ABSTRACT
Purines and their derivatives control intracellular energy homeostasis and nucleotide synthesis, and act as signaling molecules. Here, we combine structural and sequence information to define a purine-binding motif that is present in sensor domains of thousands of bacterial receptors that modulate motility, gene expression, metabolism, and second-messenger turnover. Microcalorimetric titrations of selected sensor domains validate their ability to specifically bind purine derivatives, and evolutionary analyses indicate that purine sensors share a common ancestor with amino-acid receptors. Furthermore, we provide experimental evidence of physiological relevance of purine sensing in a second-messenger signaling system that modulates c-di-GMP levels.
Subject(s)
Bacterial Proteins , Purines , Signal Transduction , Purines/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Cyclic GMP/metabolism , Cyclic GMP/analogs & derivatives , Gene Expression Regulation, Bacterial , Bacteria/metabolism , Bacteria/genetics , Escherichia coli/metabolism , Escherichia coli/genetics , Second Messenger SystemsABSTRACT
Bacterial receptors feed into multiple signal transduction pathways that regulate a variety of cellular processes including gene expression, second messenger levels and motility. Receptors are typically activated by signal binding to ligand binding domains (LBD). Cache domains are omnipresent LBDs found in bacteria, archaea, and eukaryotes, including humans. They form the predominant family of extracytosolic bacterial LBDs and were identified in all major receptor types. Cache domains are composed of either a single (sCache) or a double (dCache) structural module. The functional relevance of bimodular LBDs remains poorly understood. Here, we identify the PacF chemoreceptor in the phytopathogen Pectobacterium atrosepticum that recognizes formate at the membrane distal module of its dCache domain, triggering chemoattraction. We further demonstrate that a family of formate-specific sCache domains has evolved from a dCache domain, exemplified by PacF, by losing the membrane proximal module. By solving high-resolution structures of two family members in complex with formate, we show that the molecular basis for formate binding at sCache and dCache domains is highly similar, despite their low sequence identity. The apparent loss of the membrane proximal module may be related to the observation that dCache domains bind ligands typically at the membrane distal module, whereas the membrane proximal module is not involved in signal sensing. This work advances our understanding of signal sensing in bacterial receptors and suggests that evolution by reducing complexity may be a common trend shaping their diversity. Significance: Many bacterial receptors contain multi-modular sensing domains indicative of complex sensory processes. The presence of more than one sensing module likely permits the integration of multiple signals, although, the molecular detail and functional relevance for these complex sensors remain poorly understood. Bimodular sensory domains are likely to have arisen from the fusion or duplication of monomodular domains. Evolution by increasing complexity is generally believed to be a dominant force. Here we reveal the opposite - how a monomodular sensing domain has evolved from a bimodular one. Our findings will thus motivate research to establish whether evolution by decreasing complexity is typical of other sensory domains.
ABSTRACT
The human NQO1 (hNQO1) is a flavin adenine nucleotide (FAD)-dependent oxidoreductase that catalyzes the two-electron reduction of quinones to hydroquinones, being essential for the antioxidant defense system, stabilization of tumor suppressors, and activation of quinone-based chemotherapeutics. Moreover, it is overexpressed in several tumors, which makes it an attractive cancer drug target. To decipher new structural insights into the flavin reductive half-reaction of the catalytic mechanism of hNQO1, we have carried serial crystallography experiments at new ID29 beamline of the ESRF to determine, to the best of our knowledge, the first structure of the hNQO1 in complex with NADH. We have also performed molecular dynamics simulations of free hNQO1 and in complex with NADH. This is the first structural evidence that the hNQO1 functional cooperativity is driven by structural communication between the active sites through long-range propagation of cooperative effects across the hNQO1 structure. Both structural results and MD simulations have supported that the binding of NADH significantly decreases protein dynamics and stabilizes hNQO1 especially at the dimer core and interface. Altogether, these results pave the way for future time-resolved studies, both at x-ray free-electron lasers and synchrotrons, of the dynamics of hNQO1 upon binding to NADH as well as during the FAD cofactor reductive half-reaction. This knowledge will allow us to reveal unprecedented structural information of the relevance of the dynamics during the catalytic function of hNQO1.
