Structures and coordination chemistry of transporters involved in manganese and iron homeostasis.

A repertoire of transporters plays a crucial role in maintaining homeostasis of biologically essential transition metals, manganese, and iron, thus ensuring cell viability. Elucidating the structure and function of many of these transporters has provided substantial understanding into how these proteins help maintain the optimal cellular concentrations of these metals. In particular, recent high-resolution structures of several transporters bound to different metals enable an examination of how the coordination chemistry of metal ion-protein complexes can help us understand metal selectivity and specificity. In this review, we first provide a comprehensive list of both specific and broad-based transporters that contribute to cellular homeostasis of manganese (Mn2+) and iron (Fe2+ and Fe3+) in bacteria, plants, fungi, and animals. Furthermore, we explore the metal-binding sites of the available high-resolution metal-bound transporter structures (Nramps, ABC transporters, P-type ATPase) and provide a detailed analysis of their coordination spheres (ligands, bond lengths, bond angles, and overall geometry and coordination number). Combining this information with the measured binding affinity of the transporters towards different metals sheds light into the molecular basis of substrate selectivity and transport. Moreover, comparison of the transporters with some metal scavenging and storage proteins, which bind metal with high affinity, reveal how the coordination geometry and affinity trends reflect the biological role of individual proteins involved in the homeostasis of these essential transition metals.

High-resolution structures with bound Mn2+ and Cd2+ map the metal import pathway in an Nramp transporter.

Transporters of the Nramp (Natural resistance-associated macrophage protein) family import divalent transition metal ions into cells of most organisms. By supporting metal homeostasis, Nramps prevent diseases and disorders related to metal insufficiency or overload. Previous studies revealed that Nramps take on a LeuT fold and identified the metal-binding site. We present high-resolution structures of Deinococcus radiodurans (Dra)Nramp in three stable conformations of the transport cycle revealing that global conformational changes are supported by distinct coordination geometries of its physiological substrate, Mn2+, across conformations, and by conserved networks of polar residues lining the inner and outer gates. In addition, a high-resolution Cd2+-bound structure highlights differences in how Cd2+ and Mn2+ are coordinated by DraNramp. Complementary metal binding studies using isothermal titration calorimetry with a series of mutated DraNramp proteins indicate that the thermodynamic landscape for binding and transporting physiological metals like Mn2+ is different and more robust to perturbation than for transporting the toxic Cd2+ metal. Overall, the affinity measurements and high-resolution structural information on metal substrate binding provide a foundation for understanding the substrate selectivity of essential metal ion transporters like Nramps.

Phenol sensing in nature is modulated via a conformational switch governed by dynamic allostery.

The NtrC family of proteins senses external stimuli and accordingly stimulates stress and virulence pathways via activation of associated σ54-dependent RNA polymerases. However, the structural determinants that mediate this activation are not well understood. Here, we establish using computational, structural, biochemical, and biophysical studies that MopR, an NtrC protein, harbors a dynamic bidirectional electrostatic network that connects the phenol pocket to two distal regions, namely the "G-hinge" and the "allosteric linker." While the G-hinge influences the entry of phenol into the pocket, the allosteric linker passes the signal to the downstream ATPase domain. We show that phenol binding induces a rewiring of the electrostatic connections by eliciting dynamic allostery and demonstrates that perturbation of the core relay residues results in a complete loss of ATPase stimulation. Furthermore, we found a mutation of the G-hinge, ∼20 Å from the phenol pocket, promotes altered flexibility by shifting the pattern of conformational states accessed, leading to a protein with 7-fold enhanced phenol binding ability and enhanced transcriptional activation. Finally, we conducted a global analysis that illustrates that dynamic allostery-driven conserved community networks are universal and evolutionarily conserved across species. Taken together, these results provide insights into the mechanisms of dynamic allostery-mediated conformational changes in NtrC sensor proteins.

Tunable Multiplexed Whole-Cell Biosensors as Environmental Diagnostics for ppb-Level Detection of Aromatic Pollutants.

Aromatics such as phenols, benzene, and toluene are carcinogenic xenobiotics which are known to pollute water resources. By employing synthetic biology approaches combined with a structure-guided design, we created a tunable array of whole-cell biosensors (WCBs). The MopR genetic system that has the natural ability to sense and degrade phenol was adapted to detect phenol down to ∼1 ppb, making this sensor capable of directly detecting phenol in permissible limits in drinking water. Importantly, by using a single WCB design, we engineered mutations into the MopR gene that enabled generation of a battery of sensors for a wide array of pollutants. The engineered WCBs were able to sense inert compounds like benzene and xylene which lack active functional groups, without any loss in sensitivity. Overall, this universal programmable biosensor platform can be used to create WCBs that can be deployed on field for rapid testing and screening of suitable drinking water sources.

Design of Protein-Based Biosensors for Selective Detection of Benzene Groups of Pollutants.

Benzene and its derivatives form a class of priority pollutants whose exposure poses grave risk to human health. Since benzene lacks active functional groups, devising specific sensors for its direct detection from a milieu of aromatics has remained a daunting task. Here, we report three engineered protein-based biosensors that exclusively and specifically detect benzene and its derivatives up to a detection limit of 0.3 ppm. Further, the biosensor design has been engineered to create templates that possess the ability to specifically discriminate between alkyl substituted benzene derivatives; such as toluene, m-xylene, and mesitylene. Interference tests with simulated wastewater samples reveal that the engineered biosensors can selectively detect a specific benzene compound in water samples containing a milieu of high concentrations of commonly occurring pollutants. This work demonstrates the potential of structure guided protein engineering as a competent strategy toward design of selective biosensors for direct detection of benzene group of pollutants from real time environmental samples.

