higher binding energy for thanatin and LptD of Escherichia coli was much better than Pseudomonas aeruginosa. But the site and quality of interaction for thanatin and LptA was almost the same for Escherichia coli and Pseudomonas aeruginosa. Accordingly, thanatin can prevent the assembly of LptA periplasmic bridge in both pathogens. The antibacterial and thermal stability of the thanatin peptide suggested that thanatin peptide might serve as a natural alternative instead of common antibiotics in the veterinary medicine. The outcome of this in silico study supports the MIC results. Therefore, a probable reason for different level of activity of thanatin against Escherichia coli and Pseudomonas aeruginosa might be the quality of LptA/LptD-thanatin interaction.

BENZ WS: the Bologna ENZyme Web Server for four-level EC number annotation.

The Bologna ENZyme Web Server (BENZ WS) annotates four-level Enzyme Commission numbers (EC numbers) as defined by the International Union of Biochemistry and Molecular Biology (IUBMB). BENZ WS filters a target sequence with a combined system of Hidden Markov Models, modelling protein sequences annotated with the same molecular function, and Pfams, carrying along conserved protein domains. BENZ returns, when successful, for any enzyme target sequence an associated four-level EC number. Our system can annotate both monofunctional and polyfunctional enzymes, and it can be a valuable resource for sequence functional annotation.

Evidence for the preferential reuse of sub-domain motifs in primordial protein folds.

A comparison of protein backbones makes clear that not more than approximately 1400 different folds exist, each specifying the three-dimensional topology of a protein domain. Large proteins are composed of specific domain combinations and many domains can accommodate different functions. These findings confirm that the reuse of domains is key for the evolution of multi-domain proteins. If reuse was also the driving force for domain evolution, ancestral fragments of sub-domain size exist that are shared between domains possessing significantly different topologies. For the fully automated detection of putatively ancestral motifs, we developed the algorithm Fragstatt that compares proteins pairwise to identify fragments, that is, instantiations of the same motif. To reach maximal sensitivity, Fragstatt compares sequences by means of cascaded alignments of profile Hidden Markov Models. If the fragment sequences are sufficiently similar, the program determines and scores the structural concordance of the fragments. By analyzing a comprehensive set of proteins from the CATH database, Fragstatt identified 12 532 partially overlapping and structurally similar motifs that clustered to 134 unique motifs. The dissemination of these motifs is limited: We found only two domain topologies that contain two different motifs and generally, these motifs occur in not more than 18% of the CATH topologies. Interestingly, motifs are enriched in topologies that are considered ancestral. Thus, our findings suggest that the reuse of sub-domain sized fragments was relevant in early phases of protein evolution and became less important later on.