Subject(s)
Antineoplastic Agents , Neoplasms , Humans , Crystallography , Temperature , NAD , Antineoplastic Agents/chemistry , Flavins , Crystallography, X-Ray , NAD(P)H Dehydrogenase (Quinone)ABSTRACT
Purines and their derivatives are key molecules for controlling intracellular energy homeostasis and nucleotide synthesis. In eukaryotes, including humans, purines also act as signaling molecules that mediate extracellular communication and control key cellular processes, such as proliferation, migration, differentiation, and apoptosis. However, the signaling role of purines in bacteria is largely unknown. Here, by combining structural and sequence information, we define a purine-binding motif, which is present in sensor domains of thousands of bacterial receptors that modulate motility, gene expression, metabolism and second messenger turnover. The screening of compound libraries and microcalorimetric titrations of selected sensor domains validated their ability to specifically bind purine derivatives. The physiological relevance of purine sensing was demonstrated in a second messenger signaling system that modulates c-di-GMP levels.
ABSTRACT
Microorganisms are exposed in their natural niches to a wide diversity of signal molecules. Specific detection of these signals results in alterations in microbial metabolism and physiology. Auxins like indole-3-acetic acid are key phytohormones that regulate plant growth and development. Nonetheless, auxin biosynthesis is not restricted to plants but is ubiquitous in all kingdoms of life. This wide phylogenetic distribution of auxins production, together with the diversity of regulated cellular processes, have made auxins key intra- and inter-kingdom signal molecules in life modulating, for example microbial physiology, metabolism and virulence. Despite their increasing importance as global signal molecules, the mechanisms by which auxins perform their regulatory functions in microorganisms are largely unknown. In this article, we outline recent research that has advanced our knowledge of the mechanisms of bacterial auxin perception. We also highlight the potential applications of this research in aspects such as antibiotic production, biosensor design, plant microbiome engineering and antivirulence therapies.
Subject(s)
Indoleacetic Acids , Plant Growth Regulators , Phylogeny , Indoleacetic Acids/metabolism , Plant Growth Regulators/metabolism , Plants/metabolism , Plant DevelopmentABSTRACT
About half of the known bacterial species perform chemotaxis that gains them access to sites that are optimal for growth and survival. The motility apparatus and chemotaxis signaling pathway impose a large energetic and metabolic burden on the cell. There is almost no limit to the type of chemoeffectors that are recognized by bacterial chemoreceptors. For example, they include hormones, neurotransmitters, quorum-sensing molecules, and inorganic ions. However, the vast majority of chemoeffectors appear to be of metabolic value. We review here the experimental evidence indicating that accessing nutrients is the main selective force that led to the evolution of chemotaxis.
Subject(s)
Bacterial Proteins , Chemotaxis , Bacterial Proteins/metabolism , Chemoreceptor Cells/metabolism , Bacteria/metabolism , Signal TransductionABSTRACT
The development of bio-MOFs or MOF biocomposites through the combination of MOFs with biopolymers offers the possibility of expanding the potential applications of MOFs, making use of more environmentally benign processes and reagents and giving rise to a new generation of greener and more bio-oriented composite materials. Now, with the increasing use of MOFs for biotechnological applications, the development of new protocols and materials to obtain novel bio-MOFs compatible with biomedical or biotechnological uses is needed. Herein, and as a proof of concept, we have explored the possibility of using short-peptide supramolecular hydrogels as media to promote the growth of MOF particles, giving rise to a new family of bio-MOFs. Short-peptide supramolecular hydrogels are very versatile materials that have shown excellent in vitro and in vivo biomedical applications such as tissue engineering and drug delivery vehicles, among others. These peptides self-assemble by noncovalent interactions, and, as such, these hydrogels are easily reversible, being more biocompatible and biodegradable. These peptides can self-assemble by a multitude of stimuli, such as changes in pH, temperature, solvent, adding salts, enzymatic activity, and so forth. In this work, we have taken advantage of this ability to promote peptide self-assembly with some of the components required to form MOF particles, giving rise to more homogeneous and well-integrated composite materials. Hydrogel formation has been triggered using Zn2+ salts, required to form ZIF-8, and formic acid, required to form MOF-808. Two different protocols for the in situ MOF growth have been developed. Finally, the MOF-808 composite hydrogel has been tested for the decontamination of water polluted with phosphate ions as well as for the catalytic degradation of toxic organophosphate methyl paraoxon in an unbuffered solution.
