Sodium/Iodide Symporter Metastable Intermediates Provide Insights into Conformational Transition between Principal Thermodynamic States.

The Sodium/Iodide Symporter (NIS), a 13-helix transmembrane protein found in the thyroid and other tissues, transports iodide, a required constituent of thyroid hormones T3 and T4. Despite extensive experimental information and clinical data, structural details of the intermediate microstates comprising the conformational transition of NIS between its inwardly and outwardly open states remain unresolved. We present data from a combination of enhanced sampling and transition path molecular dynamics (MD) simulations that elucidate the principal intermediate states comprising the inwardly to outwardly open transition of fully bound and apo NIS under an enforced ionic gradient. Our findings suggest that in both the absence and presence of bound physiological ions, NIS principally occupies a proximally inward to inwardly open state. When fully bound, NIS is also found to occupy a rare "inwardly occluded" state. The results of this work provide novel insight into the populations of NIS intermediates and the free energy landscape comprising the conformational transition, adding to a mechanistic understanding of NIS ion transport. Moreover, the knowledge gained from this approach can serve as a basis for studies of NIS mutants to target therapeutic interventions.

Structural insights into the mechanism of the sodium/iodide symporter.

The sodium/iodide symporter (NIS) is the essential plasma membrane protein that mediates active iodide (I-) transport into the thyroid gland, the first step in the biosynthesis of the thyroid hormones-the master regulators of intermediary metabolism. NIS couples the inward translocation of I- against its electrochemical gradient to the inward transport of Na+ down its electrochemical gradient1,2. For nearly 50 years before its molecular identification3, NIS was the molecule at the centre of the single most effective internal radiation cancer therapy: radioiodide (131I-) treatment for thyroid cancer2. Mutations in NIS cause congenital hypothyroidism, which must be treated immediately after birth to prevent stunted growth and cognitive deficiency2. Here we report three structures of rat NIS, determined by single-particle cryo-electron microscopy: one with no substrates bound; one with two Na+ and one I- bound; and one with one Na+ and the oxyanion perrhenate bound. Structural analyses, functional characterization and computational studies show the substrate-binding sites and key residues for transport activity. Our results yield insights into how NIS selects, couples and translocates anions-thereby establishing a framework for understanding NIS function-and how it transports different substrates with different stoichiometries and releases substrates from its substrate-binding cavity into the cytosol.

Class I PI3K Biology.

This chapter is an introduction to phosphoinositide 3-kinases (PI3K), with class I PI3Ks as the central focus. First, the various PI3K isoforms in class I are presented with emphasis on their overall structure, subunits, subunit constitutive domains, domain-domain interactions, and functional relevance. This structural analysis is followed by a comprehensive history of seminal investigations into PI3K activity. Next, we highlight the divergent roles of the isoforms: PI3Kα, PI3Kß, PI3Kδ, and PI3Kγ. This section details signaling pathways in which these PI3K isoforms are involved, including the key upstream regulators of PI3K activity and some downstream cellular effects. Nodes of the PI3K pathway are also presented. Inhibitors of some isoforms are discussed to give an overview of the basis of some immunotherapies that are being used to target cell signaling. Finally, the chapter ends with a discussion of the dysregulation of PI3Ks in diseases including APDS, asthma, arthritis, and oncogenic mutations.

Development of high-affinity nanobodies specific for NaV1.4 and NaV1.5 voltage-gated sodium channel isoforms.

Voltage-gated sodium channels, NaVs, are responsible for the rapid rise of action potentials in excitable tissues. NaV channel mutations have been implicated in several human genetic diseases, such as hypokalemic periodic paralysis, myotonia, and long-QT and Brugada syndromes. Here, we generated high-affinity anti-NaV nanobodies (Nbs), Nb17 and Nb82, that recognize the NaV1.4 (skeletal muscle) and NaV1.5 (cardiac muscle) channel isoforms. These Nbs were raised in llama (Lama glama) and selected from a phage display library for high affinity to the C-terminal (CT) region of NaV1.4. The Nbs were expressed in Escherichia coli, purified, and biophysically characterized. Development of high-affinity Nbs specifically targeting a given human NaV isoform has been challenging because they usually show undesired crossreactivity for different NaV isoforms. Our results show, however, that Nb17 and Nb82 recognize the CTNaV1.4 or CTNaV1.5 over other CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 and Nb82 bind to the CTNaV1.4 and CTNaV1.5 with high affinity (KD ∼ 40-60 nM). In addition, as proof of concept, we show that Nb82 could detect NaV1.4 and NaV1.5 channels in mammalian cells and tissues by Western blot. Furthermore, human embryonic kidney cells expressing holo NaV1.5 channels demonstrated a robust FRET-binding efficiency for Nb17 and Nb82. Our work lays the foundation for developing Nbs as anti-NaV reagents to capture NaVs from cell lysates and as molecular visualization agents for NaVs.

