Interactions between the protein barnase and co-solutes studied by NMR.

Protein solubility and stability depend on the co-solutes present. There is little theoretical basis for selection of suitable co-solutes. Some guidance is provided by the Hofmeister series, an empirical ordering of anions according to their effect on solubility and stability; and by osmolytes, which are small organic molecules produced by cells to allow them to function in stressful environments. Here, NMR titrations of the protein barnase with Hofmeister anions and osmolytes are used to measure and locate binding, and thus to separate binding and bulk solvent effects. We describe a rationalisation of Hofmeister (and inverse Hofmeister) effects, which is similar to the traditional chaotrope/kosmotrope idea but based on solvent fluctuation rather than water withdrawal, and characterise how co-solutes affect protein stability and solubility, based on solvent fluctuations. This provides a coherent explanation for solute effects, and points towards a more rational basis for choice of excipients.

Clostridioides difficile canonical L,D-transpeptidases catalyze a novel type of peptidoglycan cross-links and are not required for beta-lactam resistance.

Clostridioides difficile is the leading cause of antibiotic-associated diarrhea worldwide with significant morbidity and mortality. This organism is naturally resistant to several beta-lactam antibiotics that inhibit the polymerization of peptidoglycan, an essential component of the bacteria cell envelope. Previous work has revealed that C. difficile peptidoglycan has an unusual composition. It mostly contains 3-3 cross-links, catalyzed by enzymes called L,D-transpeptidases (Ldts) that are poorly inhibited by beta-lactams. It was therefore hypothesized that peptidoglycan polymerization by these enzymes could underpin antibiotic resistance. Here, we investigated the catalytic activity of the three canonical Ldts encoded by C. difficile (LdtCd1, LdtCd2, and LdtCd3) in vitro and explored their contribution to growth and antibiotic resistance. We show that two of these enzymes catalyze the formation of novel types of peptidoglycan cross-links using meso-diaminopimelic acid both as a donor and an acceptor, also observed in peptidoglycan sacculi. We demonstrate that the simultaneous deletion of these three genes only has a minor impact on both peptidoglycan structure and resistance to beta-lactams. This unexpected result therefore implies that the formation of 3-3 peptidoglycan cross-links in C. difficile is catalyzed by as yet unidentified noncanonical Ldt enzymes.

DUF916 and DUF3324 in the WxL protein cluster bind to WxL and link bacterial and host surfaces.

Bacterial WxL proteins contain peptidoglycan-binding WxL domains, which have a dual Trp-x-Leu motif and are involved in virulence. It was recently shown that WxL proteins occur in gene clusters, containing typically a small WxL protein (which in the mature protein consists only of a WxL domain), a large WxL protein (which contains a C-terminal WxL domain with N-terminal host-binding domains), and a conserved protein annotated as a Domain of Unknown Function (DUF). Here we analyze this DUF and show that it contains two tandem domains-DUF916 and DUF3324-which both have an IgG-like fold and together form a single functional unit, connected to a C-terminal transmembrane helix. DUF3324 is a stable domain, while DUF916 is less stable and is likely to require a stabilizing interaction with WxL. The protein is suggested to have an important role to bind and stabilize WxL on the peptidoglycan surface, via the DUF916 domain, and to bind to host cells via the DUF3324 domain. AlphaFold2 predicts that a ß-hairpin strand from DUF916 inserts into WxL adjacent to its N-terminus. We therefore propose to rename the DUF916-DUF3324 pair as WxL Interacting Protein (WxLIP), with DUF916, DUF3324 and the transmembrane helix forming the first, second and third domains of WxLIP, which we characterize as peptidoglycan binding domain (PGBD), host binding domain (HBD), and transmembrane helix (TMH) respectively.

A review on the structure of Bombyx mori silk fibroin fiber studied using solid-state NMR: An antipolar lamella with an 8-residue repeat.