Subject(s)
Metal-Organic Frameworks , Metal-Organic Frameworks/chemistry , Hydrogels/chemistry , Salts , Peptides , Drug Delivery SystemsABSTRACT
Modified surfaces like siliconized glass are commonly used to support protein crystallization and facilitate obtaining crystals. Over the years, various surfaces have been proposed to decrease the energetic penalty required for consistent protein clustering, but scarce attention has been paid to the underlying mechanisms of interactions. Here, we propose self-assembled monolayers that are surfaces exposing fine-tuned moieties with a very regular topography and subnanometer roughness, as a tool to unveil the interaction between proteins and functionalized surfaces. We studied the crystallization of three model proteins having progressively narrower metastable zones, i.e., lysozyme, catalase, and proteinase K, on monolayers exposing thiol, methacrylate, and glycidyloxy groups. Thanks to comparable surface wettability, the induction or the inhibition of nucleation was readily attributed to the surface chemistry. For example, thiol groups strongly induced the nucleation of lysozyme thanks to electrostatic pairing, whereas methacrylate and glycidyloxy groups had an effect comparable to unfunctionalized glass. Overall, the action of surfaces led to differences in nucleation kinetics, crystal habit, and even crystal form. This approach can support the fundamental understanding of the interaction between protein macromolecules and specific chemical groups, which is crucial for many technological applications in the pharmaceutical and food industry.
ABSTRACT
Bacteria have evolved a sophisticated array of signal transduction systems that allow them to adapt their physiology and metabolism to changing environmental conditions. Typically, these systems recognize signals through dedicated ligand binding domains (LBDs) to ultimately trigger a diversity of physiological responses. Nonetheless, an increasing number of reports reveal that signal transduction receptors also bind antagonists to inhibit responses mediated by agonists. The mechanisms by which antagonists block the downstream signaling cascade remain largely unknown. To advance our knowledge in this field, we used the LysR-type transcriptional regulator AdmX as a model. AdmX activates the expression of an antibiotic biosynthetic cluster in the rhizobacterium Serratia plymuthica. AdmX specifically recognizes the auxin phytohormone indole-3-acetic acid (IAA) and its biosynthetic intermediate indole-3-pyruvic acid (IPA) as signals. However, only IAA, but not IPA, was shown to regulate antibiotic production in S. plymuthica. Here, we report the high-resolution structures of the LBD of AdmX in complex with IAA and IPA. We found that IAA and IPA compete for binding to AdmX. Although IAA and IPA binding does not alter the oligomeric state of AdmX, IPA binding causes a higher degree of compactness in the protein structure. Molecular dynamics simulations revealed significant differences in the binding modes of IAA and IPA by AdmX, and the inspection of the three-dimensional structures evidenced differential agonist- and antagonist-mediated structural changes. Key residues for auxin binding were identified and an auxin recognition motif defined. Phylogenetic clustering supports the recent evolutionary emergence of this motif specifically in plant-associated enterobacteria. IMPORTANCE Although antagonists were found to bind different bacterial signal transduction receptors, we are still at the early stages of understanding the molecular details by which these molecules exert their inhibitory effects. Here, we provide insight into the structural changes resulting from the binding of an agonist and an antagonist to a sensor protein. Our data indicate that agonist and antagonist recognition is characterized by small conformational differences in the LBDs that can be efficiently transmitted to the output domain to modulate the final response. LBDs are subject to strong selective pressures and are rapidly evolving domains. An increasing number of reports support the idea that environmental factors drive the evolution of sensor domains. Given the recent evolutionary history of AdmX homologs, as well as their narrow phyletic distribution within plant-associated bacteria, our results are in accordance with a plant-mediated evolutionary process that resulted in the emergence of receptor proteins that specifically sense auxin phytohormones.