In Structural Glycobiology, Deuterium provides the Details.

In this issue of Structure, Gadjos et al. (2021b) determine the structure of a bacterial lectin in complex with L-fucose by neutron diffraction of both perdeuterated protein and carbohydrate ligand. The structure provides insight into lectin-ligand interactions, opening avenues for drug design targeting bacterial lectins for intervention in infectious disease.

The Iodide Transport Defect-Causing Y348D Mutation in the Na+/I- Symporter Renders the Protein Intrinsically Inactive and Impairs Its Targeting to the Plasma Membrane.

Background: The sodium/iodide (Na+/I-) symporter (NIS) mediates active transport of I- into the thyroid gland. Mutations in the SLC5A5 gene, which encodes NIS, cause I- transport defects (ITDs)-which, if left untreated, lead to congenital hypothyroidism and consequent cognitive and developmental deficiencies. The ITD-causing NIS mutation Y348D, located in transmembrane segment (TMS) 9, was reported in three Sudanese patients. Methods: We generated cDNAs coding for Y348D NIS and mutants with other hydrophilic and hydrophobic amino acid substitutions at position 348 and transfected them into cells. The activity of the resulting mutants was quantitated by radioiodide transport assays. NIS glycosylation was investigated by Western blotting after endoglycosidase H (Endo H) and PNGase-F glycosidase treatment. Subcellular localization of the mutant proteins was ascertained by flow cytometry analysis, cell surface biotinylation, and immunofluorescence. The intrinsic activity of Y348D was studied by measuring radioiodide transport in membrane vesicles prepared from Y348D-NIS-expressing cells. Our NIS homology models and molecular dynamics simulations were used to identify residues that interact with Y348 and investigate possible interactions between Y348 and the membrane. The sequences of several Slc5 family transporters were aligned, and a phylogenetic tree was generated in ClustalX. Results: Cells expressing Y348D NIS transport no I-. Furthermore, Y348D NIS is only partially glycosylated, is retained intracellularly, and is intrinsically inactive. Hydrophilic residues other than Asp at position 348 also yield NIS proteins that fail to be targeted to the plasma membrane (PM), whereas hydrophobic residues at this position, which we show do not interact with the membrane, rescue PM targeting and function. Conclusions: Y348D NIS does not reach the PM and is intrinsically inactive. Hydrophobic amino acid substitutions at position 348, however, preserve NIS activity. Our findings are consistent with our homology model's prediction that Y348 should face the side opposite the TMS9 residues that coordinate Na+ and participate in Na+ transport, and with the notion that Y348 interacts only with hydrophobic residues. Hydrophilic or charged residues at position 348 have deleterious effects on NIS PM targeting and activity, whereas a hydrophobic residue at this position rescues NIS activity.

Structural basis of cytoplasmic NaV1.5 and NaV1.4 regulation.

Voltage-gated sodium channels (NaVs) are membrane proteins responsible for the rapid upstroke of the action potential in excitable cells. There are nine human voltage-sensitive NaV1 isoforms that, in addition to their sequence differences, differ in tissue distribution and specific function. This review focuses on isoforms NaV1.4 and NaV1.5, which are primarily expressed in skeletal and cardiac muscle cells, respectively. The determination of the structures of several eukaryotic NaVs by single-particle cryo-electron microscopy (cryo-EM) has brought new perspective to the study of the channels. Alignment of the cryo-EM structure of the transmembrane channel pore with x-ray crystallographic structures of the cytoplasmic domains illustrates the complementary nature of the techniques and highlights the intricate cellular mechanisms that modulate these channels. Here, we review structural insights into the cytoplasmic C-terminal regulation of NaV1.4 and NaV1.5 with special attention to Ca2+ sensing by calmodulin, implications for disease, and putative channel dimerization.