Silk fibroin (SF) fiber from the silkworm Bombyx mori in the Silk II form has been used as an excellent textile fiber for over 5000 years. Recently it has been developed for a range of biomedical applications. Further expansion of these uses builds on the excellent mechanical strength of SF fiber, which derives from its structure. This relationship between strength and SF structure has been studied for over 50 years, but it is still not well understood. In this review, we report the use of solid-state NMR to study stable-isotope labeled SF fiber and stable-isotope labeled peptides including (Ala-Gly)15 and (Ala-Gly-Ser-Gly-Ala-Gly)5 as models of the crystalline fraction. We show that the crystalline fraction is a lamellar structure with a repetitive folding using ß-turns every eighth amino acid, and that the sidechains adopt an antipolar arrangement rather than the more well-known polar structure described by Marsh, Corey and Pauling (that is, the Ala methyls in each layer point in opposite directions in alternate strands). The amino acids Ser, Tyr and Val are the next most common in B. mori SF after Gly and Ala, and occur in the crystalline and semi-crystalline regions, probably defining the edges of the crystalline region. Thus, we now have an understanding of the main features of Silk II but there is still a long way to go.

Improved methodology for protein NMR structure calculation using hydrogen bond restraints and ANSURR validation: The SH2 domain of SH2B1.

Protein structures calculated using NMR data are less accurate and less well-defined than they could be. Here we use the program ANSURR to show that this deficiency is at least in part due to a lack of hydrogen bond restraints. We describe a protocol to introduce hydrogen bond restraints into the structure calculation of the SH2 domain from SH2B1 in a systematic and transparent way and show that the structures generated are more accurate and better defined as a result. We also show that ANSURR can be used as a guide to know when the structure calculation is good enough to stop.

Protein Binding: A Fuzzy Concept.

Our understanding of protein binding interactions has matured significantly over the last few years, largely as a result of trying to make sense of the binding interactions of intrinsically disordered proteins. Here, we bring together some disparate ideas that have largely developed independently, and show that they can be linked into a coherent picture that provides insight into quantitative aspects of protein interactions, in particular that transient protein interactions are often optimised for speed, rather than tight binding.

Bioinformatic analysis of WxL domain proteins.

The WxL domain is found on the cell surface of many bacteria, most of which are commensal gut bacteria. Its functions are generally identified as being related to virulence and/or peptidoglycan attachment, but there is so far no clear function or structure for this domain. Here, a range of bioinformatics tools were used to clarify the structure and function. These indicate that WxL domains occur in cell surface-associated gene clusters that always contain a small WxL, large WxL and DUF916 domain; and that the small and large WxL proteins have distinct structure despite sharing two conserved WxL motifs. The two WxL motifs form a hydrophobic surface buried inside the protein. The likely function of the WxL domain is to attach to bacterial peptidoglycan, forming a platform to allow associated domains in the cluster to interact with host proteins.

Quantitation of phosphohistidine in proteins in a mammalian cell line by 31P NMR.

There is growing evidence to suggest that phosphohistidines are present at significant levels in mammalian cells and play a part in regulating cellular activity, in particular signaling pathways related to cancer. Because of the chemical instability of phosphohistidine at neutral or acid pH, it remains unclear how much phosphohistidine is present in cells. Here we describe a protocol for extracting proteins from mammalian cells in a way that avoids loss of covalent phosphates from proteins, and use it to measure phosphohistidine concentrations in human bronchial epithelial cell (16HBE14o-) lysate using 31P NMR spectroscopic analysis. Phosphohistidine is determined on average to be approximately one third as abundant as phosphoserine and phosphothreonine combined (and thus roughly 15 times more abundant than phosphotyrosine). The amount of phosphohistidine, and phosphoserine/phosphothreonine per gram of protein from a cell lysate was determined to be 23 µmol/g and 68 µmol/g respectively. The amount of phosphohistidine, and phosphoserine/phosphothreonine per cell was determined to be 1.8 fmol/cell, and 5.8 fmol/cell respectively. Phosphorylation is largely at the N3 (tele) position. Typical tryptic digest conditions result in loss of most of the phosphohistidine present, which may explain why the amounts reported here are greater than is generally seen using mass spectroscopy assays. The results further strengthen the case for a functional role of phosphohistidine in eukaryotic cells.

Utilization and accumulation of compatible solutes in Halomonas pacifica: a species of moderately halophilic bacteria isolated from a saline lake in South Libya.

