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.

Identification of motif-based interactions between SARS-CoV-2 protein domains and human peptide ligands pinpoint antiviral targets.

The virus life cycle depends on host-virus protein-protein interactions, which often involve a disordered protein region binding to a folded protein domain. Here, we used proteomic peptide phage display (ProP-PD) to identify peptides from the intrinsically disordered regions of the human proteome that bind to folded protein domains encoded by the SARS-CoV-2 genome. Eleven folded domains of SARS-CoV-2 proteins were found to bind 281 peptides from human proteins, and affinities of 31 interactions involving eight SARS-CoV-2 protein domains were determined (KD ∼ 7-300 µM). Key specificity residues of the peptides were established for six of the interactions. Two of the peptides, binding Nsp9 and Nsp16, respectively, inhibited viral replication. Our findings demonstrate how high-throughput peptide binding screens simultaneously identify potential host-virus interactions and peptides with antiviral properties. Furthermore, the high number of low-affinity interactions suggest that overexpression of viral proteins during infection may perturb multiple cellular pathways.

Shotgun Metagenomic Analyses of Microbial Assemblages in the Aquatic Ecosystem of Winam Gulf of Lake Victoria, Kenya Reveals Multiclass Pollution.

Lake Victoria, the second-largest freshwater lake in the world, provides an important source of food and income, particularly fish for both domestic consumption and for export market. In recent years, Lake Victoria has suffered massive pollution from both industrial and wastewater discharge. Microplastic biomes, pharmaceutical residues, drugs of abuse, heavy metals, agrochemicals, and personal care products are ubiquitous in the aquatic ecosystem of Winam Gulf. These pollutants are known to alter microbial assemblages in aquatic ecosystems with far-reaching ramification including a calamitous consequence to human health. Indeed, some of these pollutants have been associated with human cancers and antimicrobial resistance. There is a paucity of data on the microbial profiles of this important but heavily polluted aquatic ecosystem. The current study sought to investigate the metagenomic profiles of microbial assemblages in the Winam Gulf ecosystem. Water and sediment samples were collected from several locations within the study sites. Total genomic DNA pooled from all sampling sites was extracted and analyzed by whole-genome shotgun sequencing. Analyses revealed three major kingdoms: bacteria, archaea and eukaryotes belonging to 3 phyla, 13 classes, 14 families, 9 orders, 14 genera, and 10 species. Proteobacteria, Betaproteobacteria, Comamonadaceae, Burkholdariales, and Arcobacter were the dominated phyla, class, family, order, genera, and species, respectively. The Kyoto Encyclopedia of Genes and Genomes indicated the highest number of genes involved in metabolism. The presence of carbohydrate metabolism genes and enzymes was used to infer organic pollutions from sewage and agricultural runoffs. Similarly, the presence of xylene and nutrotoluene degradation genes and enzyme was used to infer industrial pollution into the lake. Drug metabolism genes lend credence to the possibility of pharmaceutical pollutants in water. Taken together, there is a clear indication of massive pollution. In addition, carbohydrate-active enzymes were the most abundant and included genes in glycoside hydrolases. Shotgun metagenomic analyses conveyed an understanding of the microbial communities of the massively polluted aquatic ecosystem of Winam Gulf, Lake Vicoria, Kenya. The current study documents the presence of multiclass pollutants in Lake Victoria and reveals information that might be useful for a potential bioremediation strategy using the native microbial communities.

Atomic resolution protein allostery from the multi-state structure of a PDZ domain.

Recent methodological advances in solution NMR allow the determination of multi-state protein structures and provide insights into structurally and dynamically correlated protein sites at atomic resolution. This is demonstrated in the present work for the well-studied PDZ2 domain of protein human tyrosine phosphatase 1E for which protein allostery had been predicted. Two-state protein structures were calculated for both the free form and in complex with the RA-GEF2 peptide using the exact nuclear Overhauser effect (eNOE) method. In the apo protein, an allosteric conformational selection step comprising almost 60% of the domain was detected with an "open" ligand welcoming state and a "closed" state that obstructs the binding site by changing the distance between the ß-sheet 2, α-helix 2, and sidechains of residues Lys38 and Lys72. The observed induced fit-type apo-holo structural rearrangements are in line with the previously published evolution-based analysis covering ~25% of the domain with only a partial overlap with the protein allostery of the open form. These presented structural studies highlight the presence of a dedicated highly optimized and complex dynamic interplay of the PDZ2 domain owed by the structure-dynamics landscape.

