Structural basis of norepinephrine recognition and transport inhibition in neurotransmitter transporters

AbstractNorepinephrine is a biogenic amine neurotransmitter that has widespread effects on alertness, arousal and pain sensation. Consequently, blockers of norepinephrine uptake have served as vital tools to treat depression and chronic pain. Here, we employ the Drosophila melanogaster dopamine transporter as a surrogate for the norepinephrine transporter and determine X-ray structures of the transporter in its substrate-free and norepinephrine-bound forms. We also report structures of the transporter in complex with inhibitors of chronic pain including duloxetine, milnacipran and a synthetic opioid, tramadol. When compared to dopamine, we observe that norepinephrine binds in a different pose, in the vicinity of subsite C within the primary binding site. Our experiments reveal that this region is the binding site for chronic pain inhibitors and a determinant for norepinephrine-specific reuptake inhibition, thereby providing a paradigm for the design of specific inhibitors for catecholamine neurotransmitter transporters.

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Structural basis of norepinephrine recognition and transport inhibition in neurotransmitter transporters

10.1101/2020.04.30.070219 ◽

2020 ◽

Author(s):

Shabareesh Pidathala ◽

Aditya Kumar Mallela ◽

Deepthi Joseph ◽

Aravind Penmatsa

Keyword(s):

Chronic Pain ◽

Binding Site ◽

Dopamine Transporter ◽

Binding Pocket ◽

Norepinephrine Transporter ◽

Structural Basis ◽

List Type ◽

X Ray ◽

Neurotransmitter Transporters ◽

Reuptake Inhibition

AbstractNorepinephrine is a biogenic amine neurotransmitter that has widespread effects on cardiovascular tone, alertness and sensation of pain. As a consequence, blockers of norepinephrine uptake have served as vital tools to treat depression and chronic pain. Here, we employ a modified Drosophila melanogaster dopamine transporter as a surrogate for the human norepinephrine transporter and determine the X-ray structures of the transporter in its substrate-free and norepinephrine-bound forms. We also report structures of the transporter in complex with inhibitors of chronic pain including duloxetine, milnacipran and a synthetic opioid, tramadol. When compared to dopamine, we observe that norepinephrine binds in a different pose, in the vicinity of subsite C within the primary binding site. Our experiments reveal that this region is the binding site for chronic pain inhibitors and a determinant for norepinephrine-specific reuptake inhibition, thereby providing a paradigm for the design of specific inhibitors for catecholamine neurotransmitter transporters.HighlightsX-ray structures of the Drosophila dopamine transporter in substrate-free and norepinephrine bound forms.Norepinephrine and dopamine bind in distinct conformations within the binding pocket.Chronic pain inhibitors S-duloxetine, milnacipran and tramadol bind in the primary binding site and overlap with the norepinephrine-binding pose.Selective norepinephrine reuptake inhibition occurs through specific interactions at the subsite C in the primary binding pocket.

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Molecular recognition of sugar binding in a melibiose transporter MelB by X-ray crystallography

10.1101/2020.12.20.423627 ◽

2020 ◽

Author(s):

Lan Guan ◽

Parameswaran Hariharan

Keyword(s):