When grown in high salt concentrations, halophilic bacteria often accumulate compatible solutes, which have major applications in biotechnology because they stabilize cells and proteins. Four Gram-negative bacterial strains, belonging to the family Halomonadaceae, were isolated from Qaberoun and Um-Alma lakes in South Libya using high-salinity medium. The strains were identified using 16S rRNA gene sequencing as belonging to Halomonas pacifica (strain ABQ1), Halomonas venusta (ABQ2), Halomonas elongata (ABU1) and Halomonas salifodinae (ABU2). H. pacifica ABQ1 is a moderate halophile (salinity range 0.05 to 2.5 M NaCl), with a broad tolerance to pH (7 to 9) and temperature (25-37 °C). Addition of the compatible solutes glycine betaine (betaine) and ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) to the medium had a positive effect on growth of H. pacifica at 2 M NaCl. In rich LB medium, betaine was the major compatible solute accumulated, with ectoine only being accumulated at salinities in excess of 1 M NaCl. In minimal M9 medium, betaine was not produced, but increasing amounts of ectoine were synthesized with increasing salinity, and hydroxyectoine [(4S,5S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] was also synthesized when the cells were grown in very high salt. We have thus identified H. pacifica as a producer of ectoine and hydroxyectoine, with more being produced at higher salinities. As industrial demand for these compatible solutes continues to increase, this system has biotechnological potential.

The measurement of binding affinities by NMR chemical shift perturbation.

We have carried out chemical shift perturbation titrations on three contrasting proteins. The resulting chemical shifts have been analysed to determine the best way to fit the data, and it is concluded that a simultaneous fitting of all raw shift data to a single dissociation constant is both the most accurate and the most precise method. It is shown that the optimal weighting of 15N chemical shifts to 1H chemical shifts is protein dependent, but is around the consensus value of 0.14. We show that chemical shift changes of individual residues can be fit to give residue-specific affinities. Residues with affinities significantly stronger than average are found in close contact with the ligand and are suggested to form a rigid contact surface, but only when the binding involves little conformational change. This observation may be of value in analysing binding and conformational change.

1H, 13C, and 15N resonance assignments of a conserved putative cell wall binding domain from Enterococcus faecalis.

Enterococcus faecalis is a major causative agent of hospital acquired infections. The ability of E. faecalis to evade the host immune system is essential during pathogenesis, which has been shown to be dependent on the complete separation of daughter cells by peptidoglycan hydrolases. AtlE is a peptidoglycan hydrolase which is predicted to bind to the cell wall of E. faecalis, via six C-terminal repeat sequences. Here, we report the near complete assignment of one of these six repeats, as well as the predicted backbone structure and dynamics. This data will provide a platform for future NMR studies to explore the ligand recognition motif of AtlE and help to uncover its potential role in E. faecalis virulence.

CORM-3 induces DNA damage through Ru(II) binding to DNA.

When the 'CO-releasing molecule-3', CORM-3 (Ru(CO)3Cl(glycinate)), is dissolved in water it forms a range of ruthenium complexes. These are taken up by cells and bind to intracellular ligands, notably thiols such as cysteine and glutathione, where the Ru(II) reaches high intracellular concentrations. Here, we show that the Ru(II) ion also binds to DNA, at exposed guanosine N7 positions. It therefore has a similar cellular target to the anticancer drug cisplatin, but not identical, because Ru(II) shows no evidence of forming intramolecular crossbridges in the DNA. The reaction is slow, and with excess Ru, intermolecular DNA crossbridges are formed. The addition of CORM-3 to human colorectal cancer cells leads to strand breaks in the DNA, as assessed by the alkaline comet assay. DNA damage is inhibited by growth media containing amino acids, which bind to extracellular Ru and prevent its entry into cells. We conclude that the cytotoxicity of Ru(II) is different from that of platinum, making it a promising development target for cancer therapeutics.

The accuracy of protein structures in solution determined by AlphaFold and NMR.

In the recent Critical Assessment of Structure Prediction (CASP) competition, AlphaFold2 performed outstandingly. Its worst predictions were for nuclear magnetic resonance (NMR) structures, which has two alternative explanations: either the NMR structures were poor, implying that Alpha-Fold may be more accurate than NMR, or there is a genuine difference between crystal and solution structures. Here, we use the program Accuracy of NMR Structures Using RCI and Rigidity (ANSURR), which measures the accuracy of solution structures, and show that one of the NMR structures was indeed poor. We then compare Alpha-Fold predictions to NMR structures and show that Alpha-Fold tends to be more accurate than NMR ensembles. There are, however, some cases where the NMR ensembles are more accurate. These tend to be dynamic structures, where Alpha-Fold had low confidence. We suggest that Alpha-Fold could be used as the model for NMR-structure refinements and that Alpha-Fold structures validated by ANSURR may require no further refinement.