Small-molecule activation of OGG1 increases oxidative DNA damage repair by gaining a new function.

Oxidative DNA damage is recognized by 8-oxoguanine (8-oxoG) DNA glycosylase 1 (OGG1), which excises 8-oxoG, leaving a substrate for apurinic endonuclease 1 (APE1) and initiating repair. Here, we describe a small molecule (TH10785) that interacts with the phenylalanine-319 and glycine-42 amino acids of OGG1, increases the enzyme activity 10-fold, and generates a previously undescribed ß,δ-lyase enzymatic function. TH10785 controls the catalytic activity mediated by a nitrogen base within its molecular structure. In cells, TH10785 increases OGG1 recruitment to and repair of oxidative DNA damage. This alters the repair process, which no longer requires APE1 but instead is dependent on polynucleotide kinase phosphatase (PNKP1) activity. The increased repair of oxidative DNA lesions with a small molecule may have therapeutic applications in various diseases and aging.

Solution nuclear magnetic resonance spectroscopy of bacterial outer membrane proteins in natively excreted vesicles using engineered Escherichia coli.

Gaining structural information on membrane proteins in their native lipid environment is a long-standing challenge in molecular biology. Instead, it is common to employ membrane mimetics, which has been shown to affect protein structure, dynamics, and function severely. Here, we describe the incorporation of a bacterial outer membrane protein (OmpW) into natively excreted membrane vesicles for solution nuclear magnetic resonance (NMR) spectroscopy using a mutant Escherichia coli strain with a high outer membrane vesicle (OMV) production rate. We collected NMR spectra from both vesicles containing overexpressed OmpW and vesicles from a control strain to account for the presence of physiologically relevant outer membrane proteins in vesicles and observed distinct resonance signals from OmpW. Due to the increased production of OMVs and the use of non-uniform sampling techniques we were able to obtain high-resolution 2D (HSQC) and 3D (HNCO) NMR spectra of our target protein inside its native lipid environment. While this workflow is not yet sufficient to achieve in situ structure determination, our results pave the way for further research on vesicle-based solution NMR spectroscopy.

Structural Characterization of a Thorarchaeota Profilin Indicates Eukaryotic-Like Features but with an Extended N-Terminus.

The emergence of the first eukaryotic cell is preceded by evolutionary events, which are still highly debatable. Clues of the exact sequence of events are beginning to emerge. Recent metagenomics analyses has uncovered the Asgard super-phylum as the closest yet known archaea host of eukaryotes. Some of these have been tested and confirmed experimentally. However, the bulk of eukaryotic signature proteins predicted to be encoded by the Asgard super-phylum have not been studied, and their true functions, at least in the context of a eukaryotic cell, are still elusive. For example, there are several different variants of the profilin within each Asgardian Achaea, and there are some conflicting results of their actual roles. Here, the 3D structure of profilin from Thorarchaeota is determined by nuclear magnetic resonance spectroscopy and shows that this profilin has a eukaryotic-like profilin with a rigid core and an extended N-terminus previously implicated in polyproline binding. In addition, it is also shown that Thorarchaeota Profilin co-localizes with eukaryotic actin in cultured HeLa cells. This finding reaffirms the notion that Asgardian encoded proteins possess eukaryotic-like characteristics and strengthen the likely existence of a complex cytoskeleton already in a last eukaryotic common ancestor.

The dynamic properties of a nuclear coactivator binding domain are evolutionarily conserved.

Evolution of proteins is constrained by their structure and function. While there is a consensus that the plasticity of intrinsically disordered proteins relaxes the structural constraints on evolution there is a paucity of data on the molecular details of these processes. The Nuclear Coactivator Binding Domain (NCBD) from CREB-binding protein is a protein interaction domain, which contains a hydrophobic core but is not behaving as a typical globular domain, and has been described as 'molten-globule like'. The highly dynamic properties of NCBD makes it an interesting model system for evolutionary structure-function investigation of intrinsically disordered proteins. We have here compared the structure and biophysical properties of an ancient version of NCBD present in a bilaterian animal ancestor living around 600 million years ago with extant human NCBD. Using a combination of NMR spectroscopy, circular dichroism and kinetics we show that although NCBD has increased its thermodynamic stability, it has retained its dynamic biophysical properties in the ligand-free state in the evolutionary lineage leading from the last common bilaterian ancestor to humans. Our findings suggest that the dynamic properties of NCBD have been maintained by purifying selection and thus are important for its function, which includes mediating several distinct protein-protein interactions.