Prediction of Mycobacterium tuberculosis pyrazinamidase function based on structural stability, physicochemical and geometrical descriptors.

BACKGROUND: Pyrazinamide is an important drug against the latent stage of tuberculosis and is used in both first- and second-line treatment regimens. Pyrazinamide-susceptibility test usually takes a week to have a diagnosis to guide initial therapy, implying a delay in receiving appropriate therapy. The continued increase in multi-drug resistant tuberculosis and the prevalence of pyrazinamide resistance in several countries makes the development of assays for prompt identification of resistance necessary. The main cause of pyrazinamide resistance is the impairment of pyrazinamidase function attributed to mutations in the promoter and/or pncA coding gene. However, not all pncA mutations necessarily affect the pyrazinamidase function. OBJECTIVE: To develop a methodology to predict pyrazinamidase function from detected mutations in the pncA gene. METHODS: We measured the catalytic constant (kcat), KM, enzymatic efficiency, and enzymatic activity of 35 recombinant mutated pyrazinamidase and the wild type (Protein Data Bank ID = 3pl1). From all the 3D modeled structures, we extracted several predictors based on three categories: structural stability (estimated by normal mode analysis and molecular dynamics), physicochemical, and geometrical characteristics. We used a stepwise Akaike's information criterion forward multiple log-linear regression to model each kinetic parameter with each category of predictors. We also developed weighted models combining the three categories of predictive models for each kinetic parameter. We tested the robustness of the predictive ability of each model by 6-fold cross-validation against random models. RESULTS: The stability, physicochemical, and geometrical descriptors explained most of the variability (R2) of the kinetic parameters. Our models are best suited to predict kcat, efficiency, and activity based on the root-mean-square error of prediction of the 6-fold cross-validation. CONCLUSIONS: This study shows a quick approach to predict the pyrazinamidase function only from the pncA sequence when point mutations are present. This can be an important tool to detect pyrazinamide resistance.

Identifying Structural Determinants of Product Specificity in Leishmania major Farnesyl Diphosphate Synthase.

Farnesyl diphosphate synthase (FPPS) is an isoprenoid chain elongation enzyme that catalyzes the sequential condensation of dimethylallyl diphosphate (C5) with isopentenyl diphosphate (IPP; C5) and the resulting geranyl diphosphate (GPP; C10) with another molecule of IPP, eventually producing farnesyl diphosphate (FPP; C15), which is a precursor for the biosynthesis of a vast majority of isoprenoids. Previous studies of FPPS have highlighted the importance of the structure around the hydrophobic chain elongation path in determining product specificity. To investigate what structural features define the final chain length of the product in FPPS from Leishmania major, we designed and expressed six mutants of LmFPPS by replacing small amino acids around the binding pocket with bulky residues. Using enzymatic assays, binding kinetics, and crystallographic studies, we analyzed the effects of these mutations on the activity and product specificity of FPPS. Our results revealed that replacement of Thr-164 with tryptophan and phenylalanine completely abolished the activity of FPPS. Intriguingly, the T164Y substitution displayed dual product specificity and produced a mixture GPP and FPP as final products, with an activity for FPP synthesis that was lower than that of the wild-type enzyme. These data indicate that Thr-164 is a potential regulator of product specificity.

Author Correction: Allosteric regulation of mammalian Na+/I- symporter activity by perchlorate.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Allosteric regulation of mammalian Na+/I- symporter activity by perchlorate.

The Na+/I- symporter (NIS), the plasma membrane protein that actively transports I- (stoichiometry 2Na+:1I-) in thyroid physiology and radioiodide-based thyroid cancer treatment, also transports the environmental pollutant perchlorate (stoichiometry 1Na+:1ClO4-), which competes with I- for transport. Until now, the mechanism by which NIS transports different anion substrates with different stoichiometries has remained unelucidated. We carried out transport measurements and analyzed these using a statistical thermodynamics-based equation and electrophysiological experiments to show that the different stoichiometry of ClO4- transport is due to ClO4- binding to a high-affinity non-transport allosteric site that prevents Na+ from binding to one of its two sites. Furthermore, low concentrations of ClO4- inhibit I- transport not only by competition but also, critically, by changing the stoichiometry of I- transport to 1:1, which greatly reduces the driving force. The data reveal that ClO4- pollution in drinking water is more dangerous than previously thought.

