Discovery of periplasmic solute binding proteins with specificity for ketone bodies: ß-hydroxybutyrate binding proteins.

Polyhydroxyalkanoates are a class of biodegradable, thermoplastic polymers which represent a major carbon source for various bacteria. Proteins which mediate the translocation of polyhydroxyalkanoate breakdown products, such as ß-hydroxybutyrate (BHB)-a ketone body which in humans serves as an important biomarker, have not been well characterized. In our investigation to screen a solute-binding protein (SBP) which can act as a suitable recognition element for BHB, we uncovered insights at the intersection of bacterial metabolism and diagnostics. Herein, we identify SBPs associated with putative ATP-binding cassette transporters that specifically recognize BHB, with the potential to serve as recognition elements for continuous quantification of this analyte. Through bioinformatic analysis, we identified candidate SBPs from known metabolizers of polyhydroxybutyrate-including proteins from Cupriavidus necator, Ensifer meliloti, Paucimonas lemoignei, and Thermus thermophilus. After recombinant expression in Escherichia coli, we demonstrated with intrinsic tryptophan fluorescence spectroscopy that four candidate proteins interacted with BHB, ranging from nanomolar to micromolar affinity. Tt.2, an intrinsically thermostable protein from Thermus thermophilus, was observed to have the tightest binding and specificity for BHB, which was confirmed by isothermal calorimetry. Structural analyses facilitated by AlphaFold2, along with molecular docking and dynamics simulations, were used to hypothesize key residues in the binding pocket and to model the conformational dynamics of substrate unbinding. Overall, this study provides strong evidence identifying the cognate ligands of SBPs which we hypothesize to be involved in prokaryotic cellular translocation of polyhydroxyalkanoate breakdown products, while highlighting these proteins' promising biotechnological application.

Identification and Characterization of a Bacterial Periplasmic Solute Binding Protein That Binds l-Amino Acid Amides.

Periplasmic solute-binding proteins (SBPs) are key ligand recognition components of bacterial ATP-binding cassette (ABC) transporters that allow bacteria to import nutrients and metabolic precursors from the environment. Periplasmic SBPs comprise a large and diverse family of proteins, of which only a small number have been empirically characterized. In this work, we identify a set of 610 unique uncharacterized proteins within the SBP_bac_5 family that are found in conserved operons comprising genes encoding (i) ABC transport systems and (ii) putative amidases from the FmdA_AmdA family. From these uncharacterized SBP_bac_5 proteins, we characterize a representative periplasmic SBP from Mesorhizobium sp. A09 (MeAmi_SBP) and show that MeAmi_SBP binds l-amino acid amides but not the corresponding l-amino acids. An X-ray crystal structure of MeAmi_SBP bound to l-serinamide highlights the residues that impart distinct specificity for l-amino acid amides and reveals a structural Ca2+ binding site within one of the lobes of the protein. We show that the residues involved in ligand and Ca2+ binding are conserved among the 610 SBPs from experimentally uncharacterized FmdA_AmdA amidase-associated ABC transporter systems, suggesting these homologous systems are also likely to be involved in the sensing, uptake, and metabolism of l-amino acid amides across many Gram-negative nitrogen-fixing soil bacteria. We propose that MeAmi_SBP is involved in the uptake of such solutes to supplement pathways such as the citric acid cycle and the glutamine synthetase-glutamate synthase pathway. This work expands our currently limited understanding of microbial interactions with l-amino acid amides and bacterial nitrogen utilization.

Molecular handcraft of a well-folded protein chimera.