Influence of Detergent and Lipid Composition on Reconstituted Membrane Proteins for Structural Studies.

Membrane proteins are frequently reconstituted in different detergents as a prerequisite to create a phospholipid environment reminiscent of their native environment. Different detergent characteristics such as their chain length and bond types could affect the structure and function of proteins. Yet, they are seldom taken into account when choosing a detergent for structural studies. Here, we explore the effect of different detergents and lipids with varying degrees of double- or single-bond composition on 1H-15N transverse relaxation optimized spectroscopy spectra of the outer membrane protein W (OmpW). We observed changes in nuclear magnetic resonance chemical shifts for OmpW reconstituted in micelles, bicelles, and nanodiscs, depending on their detergent/lipid composition. These results suggest that a careful evaluation of detergents is necessary, so as not to jeopardize the structure and function of the protein.

Heimdallarchaea encodes profilin with eukaryotic-like actin regulation and polyproline binding.

It is now widely accepted that the first eukaryotic cell emerged from a merger of an archaeal host cell and an alphaproteobacterium. However, the exact sequence of events and the nature of the cellular biology of both partner cells is still contentious. Recently the structures of profilins from some members of the newly discovered Asgard superphylum were determined. In addition, it was found that these profilins inhibit eukaryotic rabbit actin polymerization and that this reaction is regulated by phospholipids. However, the interaction with polyproline repeats which are known to be crucial for the regulation of profilin:actin polymerization was found to be absent for these profilins and was thus suggested to have evolved later in the eukaryotic lineage. Here, we show that Heimdallarchaeota LC3, a candidate phylum within the Asgard superphylum, encodes a putative profilin (heimProfilin) that interacts with PIP2 and its binding is regulated by polyproline motifs, suggesting an origin predating the rise of the eukaryotes. More precisely, we determined the 3D-structure of Heimdallarchaeota LC3 profilin and show that this profilin is able to: i) inhibit eukaryotic actin polymerization in vitro; ii) bind to phospholipids; iii) bind to polyproline repeats from enabled/vasodilator-stimulated phosphoprotein; iv) inhibit actin from Heimdallarchaeota from polymerizing into filaments. Our results therefore provide hints of the existence of a complex cytoskeleton already in last eukaryotic common ancestor.

NMR resonance assignment and dynamics of profilin from Heimdallarchaeota.

The origin of the eukaryotic cell is an unsettled scientific question. The Asgard superphylum has emerged as a compelling target for studying eukaryogenesis due to the previously unseen diversity of eukaryotic signature proteins. However, our knowledge about these proteins is still relegated to metagenomic data and very little is known about their structural properties. Additionally, it is still unclear if these proteins are functionally homologous to their eukaryotic counterparts. Here, we expressed, purified and structurally characterized profilin from Heimdallarchaeota in the Asgard superphylum. The structural analysis shows that while this profilin possesses similar secondary structural elements as eukaryotic profilin, it contains additional secondary structural elements that could be critical for its function and an indication of divergent evolution.

Protein Allostery at Atomic Resolution.

Protein allostery is a phenomenon involving the long range coupling between two distal sites in a protein. In order to elucidate allostery at atomic resoluion on the ligand-binding WW domain of the enzyme Pin1, multistate structures were calculated from exact nuclear Overhauser effect (eNOE). In its free form, the protein undergoes a microsecond exchange between two states, one of which is predisposed to interact with its parent catalytic domain. In presence of the positive allosteric ligand, the equilibrium between the two states is shifted towards domain-domain interaction, suggesting a population shift model. In contrast, the allostery-suppressing ligand decouples the side-chain arrangement at the inter-domain interface thereby reducing the inter-domain interaction. As such, this mechanism is an example of dynamic allostery. The presented distinct modes of action highlight the power of the interplay between dynamics and function in the biological activity of proteins.

Protein-fragment complex structures derived by NMR molecular replacement.

