Identification and Characterization of a Secondary Sodium-Binding Site and the Main Selectivity Determinants in the Human Concentrative Nucleoside Transporter 3.

The family of concentrative Na+/nucleoside cotransporters in humans is constituted by three subtypes, namely, hCNT1, hCNT2, and hCNT3. Besides their different nucleoside selectivity, hCNT1 and hCNT2 have a Na+/nucleoside stoichiometry of 1:1, while for hCNT3 it is 2:1. This distinct stoichiometry of subtype 3 might hint the existence of a secondary sodium-binding site that is not present in the other two subtypes, but to date their three-dimensional structures remain unknown and the residues implicated in Na+ binding are unclear. In this work, we have identified and characterized the Na+ binding sites of hCNT3 by combining molecular modeling and mutagenesis studies. A model of the transporter was obtained by homology modeling, and key residues of two sodium-binding sites were identified and verified with a mutagenesis strategy. The structural model explains the altered sodium-binding properties of the hCNT3C602R polymorphic variant and supports previously generated data identifying the determinant residues of nucleoside selectivity, paving the way to understand how drugs can target this plasma membrane transporter.

Structural Plasticity in Globins: Role of Protein Dynamics in Defining Ligand Migration Pathways.

Globins are a family of proteins characterized by the presence of the heme prosthetic group and involved in variety of biological functions in the cell. Due to their biological relevance and widespread distribution in all kingdoms of life, intense research efforts have been devoted to disclosing the relationships between structural features, protein dynamics, and function. Particular attention has been paid to the impact of differences in amino acid sequence on the topological features of docking sites and cavities and to the influence of conformational flexibility in facilitating the migration of small ligands through these cavities. Often, tunnels are carved in the interior of globins, and ligand exchange is regulated by gating residues. Understanding the subtle intricacies that relate the differences in sequence with the structural and dynamical features of globins with the ultimate aim of rationalizing the thermodynamics and kinetics of ligand binding continues to be a major challenge in the field. Due to the evolution of computational techniques, significant advances into our understanding of these questions have been made. In this review we focus our attention on the analysis of the ligand migration pathways as well as the function of the structural cavities and tunnels in a series of representative globins, emphasizing the synergy between experimental and theoretical approaches to gain a comprehensive knowledge into the molecular mechanisms of this diverse family of proteins.

Histidine E7 dynamics modulates ligand exchange between distal pocket and solvent in AHb1 from Arabidopsis thaliana.

The distal His residue in type 1 nonsymbiotic hemoglobin AHb1 from Arabidopsis thaliana plays a fundamental role in stabilizing the bound ligand. This residue might also be important in regulating the accessibility to the distal cavity. The feasibility of this functional role has been examined using a combination of experimental and computational methods. We show that the exchange of CO between the solvent and the reaction site is modulated by a swinging motion of the distal His, which opens a channel that connects directly the distal heme pocket with the solvent. The nearby PheB10 aids the distal His in the stabilization of the bound ligand by providing additional protection against solvation. Overall, these findings provide evidence supporting the functional implications of the conformational rearrangement found for the distal His in AHb1, which mimics the gating role proposed for the same residue in myoglobin.

High pressure reveals structural determinants for globin hexacoordination: neuroglobin and myoglobin cases.

The influence of pressure on the equilibrium between five-(5c) and six-coordination (6c) forms in neuroglobin (Ngb) and myoglobin (Mb) has been examined by means of molecular dynamics (MD) simulations at normal and high pressure. The results show that the main effect of high pressure is to reduce the protein mobility without altering the structure in a significant manner. Moreover, our data suggest that the equilibrium between 5c and 6c states in globins is largely controlled by the structure and dynamics of the C-D region. Finally, in agreement with the available experimental data, the free energy profiles obtained from steered MD for both proteins indicate that high pressure enhances hexacoordination. In Ngb, the shift in equilibrium is mainly related to an increase in the 6c-->5c transition barrier, whereas in Mb such a shift is primarily due to a destabilization of the 5c state.

Donepezil-tacrine hybrid related derivatives as new dual binding site inhibitors of AChE.

