scholarly journals Unraveling the structural elements of pH sensitivity and substrate binding in the human zinc transporter SLC39A2 (ZIP2)

2019 ◽  
Vol 294 (20) ◽  
pp. 8046-8063 ◽  
Author(s):  
Gergely Gyimesi ◽  
Giuseppe Albano ◽  
Daniel G. Fuster ◽  
Matthias A. Hediger ◽  
Jonai Pujol-Giménez
Keyword(s):  
Binding Site ◽  
Metal Binding ◽  
In Silico ◽  
Metal Ion ◽  
Substrate Binding ◽  
Functional Characterization ◽  
Ph Sensitivity ◽  
Hek293 Cells ◽  
Substrate Binding Site

The transport and ion-coupling mechanisms of ZIP transporters remain largely uncharacterized. Previous work in our laboratory has revealed that the solute carrier family 39 member A2 (SLC39A2/ZIP2) increases its substrate transport rate in the presence of extracellular H+. Here, we used a combination of in silico and in vitro techniques involving structural modeling, mutagenesis, and functional characterization in HEK293 cells to identify amino acid residues potentially relevant for both the ZIP2–H+ interaction and substrate binding. Our ZIP2 models revealed a cluster of charged residues close to the substrate–translocation pore. Interestingly, the H63A substitution completely abrogated pH sensitivity, and substitutions of Glu-67 and Phe-269 altered the pH and voltage modulation of transport. In contrast, substitution of Glu-106, which might be part of a dimerization interface, altered pH but not voltage modulation. Substitution of Phe-269, located close to the substrate-binding site, also affected substrate selectivity. These findings were supported by an additional model of ZIP2 that was based on the structure of a prokaryotic homolog, Bordetella bronchiseptica ZrT/Irt-like protein (bbZIP), and in silico pKa calculations. We also found that residues Glu-179, His-175, His-202, and Glu-276 are directly involved in the coordination of the substrate metal ion. We noted that, unlike bbZIP, human ZIP2 is predicted to harbor a single divalent metal-binding site, with the charged side chain of Lys-203 replacing the second bound ion. Our results provide the first structural evidence for the previously observed pH and voltage modulation of ZIP2-mediated metal transport, identify the substrate-binding site, and suggest a structure-based transport mechanism for the ZIP2 transporter.

Comprehensive in silico Study of GLUT10: Prediction of Possible Substrate Binding Sites and Interacting Molecules

2020 ◽  
Vol 21 (2) ◽  
pp. 117-130 ◽  
Author(s):  
Mohammad J. Hosen ◽  
Mahmudul Hasan ◽  
Sourav Chakraborty ◽  
Ruhshan A. Abir ◽  
Abdullah Zubaer ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Binding Site ◽  
Glucose Transporter ◽  
Substrate Binding ◽  
Mutation Screening ◽  
Homology Model ◽  
Connective Tissue Disorder ◽  
Crystallographic Structure ◽  
Substrate Binding Site

Objectives: The Arterial Tortuosity Syndrome (ATS) is an autosomal recessive connective tissue disorder, mainly characterized by tortuosity and stenosis of the arteries with a propensity towards aneurysm formation and dissection. It is caused by mutations in the SLC2A10 gene that encodes the facilitative glucose transporter GLUT10. The molecules transported by and interacting with GLUT10 have still not been unambiguously identified. Hence, the study attempts to identify both the substrate binding site of GLUT10 and the molecules interacting with this site. Methods: As High-resolution X-ray crystallographic structure of GLUT10 was not available, 3D homology model of GLUT10 in open conformation was constructed. Further, molecular docking and bioinformatics investigation were employed. Results and Discussion: Blind docking of nine reported potential in vitro substrates with this 3D homology model revealed that substrate binding site is possibly made with PRO531, GLU507, GLU437, TRP432, ALA506, LEU519, LEU505, LEU433, GLN525, GLN510, LYS372, LYS373, SER520, SER124, SER533, SER504, SER436 amino acid residues. Virtual screening of all metabolites from the Human Serum Metabolome Database and muscle metabolites from Human Metabolite Database (HMDB) against the GLUT10 revealed possible substrates and interacting molecules for GLUT10, which were found to be involved directly or partially in ATS progression or different arterial disorders. Reported mutation screening revealed that a highly emergent point mutation (c. 1309G>A, p. Glu437Lys) is located in the predicted substrate binding site region. Conclusion: Virtual screening expands the possibility to explore more compounds that can interact with GLUT10 and may aid in understanding the mechanisms leading to ATS.