Allosteric Activation of PI3Kα Results in Dynamic Access to Catalytically Competent Conformations.

Class I phosphoinositide-3-kinases (PI3Ks) phosphorylate PIP2 at its 3' inositol position to generate PIP3, a second messenger that influences signaling cascades regulating cellular growth, survival, and proliferation. Previous studies have suggested that PI3Kα activation involves dislodging the p85α nSH2 domain from the p110α catalytic subunit by binding activated receptor tyrosine kinases. We carried out molecular dynamics simulations to determine, mechanistically and structurally, how PI3Kα conformations are influenced by physiological effectors and the nSH2 domain. We demonstrate that changes in protein dynamics mediated by allosteric regulation significantly increase the population of catalytically competent states without changing the enzyme ground-state structure. Furthermore, we demonstrate that modulation of active-site residue interactions with enzyme substrates can reciprocally influence nSH2 domain dynamics. Together, these results suggest that dynamic allostery plays a role in populating the catalytically competent conformation of PI3Kα, and provide a key platform for the design of novel chemotherapeutic PI3Kα inhibitors.

Lacking catalase, a protistan parasite draws on its photosynthetic ancestry to complete an antioxidant repertoire with ascorbate peroxidase.

BACKGROUND: Antioxidative enzymes contribute to a parasite's ability to counteract the host's intracellular killing mechanisms. The facultative intracellular oyster parasite, Perkinsus marinus, a sister taxon to dinoflagellates and apicomplexans, is responsible for mortalities of oysters along the Atlantic coast of North America. Parasite trophozoites enter molluscan hemocytes by subverting the phagocytic response while inhibiting the typical respiratory burst. Because P. marinus lacks catalase, the mechanism(s) by which the parasite evade the toxic effects of hydrogen peroxide had remained unclear. We previously found that P. marinus displays an ascorbate-dependent peroxidase (APX) activity typical of photosynthetic eukaryotes. Like other alveolates, the evolutionary history of P. marinus includes multiple endosymbiotic events. The discovery of APX in P. marinus raised the questions: From which ancestral lineage is this APX derived, and what role does it play in the parasite's life history? RESULTS: Purification of P. marinus cytosolic APX activity identified a 32 kDa protein. Amplification of parasite cDNA with oligonucleotides corresponding to peptides of the purified protein revealed two putative APX-encoding genes, designated PmAPX1 and PmAPX2. The predicted proteins are 93% identical, and PmAPX2 carries a 30 amino acid N-terminal extension relative to PmAPX1. The P. marinus APX proteins are similar to predicted APX proteins of dinoflagellates, and they more closely resemble chloroplastic than cytosolic APX enzymes of plants. Immunofluorescence for PmAPX1 and PmAPX2 shows that PmAPX1 is cytoplasmic, while PmAPX2 is localized to the periphery of the central vacuole. Three-dimensional modeling of the predicted proteins shows pronounced differences in surface charge of PmAPX1 and PmAPX2 in the vicinity of the aperture that provides access to the heme and active site. CONCLUSIONS: PmAPX1 and PmAPX2 phylogenetic analysis suggests that they are derived from a plant ancestor. Plant ancestry is further supported by the presence of ascorbate synthesis genes in the P. marinus genome that are similar to those in plants. The localizations and 3D structures of the two APX isoforms suggest that APX fulfills multiple functions in P. marinus within two compartments. The possible role of APX in free-living and parasitic stages of the life history of P. marinus is discussed.

Ca2+-dependent regulation of sodium channels NaV1.4 and NaV1.5 is controlled by the post-IQ motif.

