Structural Insights into the Lipid A Transport Pathway in MsbA

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Substrate binds in the S1 site of the F253A mutant of LeuT, a neurotransmitter sodium symporter homologue

LeuT serves as the model protein for understanding the relationships between structure, mechanism and pharmacology in neurotransmitter sodium symporters (NSSs). At the present time, however, there is a vigorous debate over whether there is a single high-affinity substrate site (S1) located at the original, crystallographically determined substrate site or whether there are two high-affinity substrates sites, one at the primary or S1 site and the other at a second site (S2) located at the base of the extracellular vestibule. In an effort to address the controversy over the number of high-affinity substrate sites in LeuT, one group studied the F253A mutant of LeuT and asserted that in this mutant substrate binds exclusively to the S2 site and that 1 mM clomipramine entirely ablates substrate binding to the S2 site. Here we study the binding of substrate to the F253A mutant of LeuT using ligand binding and X-ray crystallographic methods. Both experimental methods unambiguously show that substrate binds to the S1 site of the F253A mutant and that binding is retained in the presence of 1 mM clomipramine. These studies, in combination with previous work, are consistent with a mechanism for LeuT that involves a single high-affinity substrate binding site.     

Insights into the ClC-4 transport mechanism from studies of Zn2+ inhibition

The CLC family of chloride channels and transporters is a functionally diverse group of proteins important in a wide range of physiological processes. ClC-4 and ClC-5 are localized to endosomes and seem to play roles in the acidification of these compartments. These proteins were recently shown to function as Cl⁻/H⁺ antiporters. However, relatively little is known about the detailed mechanism of CLC-mediated Cl⁻/H⁺ antiport, especially for mammalian isoforms. We attempted to identify molecular tools that might be useful in probing structure-function relationships in these proteins. Here, we record currents from human ClC-4 (hClC-4) expressed in Xenopus oocytes, and find that Zn²⁺ inhibits these currents, with an apparent affinity of ∼50 μM. Although Cd²⁺ has a similar effect, Co²⁺ and Mn²⁺ do not inhibit hClC-4 currents. In contrast, the effect of Zn²⁺ on the ClC-0 channel, Zn²⁺-mediated inhibition of hClC-4 is minimally voltage-dependent, suggesting an extracellular binding site for the ion. Nine candidate external residues were tested; only mutations of three consecutive histidine residues, located in a single extracellular loop, significantly reduced the effect of Zn²⁺, with one of these making a larger contribution than the other two. An analogous tri-His sequence is absent from ClC-0, suggesting a fundamentally different inhibitory mechanism for the ion on hClC-4. Manipulations that alter transport properties of hClC-4, varying permeant ions as well as mutating the “gating glutamate”, dramatically affect Zn²⁺ inhibition, suggesting the involvement of a heretofore unexplored part of the protein in the transport process.     

Spatial structure and the mechanism of tyrosine phenol-lyase and tryptophan indole-lyase

Bacterial tyrosine phenol-lyase [EC 4.1.99.2] and tryptophan indole-lyase [EC 4.1.99.1] are pyridoxal 5′-phosphate dependent β-eliminating lyases that catalyze the reversible decomposition of L-tyrosine and L-tryptophan to pyruvate, ammonia, and phenol or indole, respectively. This review considers the three-dimensional structures of the holoenzymes of tyrosine phenol-lyase and tryptophan indole-lyase and several enzyme-inhibitor complexes that model distinct reaction stages of the β-elimination of L-tyrosine. The structural basis of the influence of monovalent cations on enzymatic activity is discussed. Studies of the spectral and catalytic properties of mutant enzymes made it possible to elucidate the catalytic functions of a number of amino acid residues and to conclude that the acid-base properties of the catalytic groups are optimal for catalysis in the hydrophobic active sites of tyrosine phenol-lyase and tryptophan indol-lyase and differ from those in water solutions. A study of the mechanisms of labilization of the Cα proton of the bound amino acids and activation of the leaving groups of the substrates during the catalytic process demonstrated that the reaction proceeds via concerted, rather than stepwise, pathways in certain cases.     