Modular assembly is a compelling pathway to create new proteins, a concept supported by protein engineering and millennia of evolution. Natural evolution provided a repository of building blocks, known as domains, which trace back to even shorter segments that underwent numerous 'copy-paste' processes culminating in the scaffolds we see today. Utilizing the subdomain-database Fuzzle, we constructed a fold-chimera by integrating a flavodoxin-like fragment into a periplasmic binding protein. This chimera is well-folded and a crystal structure reveals stable interfaces between the fragments. These findings demonstrate the adaptability of α/ß-proteins and offer a stepping stone for optimization. By emphasizing the practicality of fragment databases, our work pioneers new pathways in protein engineering. Ultimately, the results substantiate the conjecture that periplasmic binding proteins originated from a flavodoxin-like ancestor.

Interactive computational and experimental approaches improve the sensitivity of periplasmic binding protein-based nicotine biosensors for measurements in biofluids.

We developed fluorescent protein sensors for nicotine with improved sensitivity. For iNicSnFR12 at pH 7.4, the proportionality constant for ∆F/F0vs [nicotine] (δ-slope, 2.7 µM-1) is 6.1-fold higher than the previously reported iNicSnFR3a. The activated state of iNicSnFR12 has a fluorescence quantum yield of at least 0.6. We measured similar dose-response relations for the nicotine-induced absorbance increase and fluorescence increase, suggesting that the absorbance increase leads to the fluorescence increase via the previously described nicotine-induced conformational change, the 'candle snuffer' mechanism. Molecular dynamics (MD) simulations identified a binding pose for nicotine, previously indeterminate from experimental data. MD simulations also showed that Helix 4 of the periplasmic binding protein (PBP) domain appears tilted in iNicSnFR12 relative to iNicSnFR3a, likely altering allosteric network(s) that link the ligand binding site to the fluorophore. In thermal melt experiments, nicotine stabilized the PBP of the tested iNicSnFR variants. iNicSnFR12 resolved nicotine in diluted mouse and human serum at 100 nM, the peak [nicotine] that occurs during smoking or vaping, and possibly at the decreasing levels during intervals between sessions. NicSnFR12 was also partially activated by unidentified endogenous ligand(s) in biofluids. Improved iNicSnFR12 variants could become the molecular sensors in continuous nicotine monitors for animal and human biofluids.

Retracing the evolution of a modern periplasmic binding protein.

Investigating the evolution of structural features in modern multidomain proteins helps to understand their immense diversity and functional versatility. The class of periplasmic binding proteins (PBPs) offers an opportunity to interrogate one of the main processes driving diversification: the duplication and fusion of protein sequences to generate new architectures. The symmetry of their two-lobed topology, their mechanism of binding, and the organization of their operon structure led to the hypothesis that PBPs arose through a duplication and fusion event of a single common ancestor. To investigate this claim, we set out to reverse the evolutionary process and recreate the structural equivalent of a single-lobed progenitor using ribose-binding protein (RBP) as our model. We found that this modern PBP can be deconstructed into its lobes, producing two proteins that represent possible progenitor halves. The isolated halves of RBP are well folded and monomeric proteins, albeit with a lower thermostability, and do not retain the original binding function. However, the two entities readily form a heterodimer in vitro and in-cell. The x-ray structure of the heterodimer closely resembles the parental protein. Moreover, the binding function is fully regained upon formation of the heterodimer with a ligand affinity similar to that observed in the modern RBP. This highlights how a duplication event could have given rise to a stable and functional PBP-like fold and provides insights into how more complex functional structures can evolve from simpler molecular components.

Thermostable homologues of the periplasmic siderophore-binding protein CeuE from Geobacillus stearothermophilus and Parageobacillus thermoglucosidasius.

Siderophore-binding proteins from two thermophilic bacteria, Geobacillus stearothermophilus and Parageobacillus thermoglucosidasius, were identified from a search of sequence databases, cloned and overexpressed. They are homologues of the well characterized protein CjCeuE from Campylobacter jejuni. The iron-binding histidine and tyrosine residues are conserved in both thermophiles. Crystal structures were determined of the apo proteins and of their complexes with iron(III)-azotochelin and its analogue iron(III)-5-LICAM. The thermostability of both homologues was shown to be about 20°C higher than that of CjCeuE. Similarly, the tolerance of the homologues to the organic solvent dimethylformamide (DMF) was enhanced, as reflected by the respective binding constants for these ligands measured in aqueous buffer at pH 7.5 in the absence and presence of 10% and 20% DMF. Consequently, these thermophilic homologues offer advantages in the development of artificial metalloenzymes using the CeuE family.