Subject(s)
Indoleacetic Acids , Plant Growth Regulators , Phylogeny , Indoleacetic Acids/metabolism , Plant Growth Regulators/metabolism , Plants/metabolism , Bacteria/metabolism , Anti-Bacterial AgentsABSTRACT
Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 is an effector protein that targets invading DNA and plays a major role in the prokaryotic adaptive immune system. Although Streptococcus pyogenes CRISPR-Cas9 has been widely studied and repurposed for applications including genome editing, its origin and evolution are poorly understood. Here, we investigate the evolution of Cas9 from resurrected ancient nucleases (anCas) in extinct firmicutes species that last lived 2.6 billion years before the present. We demonstrate that these ancient forms were much more flexible in their guide RNA and protospacer-adjacent motif requirements compared with modern-day Cas9 enzymes. Furthermore, anCas portrays a gradual palaeoenzymatic adaptation from nickase to double-strand break activity, exhibits high levels of activity with both single-stranded DNA and single-stranded RNA targets and is capable of editing activity in human cells. Prediction and characterization of anCas with a resurrected protein approach uncovers an evolutionary trajectory leading to functionally flexible ancient enzymes.
Subject(s)
CRISPR-Cas Systems , Endonucleases , Firmicutes , CRISPR-Associated Protein 9/genetics , CRISPR-Associated Protein 9/metabolism , Endonucleases/genetics , Endonucleases/metabolism , Gene Editing , Firmicutes/enzymology , Firmicutes/genetics , RNA, Guide, CRISPR-Cas SystemsABSTRACT
SARS-CoV-2 spike (S) protein mediates virus attachment to the cells and fusion between viral and cell membranes. Membrane fusion is driven by mutual interaction between the highly conserved heptad-repeat regions 1 and 2 (HR1 and HR2) of the S2 subunit of the spike. For this reason, these S2 regions are interesting therapeutic targets for COVID-19. Although HR1 and HR2 have been described as transiently exposed during the fusion process, no significant antibody responses against these S2 regions have been reported. Here we designed chimeric proteins that imitate highly stable HR1 helical trimers and strongly bind to HR2. The proteins have broad inhibitory activity against WT B.1 and BA.1 viruses. Sera from COVID-19 convalescent donors showed significant levels of reactive antibodies (IgG and IgA) against the HR1 mimetic proteins, whereas these antibody responses were absent in sera from uninfected donors. Moreover, both inhibitory activity and antigenicity of the proteins correlate positively with their structural stability but not with the number of amino acid changes in their HR1 sequences, indicating a conformational and conserved nature of the involved epitopes. Our results reveal previously undetected spike epitopes that may guide the design of new robust COVID-19 vaccines and therapies.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Spike Glycoprotein, Coronavirus/chemistry , Viral Envelope Proteins/chemistry , Epitopes , COVID-19 Vaccines , Membrane Glycoproteins/chemistry , Recombinant Fusion Proteins/geneticsABSTRACT
Sonocrystallization implies the application of ultrasound radiation to control the nucleation and crystal growth depending on the actuation time and intensity. Its application allows to induce nucleation at lower supersaturations than required under standard conditions. Although extended in inorganic and organic crystallization, it has been scarcely explored in protein crystallization. Now, that industrial protein crystallization is gaining momentum, the interest on new ways to control protein nucleation and crystal growth is advancing. In this work we present the development of a novel ultrasound bioreactor to study its influence on protein crystallization in agarose gel. Gel media minimize convention currents and sedimentation, favoring a more homogeneous and stable conditions to study the effect of an externally generated low energy ultrasonic irradiation on protein crystallization avoiding other undesired effects such as temperature increase, introduction of surfaces which induce nucleation, destructive cavitation phenomena, etc. In-depth statistical analysis of the results has shown that the impact of ultrasound in gel media on crystal size populations are statistically significant and reproducible.