Molecular Recognition ◽

Binding Site ◽

Crystal Structures ◽

Mammalian Cells ◽

Substrate Binding ◽

Structural Basis ◽

Cooperative Binding ◽

Recognition Mechanism ◽

Substrate Binding Site ◽

X Ray

AbstractThe symporter melibiose permease MelB is the best-studied representative from MFS_2 family and the only protein in this large family with crystal structure determined. Previous thermodynamic studies show that MelB utilizes a cooperative binding as the core mechanism for its obligatory symport. Here we present two sugar-bound X-ray crystal structures of a Salmonella typhimurium MelB D59C uniport mutant that binds and catalyzes melibiose transport uncoupled to either cation, as determined by biochemical and biophysical characterizations. The two structures with bound nitrophenyl-α-D-galactoside or dodecyl-β-D-melibioside, which were refined to a resolution of 3.05 or 3.15 Å, respectively, are virtually identical at an outward-facing conformation; each one contains a α-galactoside molecule in the middle of protein. In the substrate-binding site, the galactosyl moiety on both ligands are at an essentially same configuration, so a galactoside specificity determinant pocket can be recognized, and hence the molecular recognition mechanism for the binding of sugar in MelB is deciphered. The data also allow to assign the conserved cation-binding pocket, which is directly connected to the sugar specificity determinant pocket. The intimate connection between the two selection sites lays the structural basis for the cooperative binding and coupled transport. This key structural finding answered the long-standing question on the substrate binding for the Na+-coupled MFS family of transporters.SignificanceMajor facilitator superfamily_2 transporters contain >10,000 members that are widely expressed from bacteria to mammalian cells, and catalyze uptake of varied nutrients from sugars to phospholipids. While several crystal structures with bound sugar for other MFS permeases have been determined, they are either uniporters or symporters coupled solely to H+. MelB catalyzes melibiose symport with either Na+, Li+, or H+, a prototype for Na+-coupled MFS transporters, but its sugar recognition has been a long-unsolved puzzle. Two high-resolution crystal structures presented here clearly reveal the molecular recognition mechanism for the binding of sugar in MelB. The substrate-binding site is characterized with a small specificity groove adjoining a large nonspecific cavity, which could offer a potential for future exploration of active transporters for drug delivery.

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Activating Janus kinase pseudokinase domain mutations in myeloproliferative and other blood cancers

Biochemical Society Transactions ◽

10.1042/bst20130084 ◽

2013 ◽

Vol 41 (4) ◽

pp. 1048-1054 ◽

Cited By ~ 10

Author(s):

Stefan N. Constantinescu ◽

Emilie Leroy ◽

Vitalina Gryshkova ◽

Christian Pecquet ◽

Alexandra Dusa

Keyword(s):

Structural Information ◽

Myeloproliferative Neoplasms ◽

Janus Kinase ◽

Cytokine Receptors ◽

Structural Basis ◽

X Ray ◽

Activating Mutations ◽

Tyrosine Kinase 2 ◽

V617f Mutation ◽

Specific Inhibitors

The discovery of the highly prevalent activating JAK (Janus kinase) 2 V617F mutation in myeloproliferative neoplasms, and of other pseudokinase domain-activating mutations in JAK2, JAK1 and JAK3 in blood cancers, prompted great interest in understanding how pseudokinase domains regulate kinase domains in JAKs. Recent functional and mutagenesis studies identified residues required for the V617F mutation to induce activation. Several X-ray crystal structures of either kinase or pseudokinase domains including the V617F mutant of JAK2 pseudokinase domains are now available, and a picture has emerged whereby the V617F mutation induces a defined conformational change around helix C of JH (JAK homology) 2. Effects of mutations on JAK2 can be extrapolated to JAK1 and TYK2 (tyrosine kinase 2), whereas JAK3 appears to be different. More structural information of the full-length JAK coupled to cytokine receptors might be required in order to define the structural basis of JH1 activation by JH2 mutants and eventually obtain mutant-specific inhibitors.

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Structural basis for recognition of diverse antidepressants by the human serotonin transporter

10.1101/204859 ◽

2017 ◽

Author(s):

Jonathan A. Coleman ◽

Eric Gouaux

Keyword(s):

Binding Site ◽

Serotonin Transporter ◽

Serotonin Reuptake ◽

Selective Serotonin Reuptake Inhibitors ◽

Structural Basis ◽

Local Concentration ◽

Substrate Binding Site ◽

X Ray ◽

Open Conformation ◽

Drug Selectivity

ABSTRACTSelective serotonin reuptake inhibitors are clinically prescribed antidepressants that act by increasing the local concentration of neurotransmitter at synapses and in extracellular spaces via blockade of the serotonin transporter. Here we report x-ray structures of engineered thermostable variants of the human serotonin transporter bound to the antidepressants sertraline, fluvoxamine, and paroxetine. The drugs prevent serotonin binding by occupying the central substrate binding site and stabilizing the transporter in an outward-open conformation. These structures explain how residues within the central site orchestrate binding of chemically diverse inhibitors and mediate transporter-drug selectivity.