Structures of permuted halves of a modern ribose-binding protein.

Periplasmic binding proteins (PBPs) are a class of proteins that participate in the cellular transport of various ligands. They have been used as model systems to study mechanisms in protein evolution, such as duplication, recombination and domain swapping. It has been suggested that PBPs evolved from precursors half their size. Here, the crystal structures of two permuted halves of a modern ribose-binding protein (RBP) from Thermotoga maritima are reported. The overexpressed proteins are well folded and show a monomer-dimer equilibrium in solution. Their crystal structures show partially noncanonical PBP-like fold type I conformations with structural deviations from modern RBPs. One of the half variants forms a dimer via segment swapping, suggesting a high degree of malleability. The structural findings on these permuted halves support the evolutionary hypothesis that PBPs arose via a duplication event of a flavodoxin-like protein and further support a domain-swapping step that might have occurred during the evolution of the PBP-like fold, a process that is necessary to generate the characteristic motion of PBPs essential to perform their functions.

Structural basis of lipoprotein recognition by the bacterial Lol trafficking chaperone LolA.

In gram-negative bacteria, lipoproteins are vital structural components of the outer membrane (OM) and crucial elements of machineries central to the physiology of the cell envelope. A dedicated apparatus, the Lol system, is required for the correct localization of OM lipoproteins and is essential for viability. The periplasmic chaperone LolA is central to this trafficking pathway, accepting triacylated lipoproteins from the inner membrane transporter LolCDE, before carrying them across the periplasm to the OM receptor LolB. Here, we report a crystal structure of liganded LolA, generated in vivo, revealing the molecular details of lipoprotein association. The structure highlights how LolA, initially primed to receive lipoprotein by interaction with LolC, further opens to accommodate the three ligand acyl chains in a precise conformation within its cavity. LolA forms extensive interactions with the acyl chains but not with any residue of the cargo, explaining the chaperone's ability to transport structurally diverse lipoproteins. Structural characterization of a ligandedLolA variant incapable of lipoprotein release reveals aberrant association, demonstrating the importance of the LolCDE-coordinated, sequential opening of LolA for inserting lipoprotein in a manner productive for subsequent trafficking. Comparison with existing structures of LolA in complex with LolC or LolCDE reveals substantial overlap of the lipoprotein and LolC binding sites within the LolA cavity, demonstrating that insertion of lipoprotein acyl chains physically disengages the chaperone protein from the transporter by perturbing interaction with LolC. Taken together, our data provide a key step toward a complete understanding of a fundamentally important trafficking pathway.

Cryo-EM reveals unique structural features of the FhuCDB Escherichia coli ferrichrome importer.

As one of the most elegant biological processes developed in bacteria, the siderophore-mediated iron uptake demands the action of specific ATP-binding cassette (ABC) importers. Although extensive studies have been done on various ABC importers, the molecular basis of these iron-chelated-siderophore importers are still not fully understood. Here, we report the structure of a ferrichrome importer FhuCDB from Escherichia coli at 3.4 Å resolution determined by cryo electron microscopy. The structure revealed a monomeric membrane subunit of FhuB with a substrate translocation pathway in the middle. In the pathway, there were unique arrangements of residues, especially layers of methionines. Important residues found in the structure were interrogated by mutagenesis and functional studies. Surprisingly, the importer's ATPase activity was decreased upon FhuD binding, which deviated from the current understanding about bacterial ABC importers. In summary, to the best of our knowledge, these studies not only reveal a new structural twist in the type II ABC importer subfamily, but also provide biological insights in the transport of iron-chelated siderophores.