Subject(s)
Hydrogels , Muramidase , Ultrasonic Waves , Crystallization/methods , Muramidase/chemistry , Proteins/chemistryABSTRACT
Acetylcholine is a central biological signal molecule present in all kingdoms of life. In humans, acetylcholine is the primary neurotransmitter of the peripheral nervous system; it mediates signal transmission at neuromuscular junctions. Here, we show that the opportunistic human pathogen Pseudomonas aeruginosa exhibits chemoattraction toward acetylcholine over a concentration range of 1 µM to 100 mM. The maximal magnitude of the response was superior to that of many other P. aeruginosa chemoeffectors. We demonstrate that this chemoattraction is mediated by the PctD (PA4633) chemoreceptor. Using microcalorimetry, we show that the PctD ligand-binding domain (LBD) binds acetylcholine with a equilibrium dissociation constant (KD) of 23 µM. It also binds choline and with lower affinity betaine. Highly sensitive responses to acetylcholine and choline, and less sensitive responses to betaine and l-carnitine, were observed in Escherichia coli expressing a chimeric receptor comprising the PctD-LBD fused to the Tar chemoreceptor signaling domain. We also identified the PacA (ECA_RS10935) chemoreceptor of the phytopathogen Pectobacterium atrosepticum, which binds choline and betaine but fails to recognize acetylcholine. To identify the molecular determinants for acetylcholine recognition, we report high-resolution structures of PctD-LBD (with bound acetylcholine and choline) and PacA-LBD (with bound betaine). We identified an amino acid motif in PctD-LBD that interacts with the acetylcholine tail. This motif is absent in PacA-LBD. Significant acetylcholine chemotaxis was also detected in the plant pathogens Agrobacterium tumefaciens and Dickeya solani. To the best of our knowledge, this is the first report of acetylcholine chemotaxis and extends the range of host signals perceived by bacterial chemoreceptors. IMPORTANCE P. aeruginosa causes a significant number of deaths annually worldwide. For many pathogens, chemotaxis plays an import role in the initial stages of infection, and deciphering the key chomoeffectors and their cognate chemoreceptors may permit the development of strategies to inhibit this process. Genome analyses have shown that many bacteria possess a large number of chemoreceptors. The chemoeffectors recognized by the large majority of chemoreceptors are unknown. However, identifying these chemoeffectors is crucial for deciphering the evolutionary forces that have shaped chemosensory signaling mechanisms in bacteria with different lifestyles. Our current understanding of the relationship between bacterial lifestyle and chemoreceptor repertoire is limited, and this work contributes to closing this gap in our knowledge. By expanding the list of known chemoeffectors and chemoreceptors, progress is made toward identifying functional receptor homologs in other bacteria.
Subject(s)
Chemotaxis , Pseudomonas aeruginosa , Acetylcholine/metabolism , Bacteria/metabolism , Bacterial Proteins/metabolism , Betaine/metabolism , Chemotaxis/genetics , Choline/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Neurotransmitter Agents/metabolism , Pseudomonas aeruginosa/geneticsABSTRACT
Obligate symbionts typically exhibit high evolutionary rates. Consequently, their proteins may differ considerably from their modern and ancestral homologs in terms of both sequence and properties, thus providing excellent models to study protein evolution. Also, obligate symbionts are challenging to culture in the lab and proteins from uncultured organisms must be produced in heterologous hosts using recombinant DNA technology. Obligate symbionts thus replicate a fundamental scenario of metagenomics studies aimed at the functional characterization and biotechnological exploitation of proteins from the bacteria in soil. Here, we use the thioredoxin from Candidatus Photodesmus katoptron, an uncultured symbiont of flashlight fish, to explore evolutionary and engineering aspects of protein folding in heterologous hosts. The symbiont protein is a standard thioredoxin in terms of 3D-structure, stability and redox activity. However, its folding outside the original host is severely impaired, as shown by a very slow refolding in vitro and an inefficient expression in E. coli that leads mostly to insoluble protein. By contrast, resurrected Precambrian thioredoxins express efficiently in E. coli, plausibly reflecting an ancient adaptation to unassisted folding. We have used a statistical-mechanical model of the folding landscape to guide back-to-ancestor engineering of the symbiont protein. Remarkably, we find that the efficiency of heterologous expression correlates with the in vitro (i.e., unassisted) folding rate and that the ancestral expression efficiency can be achieved with only 1-2 back-to-ancestor replacements. These results demonstrate a minimal-perturbation, sequence-engineering approach to rescue inefficient heterologous expression which may potentially be useful in metagenomics efforts targeting recent adaptations.