Design of Ultrasensitive Protein Biosensor Strips for Selective Detection of Aromatic Contaminants in Environmental Wastewater.

Phenol and its derivatives constitute a class of highly toxic xenobiotics that pollute both river and groundwater. Here, we use a highly stable enzyme-based in vitro biosensing scaffold to develop a chip-based environmental diagnostic for in situ accurate, direct detection of phenol with selectively down to 10 ppb. Mesoporous silica nanoparticles (MCM41) having a pore diameter of 6.5 nm was screened and found to be the optimal solid support for creation of a robust immobilized protein based sensor, which retains stability, enzyme activity, sensitivity, and selectivity at par with solution format. The sensor strip exhibits minimal cross reactivity in simulated wastewater, crowded with several common pollutants. Moreover, this design is competent towards detection of phenol content with 95% accuracy in real-time environmental samples collected from local surroundings, making it a viable candidate for commercialization. The enzyme has been further modified via evolution driven mutagenesis to generate an exclusive 2,3-dimethylphenol sensor with equivalent selectivity and sensitivity as the native phenol sensor. Thus, this approach can be extended to generate a battery of sensors for other priority aromatic pollutants, highlighting the versatility of the biosensor unit. This novel biosensor design presents promising potential for direct detection and can be integrated in a device format for on-site pollutant monitoring.

Functional insights into the mode of DNA and ligand binding of the TetR family regulator TylP from Streptomyces fradiae.

Tetracycline repressors (TetRs) modulate multidrug efflux pathways in several pathogenic bacteria. In Streptomyces, they additionally regulate secondary metabolic pathways like antibiotic production. For instance, in the antibiotic producer Streptomyces fradiae, a layered network of TetRs regulates the levels of the commercially important antibiotic tylosin, with TylP occupying the top of this cascading network. TetRs exist in two functional states, the DNA-bound and the ligand-bound form, which are allosterically regulated. Here, to develop deeper insights into the factors that govern allostery, the crystal structure of TylP was solved to a resolution of 2.3 Å. The structure revealed that TylP possesses several unique features; notably, it harbors a unique C-terminal helix-loop extension that spans the entire length of the structure. This anchor connects the DNA-binding domain (DBD) with the ligand-binding domain (LBD) via a mix of positively charged and hydrogen-bonding interactions. Supporting EMSA studies with a series of ΔC truncated versions show that a systematic deletion of this region results in complete loss of DNA binding. The structure additionally revealed that TylP is markedly different in the orientation of its DBD and LBD architecture and the dimeric geometry from its hypothesized Streptomyces homologue CprB, which is a γ-butyrolactone regulator. Rather, TylP is closer in structural design to macrolide-binding TetRs found in pathogens. Supporting molecular dynamic studies suggested that TylP binds a macrolide intermediate in the tylosin pathway. Collectively, the structure along with corroborating biochemical studies provided insights into the novel mode of regulation of TetRs in antibiotic-producing organisms.

Structure Guided Design of Protein Biosensors for Phenolic Pollutants.

Phenolic aromatic compounds are a major source of environmental pollution. Currently there are no in situ methods for specifically and selectively detecting these pollutants. Here, we exploit the nature's biosensory machinery by employing Acinetobacter calcoaceticus NCIB8250 protein, MopR, as a model system to develop biosensors for selective detection of a spectrum of these pollutants. The X-ray structure of the sensor domain of MopR was used as a scaffold for logic-based tunable biosensor design. By employing a combination of in silico structure guided approaches, mutagenesis and isothermal calorimetric studies, we were able to generate biosensor templates, that can selectively and specifically sense harmful compounds like chlorophenols, cresols, catechol, and xylenols. Furthermore, the ability of native protein to selectively sense phenol as the primary ligand was also enhanced. Overall, this methodology can be extended as a suitable framework for development of a series of exclusive biosensors for accurate and selective detection of aromatic pollutants from real time environmental samples.

Structural Basis of Selective Aromatic Pollutant Sensing by the Effector Binding Domain of MopR, an NtrC Family Transcriptional Regulator.

Phenol and its derivatives are common pollutants that are present in industrial discharge and are major xenobiotics that lead to water pollution. To monitor as well as improve water quality, attempts have been made in the past to engineer bacterial in vivo biosensors. However, due to the paucity of structural information, there is insufficiency in gauging the factors that lead to high sensitivity and selectivity, thereby impeding development. Here, we present the crystal structure of the sensor domain of MopR (MopR(AB)) from Acinetobacter calcoaceticus in complex with phenol and its derivatives to a maximum resolution of 2.5 Å. The structure reveals that the N-terminal residues 21-47 possess a unique fold, which are involved in stabilization of the biological dimer, and the central ligand binding domain belongs to the "nitric oxide signaling and golgi transport" fold, commonly present in eukaryotic proteins that bind long-chain fatty acids. In addition, MopR(AB) nests a zinc atom within a novel zinc binding motif, crucial for maintaining structural integrity. We propose that this motif is crucial for orchestrated motions associated with the formation of the effector binding pocket. Our studies reveal that residues W134 and H106 play an important role in ligand binding and are the key selectivity determinants. Furthermore, comparative analysis of MopR with XylR and DmpR sensor domains enabled the design of a MopR binding pocket that is competent in binding DmpR-specific ligands. Collectively, these findings pave way towards development of specific/broad based biosensors, which can act as useful tools for detection of this class of pollutants.