The accuracy of NMR protein structures in the Protein Data Bank.

The program ANSURR measures the accuracy of NMR structures by comparing rigidity obtained from experimental backbone chemical shifts and from structures. We report on ANSURR analysis of 7,000 PDB NMR ensembles within the Protein Data Bank, which can be found at ansurr.com. The accuracy of NMR structures progressively improved up until 2005, but since then, it has plateaued. Most structures have accurate secondary structure, but are generally too floppy, particularly in loops. Thus, there is a need for more experimental restraints in loops. Currently, the best predictors of accuracy are Ramachandran distribution and the number of NOE restraints per residue. The precision of structures within the ensemble correlates well with accuracy, as does the number of hydrogen bond restraints per residue. Structure accuracy is improved when other components (such as additional polypeptide chains or ligands) are included.

A method for validating the accuracy of NMR protein structures.

We present a method that measures the accuracy of NMR protein structures. It compares random coil index [RCI] against local rigidity predicted by mathematical rigidity theory, calculated from NMR structures [FIRST], using a correlation score (which assesses secondary structure), and an RMSD score (which measures overall rigidity). We test its performance using: structures refined in explicit solvent, which are much better than unrefined structures; decoy structures generated for 89 NMR structures; and conventional predictors of accuracy such as number of restraints per residue, restraint violations, energy of structure, ensemble RMSD, Ramachandran distribution, and clashscore. Restraint violations and RMSD are poor measures of accuracy. Comparisons of NMR to crystal structures show that secondary structure is equally accurate, but crystal structures are typically too rigid in loops, whereas NMR structures are typically too floppy overall. We show that the method is a useful addition to existing measures of accuracy.

Characterisation of Bacteriocins Produced by Lactobacillus spp. Isolated from the Traditional Pakistani Yoghurt and Their Antimicrobial Activity against Common Foodborne Pathogens.

Lactic acid bacteria (LAB) are widely known for their probiotic activities for centuries. These bacteria synthesise some secretory proteinaceous toxins, bacteriocins, which help destroy similar or interrelated bacterial strains. This study was aimed at characterising bacteriocins extracted from Lactobacillus spp. found in yoghurt and assessing their bactericidal effect on foodborne bacteria. Twelve isolated Lactobacillus spp. were examined to produce bacteriocins by the organic solvent extraction method. Bacteriocins produced by two of these strains, Lactobacillus helveticus (BLh) and Lactobacillus plantarum (BLp), showed the most significant antimicrobial activity, especially against Staphylococcus aureus and Acinetobacter baumannii. Analysis of SDS-PAGE showed that L. plantarum and L. helveticus bacteriocins have a molecular weight of ~10 kDa and ~15 kDa, respectively. L. plantarum (BLp) bacteriocin was heat stable while L. helveticus (BLh) bacteriocin was heat labile. Both bacteriocins have shown activity at acidic pH. Exposure to a UV light enhances the activity of the BLh; however, it had negligible effects on the BLp. Different proteolytic enzymes confirmed the proteinaceous nature of both the bacteriocins. From this study, it was concluded that bacteriocin extracts from L. helveticus (BLh) can be considered a preferable candidate against foodborne pathogens as compared to L. plantarum (BLp). These partially purified bacteriocins should be further processed to attain purified product that could be useful for food spoilage and preservation purposes.

Probiotic Properties of Lactobacillus helveticus and Lactobacillus plantarum Isolated from Traditional Pakistani Yoghurt.