Subject(s)
Bacterial Proteins/biosynthesis , Fishes/microbiology , Protein Folding , Recombinant Proteins/biosynthesis , Vibrionaceae/metabolism , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Escherichia coli/metabolism , Metagenomics , Protein Engineering , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Symbiosis , Thioredoxins/biosynthesis , Thioredoxins/chemistry , Vibrionaceae/geneticsABSTRACT
The autoimmobilization of enzymes via cross-linked enzyme crystals (CLECs) has regained interest in recent years, boosted by the extensive knowledge gained in protein crystallization, the decrease of cost and laboriousness of the process, and the development of potential applications. In this work, we present the crystallization and preparative-scale production of reinforced cross-linked lipase crystals (RCLLCs) using a commercial detergent additive as a raw material. Bulk crystallization was carried out in 500 mL of agarose media using the batch technique. Agarose facilitates the homogeneous production of crystals, their cross-linking treatment, and their extraction. RCLLCs were active in an aqueous solution and in hexane, as shown by the hydrolysis of p-nitrophenol butyrate and α-methylbenzyl acetate, respectively. RCLLCs presented both high thermal and robust operational stability, allowing the preparation of a packed-bed chromatographic column to work in a continuous flow. Finally, we determined the three-dimensional (3D) models of this commercial lipase crystallized with and without phosphate at 2.0 and 1.7 Å resolutions, respectively.
ABSTRACT
Bacteria have evolved sophisticated signaling mechanisms to coordinate interactions with organisms of other domains, such as plants, animals and human hosts. Several important signal molecules have been identified that are synthesized by members of different domains and that play important roles in inter-domain communication. In this article, we review recent data supporting that histamine is a signal molecule that may play an important role in inter-domain and inter-species communication. Histamine is a key signal molecule in humans, with multiple functions, such as being a neurotransmitter or modulator of immune responses. More recent studies have shown that bacteria have evolved different mechanisms to sense histamine or histamine metabolites. Histamine sensing in the human pathogen Pseudomonas aeruginosa was found to trigger chemoattraction to histamine and to regulate the expression of many virulence-related genes. Further studies have shown that many bacteria are able to synthesize and secrete histamine. The release of histamine by bacteria in the human gut was found to modulate the host immune responses and, at higher doses, to result in host pathologies. The elucidation of the role of histamine as an inter-domain signaling molecule is an emerging field of research and future investigation is required to assess its potential general nature.
Subject(s)
Bacteria/metabolism , Histamine/metabolism , Signal Transduction , Animals , Bacteria/genetics , Histamine Release , Humans , Models, Biological , Models, MolecularABSTRACT
TEM-1 ß-lactamase degrades ß-lactam antibiotics with a strong preference for penicillins. Sequence reconstruction studies indicate that it evolved from ancestral enzymes that degraded a variety of ß-lactam antibiotics with moderate efficiency. This generalist to specialist conversion involved more than 100 mutational changes, but conserved fold and catalytic residues, suggesting a role for dynamics in enzyme evolution. Here, we develop a conformational dynamics computational approach to rationally mold a protein flexibility profile on the basis of a hinge-shift mechanism. By deliberately weighting and altering the conformational dynamics of a putative Precambrian ß-lactamase, we engineer enzyme specificity that mimics the modern TEM-1 ß-lactamase with only 21 amino acid replacements. Our conformational dynamics design thus re-enacts the evolutionary process and provides a rational allosteric approach for manipulating function while conserving the enzyme active site.