A new series of donepezil-tacrine hybrid related derivatives have been synthesised as dual acetylcholinesterase inhibitors that could bind simultaneously to the peripheral and catalytic sites of the enzyme. These new hybrids combined a tacrine, 6-chlorotacrine or acridine unit as catalytic binding site and indanone (the heterocycle present in donepezil) or phthalimide moiety as peripheral binding site of the enzyme, connected through a different linker tether length. One of the synthesised compounds emerged as a potent and selective AChE inhibitor, which is able to displace propidium in a competition assay. These results seem to confirm the ability of this inhibitor to bind simultaneously to both sites of the enzyme and make it a promising lead for developing disease-modifying drugs for the future treatment of Alzheimer's disease. To gain insight into the molecular determinants that modulate the inhibitory activity of these compounds, a molecular modelling study was performed to explore their binding to the enzyme.

Translocation of jellyfish green fluorescent protein via the Tat system of Escherichia coli and change of its periplasmic localization in response to osmotic up-shock.

The bacterial twin arginine translocation (Tat) pathway is capable of exporting cofactor-containing enzymes into the periplasm. To assess the capacity of the Tat pathway to export heterologous proteins and to gain information about the property of the periplasm, we fused the twin arginine signal peptide of the trimethylamine N-oxide reductase to the jellyfish green fluorescent protein (GFP). Unlike the Sec pathway, the Tat system successfully exported correctly folded GFP into the periplasm of Escherichia coli. Interestingly, GFP appeared as a halo in most cells and occasionally showed a polar localization in wild type strains. When subjected to a mild osmotic up-shock, GFP relocalized very quickly at the two poles of the cells. The conversion from the halo structure to a periplasmic gathering at particular locations was also observed with spherical cells of the DeltarodA-pbpA mutant or of the wild type strain treated with lysozyme. Therefore, the periplasm is not a uniform compartment and the polarization of GFP is unlikely to be caused by simple invagination of the cytoplasmic membrane at the poles. Moreover, the polar gathering of GFP is reversible; the reversion was accelerated by glucose and inhibited by azide and carbonyl cyanide m-chlorophenylhydrazone, indicating an active adaptation of the bacteria to the osmolarity in the medium. These results strongly suggest a relocalization of periplasmic substances in response to environmental changes. The polar area might be the preferential zone where bacteria sense the change in the environment.

Effect of O2, H2 and redox potential on the activity and synthesis of hydrogenase 2 in Escherichia coli.

The aim of this work was to study the influence of O2 with special emphasis on low oxygen tension, the effect of H2 under various conditions of oxygen tension and the influence of the redox potential in the growth medium on hydrogenase 2 of Escherichia coli. The hydrogenase activity and the content of the large (HybC) and small (HybO) subunits of hydrogenase 2 were compared during turbidostat cultivation in a wild strain and mutant HDK103 lacking hydrogenases 1 and 3. No hydrogenase 2 activity in the mutant HDK103 was observed under aerobic conditions, but it was maximal under anaerobic conditions and half-maximal at an oxygen tension of approximately 4 mbar as is common for enzymes of anaerobic respiration. The content of hydrogenase 2 in both the strains was maximal under anaerobic conditions. In the wild strain, H2 addition enhanced hydrogenase activity and the HybO content under microaerobic conditions only. Under anaerobic conditions endogenous H2 production hindered this effect. Under aerobic conditions, the 02-related negative effect seemed to dominate over the H2-related positive effect. By contrast, in the mutant HDK103, hydrogen influenced neither hydrogenase 2 activity nor its content. A possible role of hydrogenase I in the response of hydrogenase 2 to hydrogen is discussed. Under conditions of different O2 tension, hydrogenase activity in both strains correlated inversely with the value of the redox potential of the medium. The presence of H2 changed this dependence. Thus, the value of the redox potential itself is not a controlling factor for hydrogenase 2.

Bacterial twin-arginine signal peptide-dependent protein translocation pathway: evolution and mechanism.