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Electron diffraction detection of ordliking in annealed PbTe thin film

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100178227 ◽

1990 ◽

Vol 48 (4) ◽

pp. 1018-1019

Author(s):

Karimat El-Sayed

Keyword(s):

Thin Film ◽

Electron Diffraction ◽

Lead Telluride ◽

Structural Basis ◽

Face Centered Cubic ◽

X Ray ◽

Polycrystalline Thin Films ◽

Stage Process ◽

Diffraction Patterns ◽

Face Centered

Lead telluride is an important semiconductor of many applications. Many Investigators showed that there are anamolous descripancies in most of the electrophysical properties of PbTe polycrystalline thin films on annealing. X-Ray and electron diffraction studies are being undertaken in the present work in order to explain the cause of this anamolous behaviour.Figures 1-3 show the electron diffraction of the unheated, heated in air at 100°C and heated in air at 250°C respectively of a 300°A polycrystalline PbTe thin film. It can be seen that Fig. 1 is a typical [100] projection of a face centered cubic with unmixed (hkl) indices. Fig. 2 shows the appearance of faint superlattice reflections having mixed (hkl) indices. Fig. 3 shows the disappearance of thf superlattice reflections and the appearance of polycrystalline PbO phase superimposed on the [l00] PbTe diffraction patterns. The mechanism of this three stage process can be explained on structural basis as follows :

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Faculty Opinions recommendation of X-ray crystallographic studies on butyryl-ACP reveal flexibility of the structure around a putative acyl chain binding site.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1008094.101761 ◽

2002 ◽

Author(s):

John Shanklin

Keyword(s):

Binding Site ◽

Acyl Chain ◽

X Ray

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Structural basis of the stereoselective formation of the spirooxindole ring in the biosynthesis of citrinadins

Nature Communications ◽

10.1038/s41467-021-24421-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Zhiwen Liu ◽

Fanglong Zhao ◽

Boyang Zhao ◽

Jie Yang ◽

Joseph Ferrara ◽

...

Keyword(s):

High Resolution ◽

Organic Synthesis ◽

Indole Alkaloids ◽

Site Directed Mutagenesis ◽

Structural Basis ◽

Directed Mutagenesis ◽

Computational Studies ◽

X Ray ◽

Evolutionary Branch ◽

Catalytic Mechanisms

AbstractPrenylated indole alkaloids featuring spirooxindole rings possess a 3R or 3S carbon stereocenter, which determines the bioactivities of these compounds. Despite the stereoselective advantages of spirooxindole biosynthesis compared with those of organic synthesis, the biocatalytic mechanism for controlling the 3R or 3S-spirooxindole formation has been elusive. Here, we report an oxygenase/semipinacolase CtdE that specifies the 3S-spirooxindole construction in the biosynthesis of 21R-citrinadin A. High-resolution X-ray crystal structures of CtdE with the substrate and cofactor, together with site-directed mutagenesis and computational studies, illustrate the catalytic mechanisms for the possible β-face epoxidation followed by a regioselective collapse of the epoxide intermediate, which triggers semipinacol rearrangement to form the 3S-spirooxindole. Comparing CtdE with PhqK, which catalyzes the formation of the 3R-spirooxindole, we reveal an evolutionary branch of CtdE in specific 3S spirocyclization. Our study provides deeper insights into the stereoselective catalytic machinery, which is important for the biocatalysis design to synthesize spirooxindole pharmaceuticals.

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Discovery of fungal surface NADases predominantly present in pathogenic species

Nature Communications ◽

10.1038/s41467-021-21307-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Øyvind Strømland ◽

Juha P. Kallio ◽

Annica Pschibul ◽

Renate H. Skoge ◽

Hulda M. Harðardóttir ◽

...

Keyword(s):

Cell Death ◽

Aspergillus Fumigatus ◽

Neurospora Crassa ◽

Binding Site ◽

Host Cell ◽

Nicotinamide Adenine Dinucleotide ◽

Catalytic Mechanism ◽

Pathogenic Species ◽

X Ray ◽

Cellular Bioenergetics

AbstractNicotinamide adenine dinucleotide (NAD) is a key molecule in cellular bioenergetics and signalling. Various bacterial pathogens release NADase enzymes into the host cell that deplete the host’s NAD+ pool, thereby causing rapid cell death. Here, we report the identification of NADases on the surface of fungi such as the pathogen Aspergillus fumigatus and the saprophyte Neurospora crassa. The enzymes harbour a tuberculosis necrotizing toxin (TNT) domain and are predominately present in pathogenic species. The 1.6 Å X-ray structure of the homodimeric A. fumigatus protein reveals unique properties including N-linked glycosylation and a Ca2+-binding site whose occupancy regulates activity. The structure in complex with a substrate analogue suggests a catalytic mechanism that is distinct from those of known NADases, ADP-ribosyl cyclases and transferases. We propose that fungal NADases may convey advantages during interaction with the host or competing microorganisms.

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