Subject(s)
beta-Lactamases/genetics , beta-Lactamases/metabolism , Amino Acid Sequence/genetics , Catalytic Domain/genetics , Computational Biology , Escherichia coli/drug effects , Escherichia coli/enzymology , Evolution, Molecular , Molecular Dynamics Simulation , Penicillins/metabolism , Protein Conformation , Substrate SpecificityABSTRACT
Protein therapeutics have a major role in medicine in that they are used to treat diverse pathologies. Their three-dimensional structures not only offer higher specificity and lower toxicity than small organic compounds but also make them less stable, limiting their in vivo half-life. Protein analogues obtained by recombinant DNA technology or by chemical modification and/or the use of drug delivery vehicles has been adopted to improve or modulate the in vivo pharmacological activity of proteins. Nevertheless, strategies to improve the shelf-life of protein pharmaceuticals have been less explored, which has challenged the preservation of their activity. Herein, we present a methodology that simultaneously increases the stability of proteins and modulates the release profile, and implement it with human insulin as a proof of concept. Two novel thermally stable insulin composite crystal formulations intended for the therapeutic treatment of diabetes are reported. These composite crystals have been obtained by crystallizing insulin in agarose and fluorenylmethoxycarbonyl-dialanine (Fmoc-AA) hydrogels. This process affords composite crystals, in which hydrogel fibers are occluded. The insulin in both crystalline formulations remains unaltered at 50 °C for 7 days. Differential scanning calorimetry, high-performance liquid chromatography, mass spectrometry, and in vivo studies have shown that insulin does not degrade after the heat treatment. The nature of the hydrogel modifies the physicochemical properties of the crystals. Crystals grown in Fmoc-AA hydrogel are more stable and have a slower dissolution rate than crystals grown in agarose. This methodology paves the way for the development of more stable protein pharmaceuticals overcoming some of the existing limitations.
Subject(s)
Hydrogels/chemistry , Hypoglycemic Agents/chemistry , Insulin/chemistry , Animals , Crystallization/methods , Drug Liberation , Humans , Hypoglycemic Agents/administration & dosage , Insulin/administration & dosage , Male , Peptides/chemistry , Protein Stability , Rats, WistarABSTRACT
Glycosidases are phylogenetically widely distributed enzymes that are crucial for the cleavage of glycosidic bonds. Here, we present the exceptional properties of a putative ancestor of bacterial and eukaryotic family-1 glycosidases. The ancestral protein shares the TIM-barrel fold with its modern descendants but displays large regions with greatly enhanced conformational flexibility. Yet, the barrel core remains comparatively rigid and the ancestral glycosidase activity is stable, with an optimum temperature within the experimental range for thermophilic family-1 glycosidases. None of the â¼5500 reported crystallographic structures of â¼1400 modern glycosidases show a bound porphyrin. Remarkably, the ancestral glycosidase binds heme tightly and stoichiometrically at a well-defined buried site. Heme binding rigidifies this TIM-barrel and allosterically enhances catalysis. Our work demonstrates the capability of ancestral protein reconstructions to reveal valuable but unexpected biomolecular features when sampling distant sequence space. The potential of the ancestral glycosidase as a scaffold for custom catalysis and biosensor engineering is discussed.
Subject(s)
Bacteria/enzymology , Eukaryota/enzymology , Glycoside Hydrolases/metabolism , Heme/metabolism , Allosteric Regulation , Amino Acid Sequence/genetics , Bacteria/genetics , Crystallography, X-Ray , Eukaryota/genetics , Glycoside Hydrolases/genetics , Glycoside Hydrolases/ultrastructure , Molecular Dynamics Simulation , Phylogeny , Protein Structure, Secondary , Sequence Homology, Amino AcidABSTRACT
Bacteria have evolved a variety of signal transduction mechanisms that generate different outputs in response to external stimuli. Chemosensory pathways are widespread in bacteria and are among the most complex signaling mechanisms, requiring the participation of at least six proteins. These pathways mediate flagellar chemotaxis, in addition to controlling alternative functions such as second messenger levels or twitching motility. The human pathogen Pseudomonas aeruginosa has four different chemosensory pathways that carry out different functions and are stimulated by signal binding to 26 chemoreceptors. Recent research employing a diverse range of experimental approaches has advanced enormously our knowledge on these four pathways, establishing P. aeruginosa as a primary model organism in this field. In the first part of this article, we review data on the function and physiological relevance of chemosensory pathways as well as their involvement in virulence, whereas the different transcriptional and posttranscriptional regulatory mechanisms that govern pathway function are summarized in the second part. The information presented will be of help to advance the understanding of pathway function in other organisms.