Equilibrium and Non-Equilibrium Molecular Dynamics Simulations Provide Potential Mechanisms for the Loss of Ligand Binding to αIIbβ3 Mutants Affecting the Ligand-Induced Metal Binding Site (LIMBS).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1805-1805
Author(s):  
Marta Murcia ◽  
Marketa Jirouskova ◽  
Jihong Li ◽  
Barry S. Coller ◽  
Filizola Marta
Keyword(s):  
Molecular Dynamics ◽  
Ligand Binding ◽  
Binding Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Cyclic Peptide ◽  
Hek293 Cells ◽  
Peptide Ligand ◽  
Non Equilibrium

Abstract Although the role of the β3 MIDAS metal ion in ligand binding to αIIbβ3 is well established, serving as the site of interaction of the ligand Asp residue, the role of the nearby LIMBS metal ion is less well defined. Previous studies suggested a role for the LIMBS in ligand binding. We confirmed this by showing that HEK293 cells expressing normal αIIbβ3 adhered to both immobilized fibrinogen and the RGD-containing venom echistatin in the presence of either Mg++/Ca++ or Mn++, whereas two different αIIbβ3 LIMBS mutants (β3 N215A and D217A) failed to adhere to either protein. In addition, we found that both mutations also increased the binding of mAb AP5, which recognizes a ligand-induced binding site (LIBS) in the β3 PSI domain (normal 7±4% vs N215A 46±12% and D217A 41±20% of mAb anti-αIIb (HIP8) binding; mean±SD, n=6, p<0.05 for both), indicating that the mutations caused allosteric changes in the conformation of the receptor. To define the mechanism(s) by which the LIMBS mutants affect ligand binding, we carried out equilibrium and non-equilibrium (steered) molecular dynamics (MD) simulations of the cyclic peptide ligand eptifibatide in complex with either the fully hydrated normal αIIbβ3 integrin headpiece (PDB 1TY6) or the equivalent β3 D217A mutant, with and without the LIMBS metal ion. Simulations were carried out using the GROMACS package with the OPLS all-atom force-field. During the simulation, the hybrid domain of the D217A mutant demonstrated greater structural fluctuations than the normal αIIbβ3. Although Craig et al. have reported the appearance of a new contact between the RGD peptide ligand Asp carboxyl and the LIMBS metal ion in αVβ3 after 10 ps of a 1 ns simulation, we did not observe the appearance of such an interaction between the eptifibatide carboxyl and the normal αIIbβ3 LIMBS metal ion even after 20 ns. We did, however, observe such an interaction with the LIMBS metal ion in the D217A mutant. This interaction was facilitated by the movement of the LIMBS ~ 2 Å closer to the MIDAS, and was accompanied by rearrangements of the LIMBS coordinating residues D158 and N215. When the D217A mutant simulation was performed in the absence of the LIMBS metal ion, changes in the orientation of E220 were also observed. The D217A mutant demonstrated increased fluctuations in the C177–C184 specificity-determining loop (SDL), which has been implicated in ligand binding, and decreased fluctuations in K209. Steered MD were used to investigate the pulling forces required to unbind eptifibatide from its binding site. Notably, although the unbinding force decreased modestly when the LIMBS metal ion was removed, it required removal of both the LIMBS and MIDAS metal ions to effect a marked reduction in unbinding force. The binding free energies of the association of the αIIb and β3 subunits were also calculated, and the D217A mutant in the presence of the LIMBS metal ion demonstrated much tighter binding than normal integrin αIIbβ3 (ΔGb −162±6 vs −119±6 Kcal/mol; mean±SD; n=500). We conclude that the LIMBS plays a crucial role in ligand binding to αIIbβ3, perhaps by virtue of its effects on the coordination of the MIDAS, the accentuated mobility of specific domains (e.g., the SDL and the hybrid domains), and/or the number and strength of contacts between αIIb and β3.

scholarly journals Virtual Screening for Potential Phytobioactives as Therapeutic Leads to Inhibit NQO1 for Selective Anticancer Therapy

Molecules ◽  
2021 ◽  
Vol 26 (22) ◽  
pp. 6863
Author(s):  
Bhargav Shreevatsa ◽  
Chandan Dharmashekara ◽  
Vikas Halasumane Swamy ◽  
Meghana V. Gowda ◽  
Raghu Ram Achar ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Binding Site ◽  
Anticancer Agents ◽  
Substrate Binding ◽  
Tumor Burden ◽  
Screening Tools ◽  
Tumor Promoter ◽  
Substrate Binding Site