Skeletal muscle voltage-gated Na+ channel (NaV1.4) activity is subject to calmodulin (CaM) mediated Ca2+-dependent inactivation; no such inactivation is observed in the cardiac Na+ channel (NaV1.5). Taken together, the crystal structures of the NaV1.4 C-terminal domain relevant complexes and thermodynamic binding data presented here provide a rationale for this isoform difference. A Ca2+-dependent CaM N-lobe binding site previously identified in NaV1.5 is not present in NaV1.4 allowing the N-lobe to signal other regions of the NaV1.4 channel. Consistent with this mechanism, removing this binding site in NaV1.5 unveils robust Ca2+-dependent inactivation in the previously insensitive isoform. These findings suggest that Ca2+-dependent inactivation is effected by CaM's N-lobe binding outside the NaV C-terminal while CaM's C-lobe remains bound to the NaV C-terminal. As the N-lobe binding motif of NaV1.5 is a mutational hotspot for inherited arrhythmias, the contributions of mutation-induced changes in CDI to arrhythmia generation is an intriguing possibility.

Structure of the zebrafish galectin-1-L2 and model of its interaction with the infectious hematopoietic necrosis virus (IHNV) envelope glycoprotein.

Galectins, highly conserved ß-galactoside-binding lectins, have diverse regulatory roles in development and immune homeostasis and can mediate protective functions during microbial infection. In recent years, the role of galectins in viral infection has generated considerable interest. Studies on highly pathogenic viruses have provided invaluable insight into the participation of galectins in various stages of viral infection, including attachment and entry. Detailed mechanistic and structural aspects of these processes remain undetermined. To address some of these gaps in knowledge, we used Zebrafish as a model system to examine the role of galectins in infection by infectious hematopoietic necrosis virus (IHNV), a rhabdovirus that is responsible for significant losses in both farmed and wild salmonid fish. Like other rhabdoviruses, IHNV is characterized by an envelope consisting of trimers of a glycoprotein that display multiple N-linked oligosaccharides and play an integral role in viral infection by mediating the virus attachment and fusion. Zebrafish's proto-typical galectin Drgal1-L2 and the chimeric-type galectin Drgal3-L1 interact directly with the glycosylated envelope of IHNV, and significantly reduce viral attachment. In this study, we report the structure of the complex of Drgal1-L2 with N-acetyl-d-lactosamine at 2.0 Å resolution. To gain structural insight into the inhibitory effect of these galectins on IHNV attachment to the zebrafish epithelial cells, we modeled Drgal3-L1 based on human galectin-3, as well as, the ectodomain of the IHNV glycoprotein. These models suggest mechanisms for which the binding of these galectins to the IHNV glycoprotein hinders with different potencies the viral attachment required for infection.

Getting the Most Out of Your Crystals: Data Collection at the New High-Flux, Microfocus MX Beamlines at NSLS-II.

Advances in synchrotron technology are changing the landscape of macromolecular crystallography. The two recently opened beamlines at NSLS-II-AMX and FMX-deliver high-flux microfocus beams that open new possibilities for crystallographic data collection. They are equipped with state-of-the-art experimental stations and automation to allow data collection on previously intractable crystals. Optimized data collection strategies allow users to tailor crystal positioning to optimally distribute the X-ray dose over its volume. Vector data collection allows the user to define a linear trajectory along a well diffracting volume of the crystal and perform rotational data collection while moving along the vector. This is particularly well suited to long, thin crystals. We describe vector data collection of three proteins-Akt1, PI3Kα, and CDP-Chase-to demonstrate its application and utility. For smaller crystals, we describe two methods for multicrystal data collection in a single loop, either manually selecting multiple centers (using H108A-PHM as an example), or "raster-collect", a more automated approach for a larger number of crystals (using CDP-Chase as an example).

Effects of copper occupancy on the conformational landscape of peptidylglycine α-hydroxylating monooxygenase.

The structures of metalloproteins that use redox-active metals for catalysis are usually exquisitely folded in a way that they are prearranged to accept their metal cofactors. Peptidylglycine α-hydroxylating monooxygenase (PHM) is a dicopper enzyme that catalyzes hydroxylation of the α-carbon of glycine-extended peptides for the formation of des-glycine amidated peptides. Here, we present the structures of apo-PHM and of mutants of one of the copper sites (H107A, H108A, and H172A) determined in the presence and absence of citrate. Together, these structures show that the absence of one copper changes the conformational landscape of PHM. In one of these structures, a large interdomain rearrangement brings residues from both copper sites to coordinate a single copper (closed conformation) indicating that full copper occupancy is necessary for locking the catalytically competent conformation (open). These data suggest that in addition to their required participation in catalysis, the redox-active metals play an important structural role.