Recently we have established an NMR molecular replacement method, which is capable of solving the structure of the interaction site of protein-ligand complexes in a fully automated manner. While the method was successfully applied for ligands with strong and weak binding affinities, including small molecules and peptides, its applicability on ligand fragments remains to be shown. Structures of fragment-protein complexes are more challenging for the method since fragments contain only few protons. Here we show a successful application of the NMR molecular replacement method in solving structures of complexes between three derivatives of a ligand fragment and the protein receptor PIN1. We anticipate that this approach will find a broad application in fragment-based lead discovery.

Backbone and side-chain chemical shift assignments of full-length, apo, human Pin1, a phosphoprotein regulator with interdomain allostery.

Pin1 is a human peptidyl-prolyl cis-trans isomerase important for the regulation of phosphoproteins that are implicated in many diseases including cancer and Alzheimer's. Further biophysical study of Pin1 will elucidate the importance of the two-domain system to regulate its own activity. Here, we report near-complete backbone and side-chain 1H, 13C and 15N NMR chemical shift assignments of full-length, apo Pin1 for the purpose of studying interdomain allostery and dynamics.

Structure and dynamics conspire in the evolution of affinity between intrinsically disordered proteins.

In every established species, protein-protein interactions have evolved such that they are fit for purpose. However, the molecular details of the evolution of new protein-protein interactions are poorly understood. We have used nuclear magnetic resonance spectroscopy to investigate the changes in structure and dynamics during the evolution of a protein-protein interaction involving the intrinsically disordered CREBBP (CREB-binding protein) interaction domain (CID) and nuclear coactivator binding domain (NCBD) from the transcriptional coregulators NCOA (nuclear receptor coactivator) and CREBBP/p300, respectively. The most ancient low-affinity "Cambrian-like" [540 to 600 million years (Ma) ago] CID/NCBD complex contained less secondary structure and was more dynamic than the complexes from an evolutionarily younger "Ordovician-Silurian" fish ancestor (ca. 440 Ma ago) and extant human. The most ancient Cambrian-like CID/NCBD complex lacked one helix and several interdomain interactions, resulting in a larger solvent-accessible surface area. Furthermore, the most ancient complex had a high degree of millisecond-to-microsecond dynamics distributed along the entire sequences of both CID and NCBD. These motions were reduced in the Ordovician-Silurian CID/NCBD complex and further redistributed in the extant human CID/NCBD complex. Isothermal calorimetry experiments show that complex formation is enthalpically favorable and that affinity is modulated by a largely unfavorable entropic contribution to binding. Our data demonstrate how changes in structure and motion conspire to shape affinity during the evolution of a protein-protein complex and provide direct evidence for the role of structural, dynamic, and frustrational plasticity in the evolution of interactions between intrinsically disordered proteins.

Proteome-wide analysis of phospho-regulated PDZ domain interactions.

A key function of reversible protein phosphorylation is to regulate protein-protein interactions, many of which involve short linear motifs (3-12 amino acids). Motif-based interactions are difficult to capture because of their often low-to-moderate affinities. Here, we describe phosphomimetic proteomic peptide-phage display, a powerful method for simultaneously finding motif-based interaction and pinpointing phosphorylation switches. We computationally designed an oligonucleotide library encoding human C-terminal peptides containing known or predicted Ser/Thr phosphosites and phosphomimetic variants thereof. We incorporated these oligonucleotides into a phage library and screened the PDZ (PSD-95/Dlg/ZO-1) domains of Scribble and DLG1 for interactions potentially enabled or disabled by ligand phosphorylation. We identified known and novel binders and characterized selected interactions through microscale thermophoresis, isothermal titration calorimetry, and NMR We uncover site-specific phospho-regulation of PDZ domain interactions, provide a structural framework for how PDZ domains accomplish phosphopeptide binding, and discuss ligand phosphorylation as a switching mechanism of PDZ domain interactions. The approach is readily scalable and can be used to explore the potential phospho-regulation of motif-based interactions on a large scale.

NOE-Derived Methyl Distances from a 360†kDa Proteasome Complex.