Probiotic bacteria are of utmost importance owing to their extensive utilisation in dairy products and in the prevention of various intestinal diseases. The objective of this study was to assess the probiotic properties of bacteriocin-producing isolates of Lactobacillus helveticus and Lactobacillus plantarum isolated from traditional Pakistani yoghurt. In this study, ten bacteriocin-producing isolates were selected to screen for the probiotic property. The isolates showed resistance to acidic pH (6-6.5), bile salt (0.01-1%), and 1-7% NaCl salt and showed good growth at acidic pH and antibacterial activity against ten different foodborne pathogens. Interestingly, these isolates were proved to be effective against Actinobacter baumannii but least effective against Klebsiella pneumoniae and Pseudomonas aeruginosa. A few isolates were found to be resistant to some antibiotics like vancomycim, gentamycin, erythromycin, streptomycin, and clindamycin. Our results provide strong evidence in favour of traditional Pakistani yoghurts as a potential source of bacteriocin-producing bacteria with an added benefit of the probiotic property. Specifically, LBh5 was considered a good probiotic isolate as compared to other isolates used in the study. Further extensive research should be done on isolation and characterisation of probiotic isolates from local fermented foods, and then, these isolates should be used in the development of probiotic enriched food supplements in Pakistan.

Two-site recognition of Staphylococcus aureus peptidoglycan by lysostaphin SH3b.

Lysostaphin is a bacteriolytic enzyme targeting peptidoglycan, the essential component of the bacterial cell envelope. It displays a very potent and specific activity toward staphylococci, including methicillin-resistant Staphylococcus aureus. Lysostaphin causes rapid cell lysis and disrupts biofilms, and is therefore a therapeutic agent of choice to eradicate staphylococcal infections. The C-terminal SH3b domain of lysostaphin recognizes peptidoglycans containing a pentaglycine crossbridge and has been proposed to drive the preferential digestion of staphylococcal cell walls. Here we elucidate the molecular mechanism underpinning recognition of staphylococcal peptidoglycan by the lysostaphin SH3b domain. We show that the pentaglycine crossbridge and the peptide stem are recognized by two independent binding sites located on opposite sides of the SH3b domain, thereby inducing a clustering of SH3b domains. We propose that this unusual binding mechanism allows synergistic and structurally dynamic recognition of S. aureus peptidoglycan and underpins the potent bacteriolytic activity of this enzyme.

Conformational exchange in the potassium channel blocker ShK.

ShK is a 35-residue disulfide-linked polypeptide produced by the sea anemone Stichodactyla helianthus, which blocks the potassium channels Kv1.1 and Kv1.3 with pM affinity. An analogue of ShK has been developed that blocks Kv1.3 > 100 times more potently than Kv1.1, and has completed Phase 1b clinical trials for the treatment of autoimmune diseases such as psoriasis and rheumatoid arthritis. Previous studies have indicated that ShK undergoes a conformational exchange that is critical to its function, but this has proved difficult to characterise. Here, we have used high hydrostatic pressure as a tool to increase the population of the alternative state, which is likely to resemble the active form that binds to the Kv1.3 channel. By following changes in chemical shift with pressure, we have derived the chemical shift values of the low- and high-pressure states, and thus characterised the locations of structural changes. The main difference is in the conformation of the Cys17-Cys32 disulfide, which is likely to affect the positions of the critical Lys22-Tyr23 pair by twisting the 21-24 helix and increasing the solvent exposure of the Lys22 sidechain, as indicated by molecular dynamics simulations.

Paramagnetic relaxation enhancement-assisted structural characterization of a partially disordered conformation of ubiquitin.

Nuclear magnetic resonance (NMR) is a powerful tool to study three-dimensional structures as well as protein conformational fluctuations in solution, but it is compromised by increases in peak widths and missing signals. We previously reported that ubiquitin has two folded conformations, N1 and N2 and plus another folded conformation, I, in which some amide group signals of residues 33-41 almost disappeared above 3 kbar at pH 4.5 and 273 K. Thus, well-converged structural models could not be obtained for this region owing to the absence of distance restraints. Here, we reexamine the problem using the ubiquitin Q41N variant as a model for this locally disordered conformation, I. We demonstrate that the variant shows pressure-induced loss of backbone amide group signals at residues 28, 33, 36, and 39-41 like the wild-type, with a similar but smaller effect on CαH and CßH signals. In order to characterize this I structure, we measured paramagnetic relaxation enhancement (PRE) under high pressure to obtain distance restraints, and calculated the structure assisted by Bayesian inference. We conclude that the more disordered I conformation observed at pH 4.0, 278 K, and 2.5 kbar largely retained the N2 conformation, although the amide groups at residues 33-41 have more heterogeneous conformations and more contact with water, which differ from the N1 and N2 states. The PRE-assisted strategy has the potential to improve structural characterization of proteins that lack NMR signals, especially for relatively more open and hydrated protein conformations.