Fine-tuning spermidine binding modes in the putrescine binding protein PotF.

A profound understanding of the molecular interactions between receptors and ligands is important throughout diverse research, such as protein design, drug discovery, or neuroscience. What determines specificity and how do proteins discriminate against similar ligands? In this study, we analyzed factors that determine binding in two homologs belonging to the well-known superfamily of periplasmic binding proteins, PotF and PotD. Building on a previously designed construct, modes of polyamine binding were swapped. This change of specificity was approached by analyzing local differences in the binding pocket as well as overall conformational changes in the protein. Throughout the study, protein variants were generated and characterized structurally and thermodynamically, leading to a specificity swap and improvement in affinity. This dataset not only enriches our knowledge applicable to rational protein design but also our results can further lay groundwork for engineering of specific biosensors as well as help to explain the adaptability of pathogenic bacteria.

Actinobacillus utilizes a binding protein-dependent ABC transporter to acquire the active form of vitamin B6.

Bacteria require high-efficiency uptake systems to survive and proliferate in nutrient-limiting environments, such as those found in host organisms. ABC transporters in the bacterial plasma membrane provide a mechanism for transport of many substrates. In this study, we examine an operon containing a periplasmic binding protein in Actinobacillus for its potential role in nutrient acquisition. The electron density map of 1.76 Å resolution obtained from the crystal structure of the periplasmic binding protein was best fit with a molecular model containing a pyridoxal-5'-phosphate (P5P/pyridoxal phosphate/the active form of vitamin B6) ligand within the protein's binding site. The identity of the P5P bound to this periplasmic binding protein was verified by isothermal titration calorimetry, microscale thermophoresis, and mass spectrometry, leading us to name the protein P5PA and the operon P5PAB. To illustrate the functional utility of this uptake system, we introduced the P5PAB operon from Actinobacillus pleuropneumoniae into an Escherichia coli K-12 strain that was devoid of a key enzyme required for P5P synthesis. The growth of this strain at low levels of P5P supports the functional role of this operon in P5P uptake. This is the first report of a dedicated P5P bacterial uptake system, but through bioinformatics, we discovered homologs mainly within pathogenic representatives of the Pasteurellaceae family, suggesting that this operon exists more widely outside the Actinobacillus genus.

Conformational changes and binding property of the periplasmic binding protein BtuF during vitamin B12 transport revealed by collision-induced unfolding, hydrogen-deuterium exchange mass spectrometry and molecular dynamic simulation.

The periplasmic binding protein (PBP) BtuF plays a key role in transporting vitamin B12 from periplasm to the ATP-binding cassette (ABC) transporter BtuCD. Conformational changes of BtuF during transport can hardly be captured by traditional biophysical methods and the exact mechanism regarding B12 and BtuF recognition is still under debate. In the present work, conformational changes of BtuF upon B12 binding and release were investigated using hybrid approaches including collision-induced unfolding (CIU), hydrogen deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulation. It was found that B12 binding increased the stability of BtuF. In addition, fast exchange regions of BtuF were localized. Most importantly, midpoint of hinge helix in BtuF was found highly flexible, and binding of B12 proceed in a manner similar to the Venus flytrap mechanism. Our study therefore delineates a clear view of BtuF delivering B12, and demonstrated a hybrid approach encompassing MS and computer based methods that holds great potential to the probing of conformational dynamics of proteins in action.

Stability of Ligand-induced Protein Conformation Influences Affinity in Maltose-binding Protein.