Insights into the decoding mechanism from recent ribosome structures

During the decoding process, tRNA selection by the ribosome is far more accurate than expected from codon-anticodon pairing. Antibiotics such as streptomycin and paromomycin have long been known to increase the error rate of translation, and many mutations that increase or lower accuracy have been characterized. Recent crystal structures show that the specific recognition of base-pairing geometry leads to a closure of the domains of the small subunit around cognate tRNA. This domain closure is likely to trigger subsequent steps in tRNA selection. Many antibiotics and mutations act by making the domain closure more or less favourable. In conjunction with recent cryoelectron microscopy structures of the ribosome, a comprehensive structural understanding of the decoding process is beginning to emerge.     

ABC transporter architecture and mechanism: implications from the crystal structures of BtuCD and BtuF

ABC transporters are ubiquitous membrane proteins that facilitate unidirectional substrate translocation across the lipid bilayer. Over the past five years, new crystal structures have advanced our understanding of how ABC transporters couple adenosine triphosphate (ATP) hydrolysis to substrate transport. In the following, we will briefly review the results of these structural investigations and outline their mechanistic implications.     

Insights into the catalytic mechanism of cofactor-independent phosphoglycerate mutase from X-ray crystallography,simulated dynamics and molecular modeling

Phosphoglycerate mutases catalyze the isomerization of 2 and 3-phosphoglycerates, and are essential for glucose metabolism in most organisms. Here, we further characterize the 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGM) from Bacillus stearothermophilus by determination of a high-resolution (1.4A) crystal structure of the wild-type enzyme and the crystal structure of its S62A mutant. The mutant structure surprisingly showed the replacement of one of the two catalytically essential manganese ions with a water molecule, offering an additional possible explanation for its lack of catalytic activity. Crystal structures invariably show substrate phosphoglycerate to be entirely buried in a deep cleft between the two iPGM domains. Flexibility analyses were therefore employed to reveal the likely route of substrate access to the catalytic site through an aperture created in the enzyme's surface during certain stages of the catalytic process. Several conserved residues lining this aperture may contribute to orientation of the substrate as it enters. Factors responsible for the retention of glycerate within the phosphoenzyme structure in the proposed mechanism are identified by molecular modeling of the glycerate complex of the phosphoenzyme. Taken together, these results allow for a better understanding of the mechanism of action of iPGMs. Many of the results are relevant to a series of evolutionarily related enzymes. These studies will facilitate the development of iPGM inhibitors which, due to the demonstrated importance of this enzyme in many bacteria, would be of great potential clinical significance.     

Insights into mechanism of tumour promotion by mezerein

The objective of this study was to gather insights and compare the mode of action of the non phorbol, diterpene mezerein with the phorbol ester, phorbol-12-myristate-13 acetate, in normal and transformed cells. Both phorbol-12-myristate-13 acetate and mezerein are shown to activate the signal transduction pathways involving post translational modification of proteins by poly ADP-ribosylation and by protein kinase C, but to varying extents and showed different time kinetics and cell type differences. Multiple nuclear proteins, especially histones H3d, A24 and HI served as acceptors of poly ADP-ribose in response to PMA in both NIH 3T3 and HDCS cells whereas H1 and H2B were the major acceptors in case of mezerein treatment, similarly in both NIH 3T3 and HDCS cells. The results suggest an epigenetic mechanism (s) in tumour promotion by mezerein.     