Identification of a Shared Cytochrome p4502E1 Epitope Found in Anesthetic Drug-Induced and Viral Hepatitis.

Cytochrome p4502E1 (CYP2E1) autoantibodies are biomarkers for drug-induced hepatitis and chronic hepatitis C. However, major histocompatibility-restricted CYP2E1 epitopes associated with these diseases have not been identified. We hypothesized that CYP2E1 epitopes associated with different types of hepatitis may be shared and may impact immune responses and metabolism. SYFPEITHI epitope prediction identified CYP2E1 candidate epitopes that would be recognized by MHC II haplotypes. Candidate epitopes were tested for induction of hepatitis and CYP2E1 autoantibodies in mice and recognition by sera from patients with anesthetic drug-induced and viral hepatitis. Human liver cells treated with epitope hybridoma serum were analyzed for mitochondrial stress. CYP2E1 activity was measured in human microsomes similarly treated. Epitope antibodies in viral hepatitis sera were analyzed using linear regression to uncover associations with liver pathology. A P value of <0.05 was considered significant. One epitope (Gly113-Leu135) induced hepatitis and CYP2E1 autoantibodies in mice after modification of Lys123 (P < 0.05). Gly113-Leu135 antiserum recognized mitochondria and endoplasmic reticula (P < 0.05), upregulated HSP27 (P < 0.01) and mitochondrial oxidative stress via complex 1 inhibition (P < 0.001), and inhibited CYP2E1 activity. Gly113-Leu135 IgG4 detected in viral hepatitis sera was associated with severe hepatic fibrosis (P = 0.0142). We found a novel CYP2E1 epitope that was detected in anesthetic and viral hepatitis and that triggered hepatitis in mice. Our findings may improve understanding of hepatic immune responses triggered by metabolism or viruses.IMPORTANCE Drug-induced hepatitis is the leading reason that an approved drug is removed from the commercial market. Halogenated anesthetics can induce hepatitis in susceptible persons, and cytochrome p4502E1 (CYP2E1) enzymes responsible for their metabolism induce antibodies in addition to hepatitis. CYP2E1 antibodies detected in anesthetic hepatitis patients have been detected in patients with viral hepatitis, suggesting that these different forms of hepatitis could develop immune reactions to a common segment or epitope of CYP2E1. We have found a common MHC-restricted CYP2E1 epitope in anesthetic and viral hepatitis that is a dominant epitope in anesthetic hepatitis and is significantly associated with fibrosis in patients with viral hepatitis. Along with conformational epitopes, our identification of MHC-restricted CYP2E1 epitopes can be used to develop specific diagnostic tests for drug-induced or viral hepatitis or associated fibrosis or to predict individuals at risk for developing these diseases or their sequelae.

Akt Kinase Activation Mechanisms Revealed Using Protein Semisynthesis.

Akt is a critical protein kinase that drives cancer proliferation, modulates metabolism, and is activated by C-terminal phosphorylation. The current structural model for Akt activation by C-terminal phosphorylation has centered on intramolecular interactions between the C-terminal tail and the N lobe of the kinase domain. Here, we employ expressed protein ligation to produce site-specifically phosphorylated forms of purified Akt1 that are well suited for mechanistic analysis. Using biochemical, crystallographic, and cellular approaches, we determine that pSer473-Akt activation is driven by an intramolecular interaction between the C-tail and the pleckstrin homology (PH)-kinase domain linker that relieves PH domain-mediated Akt1 autoinhibition. Moreover, dual phosphorylation at Ser477/Thr479 activates Akt1 through a different allosteric mechanism via an apparent activation loop interaction that reduces autoinhibition by the PH domain and weakens PIP3 affinity. These results provide a new framework for understanding how Akt is controlled in cell signaling and suggest distinct functions for differentially modified Akt forms.