Our understanding of what determines ligand affinity of proteins is poor, even with high-resolution structures available. Both the non-covalent ligand-protein interactions and the relative free energies of available conformations contribute to the affinity of a protein for a ligand. Distant, non-binding site residues can influence the ligand affinity by altering the free energy difference between a ligand-free and ligand-bound conformation. Our hypothesis is that when different ligands induce distinct ligand-bound conformations, it should be possible to tweak their affinities by changing the free energies of the available conformations. We tested this idea for the maltose-binding protein (MBP) from Escherichia coli. We used single-molecule Förster resonance energy transfer (smFRET) to distinguish several unique ligand-bound conformations of MBP. We engineered mutations, distant from the binding site, to affect the stabilities of different ligand-bound conformations. We show that ligand affinity can indeed be altered in a conformation-dependent manner. Our studies provide a framework for the tuning of ligand affinity, apart from modifying binding site residues.

Bioinformatics analysis and biochemical characterisation of ABC transporter-associated periplasmic substrate-binding proteins ModA and MetQ from Helicobacter pylori strain SS1.

The human gastric pathogen Helicobacter pylori relies on the uptake of host-provided nutrients for its proliferation and pathogenicity. ABC transporters that mediate import of small molecules into the cytoplasm of H. pylori employ their cognate periplasmic substrate-binding proteins (SBPs) for ligand capture in the periplasm. The genome of the mouse-adapted strain SS1 of H. pylori encodes eight ABC transporter-associated SBPs, but little is known about their specificity or structure. In this study, we demonstrated that the SBP annotated as ModA binds molybdate (MoO42-, KD = 3.8 nM) and tungstate (WO42-, KD = 7.8 nM). In addition, we showed that MetQ binds D-methionine (KD = 9.5 µM), but not L-methionine, which suggests the existence of as yet unknown pathway for L-methionine uptake. Homology modelling has led to identification of the ligand-binding residues.

Structural and Functional Investigation of the Periplasmic Arsenate-Binding Protein ArrX from Chrysiogenes arsenatis.

The anaerobic bacterium Chrysiogenes arsenatis respires using the oxyanion arsenate (AsO43-) as the terminal electron acceptor, where it is reduced to arsenite (AsO33-) while concomitantly oxidizing various organic (e.g., acetate) electron donors. This respiratory activity is catalyzed in the periplasm of the bacterium by the enzyme arsenate reductase (Arr), with expression of the enzyme controlled by a sensor histidine kinase (ArrS) and a periplasmic-binding protein (PBP), ArrX. Here, we report for the first time, the molecular structure of ArrX in the absence and presence of bound ligand arsenate. Comparison of the ligand-bound structure of ArrX with other PBPs shows a high level of conservation of critical residues for ligand binding by these proteins; however, this suite of PBPs shows different structural alterations upon ligand binding. For ArrX and its homologue AioX (from Rhizobium sp. str. NT-26), which specifically binds arsenite, the structures of the substrate-binding sites in the vicinity of a conserved and critical cysteine residue contribute to the discrimination of binding for these chemically similar ligands.

The hitchhiker's guide to the periplasm: Unexpected molecular interactions of polymyxin B1 in E. coli.

The periplasm of Gram-negative bacteria is a complex, highly crowded molecular environment. Little is known about how antibiotics move across the periplasm and the interactions they experience. Here, atomistic molecular dynamics simulations are used to study the antibiotic polymyxin B1 within models of the periplasm, which are crowded to different extents. We show that PMB1 is likely to be able to "hitchhike" within the periplasm by binding to lipoprotein carriers-a previously unreported passive transport route. The simulations reveal that PMB1 forms both transient and long-lived interactions with proteins, osmolytes, lipids of the outer membrane, and the cell wall, and is rarely uncomplexed when in the periplasm. Furthermore, it can interfere in the conformational dynamics of native proteins. These are important considerations for interpreting its mechanism of action and are likely to also hold for other antibiotics that rely on diffusion to cross the periplasm.

A comprehensive binding study illustrates ligand recognition in the periplasmic binding protein PotF.