The recovery of dipolar relaxation times from fluorescence decays as a tool to probe local dynamics in single tryptophan proteins

The dipolar relaxation process induced by the excitation of the single tryptophan residue of four proteins (staphylococcal nuclease, ribonuclease-T1, phosphofructokinase, and superoxide dismutase) has been studied by dynamic fluorescence measurements. A new algorithm taking into account the relaxation effect has been applied to the fluorescence decay function obtained by phase-shift and demodulation data. This approach only requires that fluorescence be collected through the whole emission spectrum, avoiding the time-consuming determination of the data at different emission wavelengths, as usual with time-resolved emission spectroscopy. The results nicely match those reported in the literature for staphylococcal nuclease and ribonuclease-T1, demonstrating the validity of the model. Furthermore, this new methodology provides an alternative explanation for the complex decay of phosphofructokinase and human superoxide dismutase suggesting the presence of a relaxation process even in proteins that lack a lifetime-dependent spectral shift. These findings may have important implications on the analysis of small-scale protein dynamics, since dielectric relaxation directly probes a local structural change around the excited state of tryptophan.     

On the role of alphaThr183 in the allosteric regulation and catalytic mechanism of tryptophan synthase

The catalytic activity and substrate channeling of the pyridoxal 5'-phosphate-dependent tryptophan synthase alpha(2)beta(2) complex is regulated by allosteric interactions that modulate the switching of the enzyme between open, low activity and closed, high activity states during the catalytic cycle. The highly conserved alphaThr183 residue is part of loop alphaL6 and is located next to the alpha-active site and forms part of the alpha-beta subunit interface. The role of the interactions of alphaThr183 in alpha-site catalysis and allosteric regulation was investigated by analyzing the kinetics and crystal structures of the isosteric mutant alphaThr183Val. The mutant displays strongly impaired allosteric alpha-beta communication, and the catalytic activity of the alpha-reaction is reduced one hundred fold, whereas the beta-activity is not affected. The structural work establishes that the basis for the missing inter-subunit signaling is the lack of loop alphaL6 closure even in the presence of the alpha-subunit ligands, 3-indolyl-D-glycerol 3'-phosphate, or 3-indolylpropanol 3'-phosphate. The structural basis for the reduced alpha-activity has its origins in the missing hydrogen bond between alphaThr183 and the catalytic residue, alphaAsp60.     

Insights into the molecular mechanism of nuclear trafficking using nuclear transport factor 2 (NTF2)

Nuclear transport factor 2 (NTF2) mediates the nuclear import of RanGDP. The simplicity and specialization of this system, combined with the availability of crystal structures of NTF2, RanGDP and their complex, has facilitated the investigation of the molecular mechanism of its trafficking. NTF2 binds to both RanGDP and FxFG repeat-containing nucleoporins. Mutants engineered on the basis of structural information together with determination of binding constants have been used to dissect the roles of these interactions in transport. Thus, NTF2 binds to RanGDP sufficiently strongly for the complex to remain intact during transport through NPCs, but the interaction between NTF2 and FxFG nucleoporins is much more transient, which would enable NTF2 to move through the NPC by hopping from one repeat to another. An analogous nucleoporin hopping mechanism may also be used by carrier molecules of the importin-beta family to move through NPCs.     

Insights into the catalytic mechanisms of phenylalanine and tryptophan hydroxylase from kinetic isotope effects on aromatic hydroxylation