Nuclear magnetic resonance spectroscopy is the prime tool to probe structure and dynamics of biomolecules at atomic resolution. A serious challenge for that method is the size limit imposed on molecules to be studied. Standard studies are typically restricted to ca. 30-40 kDa. More recent developments lead to spin relaxation measurements in methyl groups in single proteins or protein complexes as large as a mega-Dalton, which directly allow the extraction of angular information or experiments with paramagnetic samples. However, these probes are all of indirect nature in contrast to the most intuitive and easy-to-interpret structural/dynamics restraint, the internuclear distance, which can be measured by nuclear Overhauser enhancement (NOE). Herein, we demonstrate time-averaged distance measurements on the 360 kDa half proteasome from Thermoplasma acidophilium. The approach is based on exact quantification of the NOE (eNOE). Our findings open up an avenue for such measurements on very large molecules. These restraints will help in a detailed determination of conformational changes upon perturbation such as ligand binding.

The Sign of Nuclear Magnetic Resonance Chemical Shift Difference as a Determinant of the Origin of Binding Selectivity: Elucidation of the Position Dependence of Phosphorylation in Ligands Binding to Scribble PDZ1.

The use of nuclear magnetic resonance chemical shift perturbation to monitor changes taking place around the binding site of a ligand-protein interaction is a routine and widely applied methodology in the field of protein biochemistry. Shifts are often acquired by titrating various concentrations of ligand to a fixed concentration of the receptor and may serve the purpose, among others, of determining affinity constants, locating binding surfaces, or differentiating between binding mechanisms. Shifts are quantified by the so-called combined chemical shift difference. Although the directionality of shift changes is often used for detailed analysis of specific cases, the approach has not been adapted in standard chemical shift monitoring. This is surprising as it would not require additional effort. Here, we demonstrate the importance of the sign of the chemical shift difference induced by ligand-protein interaction. We analyze the sign of the 15N/1H shift changes of the PDZ1 domain of Scribble upon interaction with two pairs of phosphorylated and unphosphorylated peptides. We find that detailed differences in the molecular basis of this PDZ-ligand interaction can be obtained from our analysis to which the classical method of combined chemical shift perturbation analysis is insensitive. In addition, we find a correlation between affinity and millisecond motions. Application of the methodology to Cyclophilin a, a cis-trans isomerase, reveals molecular details of peptide recognition. We consider our directionality vector chemical shift analysis as a method of choice when distinguishing the molecular origin of binding specificities of a class of similar ligands, which is often done in drug discovery.

The Exact Nuclear Overhauser Enhancement: Recent Advances.

Although often depicted as rigid structures, proteins are highly dynamic systems, whose motions are essential to their functions. Despite this, it is difficult to investigate protein dynamics due to the rapid timescale at which they sample their conformational space, leading most NMR-determined structures to represent only an averaged snapshot of the dynamic picture. While NMR relaxation measurements can help to determine local dynamics, it is difficult to detect translational or concerted motion, and only recently have significant advances been made to make it possible to acquire a more holistic representation of the dynamics and structural landscapes of proteins. Here, we briefly revisit our most recent progress in the theory and use of exact nuclear Overhauser enhancements (eNOEs) for the calculation of structural ensembles that describe their conformational space. New developments are primarily targeted at increasing the number and improving the quality of extracted eNOE distance restraints, such that the multi-state structure calculation can be applied to proteins of higher molecular weights. We then review the implications of the exact NOE to the protein dynamics and function of cyclophilin A and the WW domain of Pin1, and finally discuss our current research and future directions.

eNORA2 Exact NOE Analysis Program.

We have recently developed an NMR protocol to extract exact distances between nuclei in proteins from an exact interpretation of NOESY buildup intensities (eNOEs). This enabled us to calculate multistate structural ensembles that exhibit realistic spatial sampling and long-range correlations. Our initial studies were laborious and required a deep understanding of the underlying spin dynamics. Here, we present a MatLab package that integrates all data processing steps required to convert intensities of assigned peaks in NOESY series into upper and lower distance limits for structure calculation. Those steps include organization of the data in object format, extraction of autorelaxation and cross-relaxation rate constants by fitting of diagonal peak decays and cross peak buildups, validation of the data, correction for spin diffusion, graphical display of the results, and generation of distance limits in CYANA compatible format. The analysis may be carried out using a full relaxation matrix or a simplified "divide and conquer" approach that allows for partial deuteration of protons. As the program does not require expertise beyond that of standard resonance assignment/structure calculation, it is suitable for experts and nonexperts alike.