Periplasmic binding proteins (PBPs) are ubiquitous receptors in gram-negative bacteria. They sense solutes and play key roles in nutrient uptake. Escherichia coli's putrescine receptor PotF has been reported to bind putrescine and spermidine. We reveal that several similar biogenic polyamines are recognized by PotF. Using isothermal titration calorimetry paired with X-ray crystallography of the different complexes, we unveil PotF's binding modes in detail. The binding site for PBPs is located between two lobes that undergo a large conformational change upon ligand recognition. Hence, analyzing the influence of ligands on complex formation is crucial. Therefore, we solved crystal structures of an open and closed apo state and used them as a basis for molecular dynamics simulations. In addition, we accessed structural behavior in solution for all complexes by 1H-15N HSQC NMR spectroscopy. This combined analysis provides a robust framework for understanding ligand binding for future developments in drug design and protein engineering.

Design of a mediator-free, non-enzymatic electrochemical biosensor for glutamate detection.

A mediator-free, non-enzymatic electrochemical biosensor was constructed by covalent immobilization of a genetically engineered periplasmic glutamate binding protein onto gold nanoparticle-modified, screen-printed carbon electrodes (GluBP/AuNP/SPCE) for the purpose of direct measurement of glutamate levels. Glutamate serves as the predominant excitatory neurotransmitter in the central nervous system. As high levels of glutamate are an indicator of many neurologic disorders, there is a need for advancements in glutamate detection technologies. The biosensor was evaluated for glutamate detection by cyclic voltammetry. Binding of glutamate to the immobilized glutamate binding protein results in a conformational change of the latter that alters the microenvironment on the surface of the sensor, which is manifested as a change in signal. Dose-response plots correlating the electrochemical signal to glutamate concentration revealed a detection limit of 0.15 µM with a linear range of 0.1-0.8 µM. Selectivity studies confirmed a strong preferential response of the biosensor for glutamate against common interfering compounds.

129Xe NMR-Protein Sensor Reveals Cellular Ribose Concentration.

Dysregulation of cellular ribose uptake can be indicative of metabolic abnormalities or tumorigenesis. However, analytical methods are currently limited for quantifying ribose concentration in complex biological samples. Here, we utilize the highly specific recognition of ribose by ribose-binding protein (RBP) to develop a single-protein ribose sensor detectable via a sensitive NMR technique known as hyperpolarized 129Xe chemical exchange saturation transfer (hyper-CEST). We demonstrate that RBP, with a tunable ribose-binding site and further engineered to bind xenon, enables the quantitation of ribose over a wide concentration range (nM to mM). Ribose binding induces the RBP "closed" conformation, which slows Xe exchange to a rate detectable by hyper-CEST. Such detection is remarkably specific for ribose, with the minimal background signal from endogenous sugars of similar size and structure, for example, glucose or ribose-6-phosphate. Ribose concentration was measured for mammalian cell lysate and serum, which led to estimates of low-mM ribose in a HeLa cell line. This highlights the potential for using genetically encoded periplasmic binding proteins such as RBP to measure metabolites in different biological fluids, tissues, and physiologic states.

Structural features of the glutamate-binding protein from Corynebacterium glutamicum.

L-glutamate (Glu) is the major excitatory transmitter in mammalian brain. Inadequate concentration of Glu in the brain correlates to mood disorder. In industry, Glu is used as a flavour enhancer in food and in foodstuff processing. A high concentration of Glu has several effects on human health such as hypersensitive effects, headache and stomach pain. The presence of Glu in food can be detected by different analytical methods based on chromatography, or capillary electrophoresis or amperometric techniques. We have isolated and characterized a glutamate-binding protein (GluB) from the Gram-positive bacteria Corynebacterium glutamicum. Together with GluC protein, GluD protein and the cytoplasmic protein GluA, GluB permits the transport of Glu in/out of cell. In this study, we have investigated the binding features of GluB as well as the effect of temperature on its structure both in the absence and in the presence of Glu. The results have showed that GluB has a high affinity and selectivity versus Glu (nanomolar range) and the presence of the ligand induces a higher thermal stability of the protein structure.