Phenylalanine hydroxylase (PheH) and tryptophan hydroxylase (TrpH) catalyze the aromatic hydroxylation of phenylalanine and tryptophan, forming tyrosine and 5-hydroxytryptophan, respectively. The reactions of PheH and TrpH have been investigated with [4-(2)H]-, [3,5-(2)H(2)]-, and (2)H(5)-phenylalanine as substrates. All (D)k(cat) values are normal with Delta117PheH, the catalytic core of rat phenylalanine hydroxylase, ranging from 1.12-1.41. In contrast, for Delta117PheH V379D, a mutant protein in which the stoichiometry between tetrahydropterin oxidation and amino acid hydroxylation is altered, the (D)k(cat) value with [4-(2)H]-phenylalanine is 0.92 but is normal with [3,5-(2)H(2)]-phenylalanine. The ratio of tetrahydropterin oxidation to amino acid hydroxylation for Delta117PheH V379D shows a similar inverse isotope effect with [4-(2)H]-phenylalanine. Intramolecular isotope effects, determined from the deuterium contents of the tyrosine formed from [4-(2)H]-and [3,5(2)H(2)]-phenylalanine, are identical for Delta117PheH and Delta117PheH V379D, suggesting that steps subsequent to oxygen addition are unaffected in the mutant protein. The inverse effects are consistent with the reaction of an activated ferryl-oxo species at the para position of the side chain of the amino acid to form a cationic intermediate. The normal effects on the (D)k(cat) value for the wild-type enzyme are attributed to an isotope effect of 5.1 on the tautomerization of a dienone intermediate to tyrosine with a rate constant 6- to7-fold that for hydroxylation. In addition, there is a slight ( approximately 34%) preference for the loss of the hydrogen originally at C4 of phenylalanine. With (2)H(5)-indole-tryptophan as a substrate for Delta117PheH, the (D)k(cat) value is 0.89, consistent with hydroxylation being rate-limiting in this case. When deuterated phenylalanines are used as substrates for TrpH, the (D)k(cat) values are within error of those for Delta117PheH V379D. Overall, these results are consistent with the aromatic amino acid hydroxylases all sharing the same chemical mechanism, but with the isotope effect for hydroxylation by PheH being masked by tautomerization of an enedione intermediate to tyrosine.     

Insights into ALS pathomechanisms: from flies to humans

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease causing the death of motor neurons with consequent muscle atrophy and paralysis. Several neurodegenerative diseases have been modeled in Drosophila and genetic studies on this model organism led to the elucidation of crucial aspects of disease mechanisms. ALS, however, has lagged somewhat behind possibly because of the lack of a suitable genetic model. We were the first to develop a fly model for ALS and over the last few years, we have implemented and used this model for a large scale, unbiased modifier screen. We also report an extensive bioinformatic analysis of the genetic modifiers and we show that most of them are associated in a network of interacting genes controlling known as well as novel cellular processes involved in ALS pathogenesis. A similar analysis for the human homologues of the Drosophila modifiers and the validation of a subset of them in human tissues confirm and expand the significance of the data for the human disease. Finally, we analyze a possible application of the model in the process of therapeutic discovery in ALS and we discuss the importance of novel “non-obvious” models for the disease.     

Chemically defined media modifications to lower tryptophan oxidation of biopharmaceuticals

Oxidation of biopharmaceuticals is a major product quality issue with potential impacts on activity and immunogenicity. At Eli Lilly and Company, high tryptophan oxidation was observed for two biopharmaceuticals in development produced in Chinese hamster ovary cells. A switch from historical hydrolysate‐containing media to chemically defined media with a reformulated basal powder was thought to be responsible, so mitigation efforts focused on media modification. Shake flask studies identified that increasing tryptophan, copper, and manganese and decreasing cysteine concentrations were individual approaches to lower tryptophan oxidation. When amino acid and metal changes were combined, the modified formulation had a synergistic impact that led to substantially less tryptophan oxidation for both biopharmaceuticals. Similar results were achieved in shake flasks and benchtop bioreactors, demonstrating the potential to implement these modifications at manufacturing scale. The modified formulation did not negatively impact cell growth and viability, product titer, purity, charge variants, or glycan profile. A potential mechanism of action is presented for each amino acid or metal factor based on its role in oxidation chemistry. This work served not only to mitigate the tryptophan oxidation issue in two Lilly biopharmaceuticals in development, but also to increase our knowledge and appreciation for the impact of media components on product quality. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:178–188, 2016     

Insights into water accessible pathways and the inactivation mechanism of proton translocation by the membrane-embedded domain of V-type ATPases