The recently identified bacterial Tat pathway is capable of exporting proteins with a peculiar twin-arginine signal peptide in folded conformation independently of the Sec machinery. It is structurally and mechanistically similar to the delta pH-dependent pathway used for importing chloroplast proteins into the thylakoid. The tat genes are not ubiquitously present and are absent from half of the completely sequenced bacterial genomes. The presence of the tat genes seems to correlate with genome size and with the presence of important enzymes with a twin-arginine signal peptide. A minimal Tat system requires a copy of tatA and a copy of tatC. The composition and gene order of a tat locus are generally conserved within the same taxonomy group but vary considerably to other groups, which would exclude an acquisition of the Tat system by recent horizontal gene transfer. The tat genes are also found in the genomes of chloroplasts and plant mitochondria but are absent from animal mitochondrial genomes. The topology of evolution trees suggests a bacterial origin of the Tat system. In general, the twin-arginine signal peptide is capable of targeting any passenger protein to the Tat pathway. However, a structural signal carried by the mature part of a passenger protein can override targeting information in a signal peptide under certain circumstances. Tat systems show a substrate-Tat component specificity and a species specificity. The pore size of the Tat channel is estimated as being between 5 and 9 nm. Operational models of the Tat system are proposed.

Membrane targeting and translocation of bacterial hydrogenases.

Periplasmic or membrane-bound bacterial hydrogenases are generally composed of a small subunit and a large subunit. The small subunit contains a peculiar N-terminal twin-arginine signal peptide, whereas the large subunit lacks any known targeting signal for export. Genetic and biochemistry data support the assumption that the large subunit is cotranslocated with the small subunit across the cytoplasmic membrane. Indeed, the signal peptide carried by the small subunit directs both the small and the large subunits to the recently identified Mtt/Tat pathway, independently of the Sec machinery. In addition, the twin-arginine signal peptide of hydrogenase is capable of directing protein import into the thylakoidal lumen of chloroplasts via the homologous deltapH-driven pathway, which is independent of the Sec machinery. Therefore, the translocation of hydrogenase shares characteristics with the deltapH-driven import pathway in terms of Sec-independence and requirement for the twin-arginine signal peptide, and with protein import into peroxisomes in a "piggyback" fashion.

Co-translocation of a periplasmic enzyme complex by a hitchhiker mechanism through the bacterial tat pathway.

Bacterial periplasmic nickel-containing hydrogenases are composed of a small subunit containing a twin-arginine signal sequence and a large subunit devoid of an export signal. To understand how the large subunit is translocated into the periplasm, we cloned the hyb operon encoding the hydrogenase 2 of Escherichia coli, constructed a deletion mutant, and studied the mechanism of translocation of hydrogenase 2. The small subunit (HybO) or the large subunit (HybC) accumulated in the cytoplasm as a precursor when either of them was expressed in the absence of the other subunit. Therefore, contrary to most classical secretory proteins, the signal sequence of the small subunit itself is not sufficient for membrane targeting and translocation if the large subunit is missing. On the other hand, the small subunit was required not only for membrane targeting of the large subunit, but also for the acquisition of nickel by the large subunit. Most interestingly, the signal sequence of the small subunit determines whether the large subunit follows the Sec or the twin-arginine translocation pathway. Taken together, these results provide for the first time compelling evidence for a naturally occurring hitchhiker co-translocation mechanism in bacteria.

A novel sec-independent periplasmic protein translocation pathway in Escherichia coli.

The trimethylamine N-oxide (TMAO) reductase of Escherichia coli is a soluble periplasmic molybdoenzyme. The precursor of this enzyme possesses a cleavable N-terminal signal sequence which contains a twin-arginine motif. By using various moa, mob and mod mutants defective in different steps of molybdocofactor biosynthesis, we demonstrate that acquisition of the molybdocofactor in the cytoplasm is a prerequisite for the translocation of the TMAO reductase. The activation and translocation of the TMAO reductase precursor are post-translational processes, and activation is dissociable from translocation. The export of the TMAO reductase is driven mainly by the proton motive force, whereas sodium azide exhibits a limited effect on the export. The most intriguing observation is that translocation of the TMAO reductase across the cytoplasmic membrane is independent of the SecY, SecE, SecA and SecB proteins. Depletion of Ffh, a core component of the signal recognition particle of E. coli, appears to have a slight effect on the export of the TMAO reductase. These results strongly suggest that the translocation of the molybdoenzyme TMAO reductase into the periplasm uses a mechanism fundamentally different from general protein translocation.