V-type ATPases are multi-protein complexes, which acidify cellular compartments in eukaryotes. They pump protons against an ion gradient, driven by a mechano-chemical framework that exploits ATP hydrolysis as an energy source. This process drives the rotation of the so-called c-ring, a membrane embedded complex in the V_o-domain of the V-type ATPase, resulting in translocation of protons across the membrane. One way in which the enzyme is regulated is by disassembly and reassembly of the V₁-domain with the V_o-domain, which inactivates and reactivates the enzyme, respectively. Recently, structural data for the isolated V_o-domain from S. cerevisiae in an inactivated state were reported, suggesting the location of previously unobserved proton access pathways within the cytoplasmic and luminal compartments of the stator subunit a in V_o. However, the structural rationale for this inactivation remained unclear. In this study, the water accessibility pathway at the cytoplasmic side is confirmed, and novel insights into the role of the luminal channel with respect to the inactivation mechanism are obtained, using atomic-resolution molecular dynamics simulations. The results show that protonation of the key-glutamate, located in the c-ring of the V_o-domain, and facing the luminal compartment is preserved, when residing in the V₁-depleted state. Maintaining the protonation of this essential glutamate is necessary to lock the luminal channel in the inactive, solvent-free state. Based on these theoretical observations and previous experimental results, a model of the proton translocation mechanism in the V_o-domain from V-type ATPases is proposed.     

Insights into protein kinase regulation and inhibition by large scale structural comparison

Protein structure determination of soluble globular protein domains has developed into an efficient routine technology which can now be applied to generate and analyze structures of entire human protein families. In the kinase area, several kinase families still lack comprehensive structural analysis. Nevertheless, Structural Genomics (SG) efforts contributed more than 40 kinase catalytic domain structures during the past 4 years providing a rich resource of information for large scale comparisons of kinase active sites. Moreover, many of the released structures are inhibitor complexes that offer chemical starting points for development of selective and potent inhibitors. Here we discuss the currently available structural data and strategies that can be utilized for the development of highly selective inhibitors.     

Insights into the biology of Escherichia coli through structural proteomics

Escherichia coli has historically been an important organism for understanding a multitude of biological processes, and represents a model system as we attempt to simulate the workings of living cells. Many E. coli strains are also important human and animal pathogens for which new therapeutic strategies are required. For both reasons, a more complete and comprehensive understanding of the protein structure complement of E. coli is needed at the genome level. Here, we provide examples of insights into the mechanism and function of bacterial proteins that we have gained through the Bacterial Structural Genomics Initiative (BSGI), focused on medium-throughput structure determination of proteins from E. coli. We describe the structural characterization of several enzymes from the histidine biosynthetic pathway, the structures of three pseudouridine synthases, enzymes that synthesize one of the most abundant modified bases in RNA, as well as the combined use of protein structure and focused functional analysis to decipher functions for hypothetical proteins. Together, these results illustrate the power of structural genomics to contribute to a deeper biological understanding of bacterial processes.     

Insights into membrane translocation of the cell-penetrating peptide pVEC from molecular dynamics calculations

Discovery of cargo carrying cell-penetrating peptides has opened a new gate in the development of peptide-based drugs that can effectively target intracellular enzymes. Success in application and development of cell-penetrating peptides in drug design depends on understanding their translocation mechanisms. In this study, our aim was to examine the bacterial translocation mechanism of the cell-penetrating pVEC peptide (LLIILRRRIRKQAHAHSK) using steered molecular dynamics (SMD) simulations. The significance of specific residues or regions for translocation was studied by performing SMD simulations on the alanine mutants and other variants of pVEC. Residue-based analysis showed that positively charged residues contribute to adsorption to the lipid bilayer and to electrostatic interactions with the lipid bilayer as peptides are translocated. Translocation takes place in three main stages; the insertion of the N-terminus into the bilayer, the inclusion of the whole peptide inside the membrane and the exit of the N-terminus from the bilayer. These three stages mirror the three regions on pVEC; namely, the hydrophobic N-terminus, the cationic midsection, and the hydrophilic C-terminus. The N-terminal truncated pVEC, I3A, L5A, R7A mutants and scramble-pVEC make weaker interactions with the lipids during translocation highlighting the contribution of the N-terminal residues and the sequence of the structural regions to the translocation mechanism. This study provides atomistic detail about the mechanism of pVEC peptide translocation and can guide future peptide-based drug design efforts.