NAD(P)H:quinone acceptor oxidoreductase-1 (NQO1) is a ubiquitous flavin adenine dinucleotide-dependent flavoprotein that promotes obligatory two-electron reductions of quinones, quinonimines, nitroaromatics, and azo dyes. NQO1 is a multifunctional antioxidant enzyme whose expression and deletion are linked to reduced and increased oxidative stress susceptibilities. NQO1 acts as both a tumor suppressor and tumor promoter; thus, the inhibition of NQO1 results in less tumor burden. In addition, the high expression of NQO1 is associated with a shorter survival time of cancer patients. Inhibiting NQO1 also enables certain anticancer agents to evade the detoxification process. In this study, a series of phytobioactives were screened based on their chemical classes such as coumarins, flavonoids, and triterpenoids for their action on NQO1. The in silico evaluations were conducted using PyRx virtual screening tools, where the flavone compound, Orientin showed a better binding affinity score of −8.18 when compared with standard inhibitor Dicumarol with favorable ADME properties. An MD simulation study found that the Orientin binding to NQO1 away from the substrate-binding site induces a potential conformational change in the substrate-binding site, thereby inhibiting substrate accessibility towards the FAD-binding domain. Furthermore, with this computational approach we are offering a scope for validation of the new therapeutic components for their in vitro and in vivo efficacy against NQO1.

Catalytic Roles of the Metal Ion in the Substrate-Binding Site of Coenzyme B12-Dependent Diol Dehydratase

2011 ◽  
Vol 50 (7) ◽  
pp. 2944-2952 ◽  
Author(s):  
Takashi Kamachi ◽  
Kazuki Doitomi ◽  
Masanori Takahata ◽  
Tetsuo Toraya ◽  
Kazunari Yoshizawa
Keyword(s):  
Binding Site ◽  
Metal Ion ◽  
Substrate Binding ◽  
Coenzyme B12 ◽  
Substrate Binding Site ◽  
Diol Dehydratase

The in vitro inhibition of hepatic ferrochelatase by divalent lead and other soft metal ions

1983 ◽  
Vol 61 (4) ◽  
pp. 214-222 ◽  
Author(s):  
Reimer R. W. Gaertner ◽  
Bryan R. Hollebone
Keyword(s):  
Binding Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Substrate Inhibition ◽  
Thiol Group ◽  
Sodium Ascorbate ◽  
Sprague Dawley ◽  
Sprague Dawley Rats ◽  
Soft Metal

A solubilized protein with ferrochelatase activity has been extracted from hepatic mitochondria of Sprague–Dawley rats. Under anerobic conditions in the presence of sodium ascorbate the ferrochelatase velocity was typically 1.8 nM∙min−1∙mg−1. The extract also displayed zinc chelatase activity of 1.2 nM∙min−1∙mg−1 without either anerobic conditions or ascorbate. In both cases substrate inhibition occurred for metal ion and deuteroporphyrin, but in the linear range a noncompetitive two-site mechanism was observed. The ferrochelatase activity is inhibited by divalent copper, mercury, and lead ions and the sodium salt of p-chloromercuribenzoate and is replaced by zinc chelation activity. This evidence suggests that the metal-binding site includes a thiol group. The inhibition of the site is greatest with Cu2+ and decreases with increasing ionic radius to Pb2+. This observation implies that the binding site is stereochemically adapted to the small Fe2+ ion and to some extent protected from larger, sulfur-binding ions which can inhibit ferrochelatase activity.

scholarly journals Characterization of the substrate binding site of an iron detoxifying membrane transporter from Plasmodium falciparum

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Pragya Sharma ◽  
Veronika Tóth ◽  
Edel M. Hyland ◽  
Christopher J. Law
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Metal Binding ◽  
Ferrous Iron ◽  
Malaria Parasite ◽  
Substrate Binding ◽  
Structural Basis ◽  
Membrane Transporter ◽  
Substrate Binding Site ◽  
Metal Binding Domain

Abstract Background Plasmodium species are entirely dependent upon their host as a source of essential iron. Although it is an indispensable micronutrient, oxidation of excess ferrous iron to the ferric state in the cell cytoplasm can produce reactive oxygen species that are cytotoxic. The malaria parasite must therefore carefully regulate the processes involved in iron acquisition and storage. A 273 amino acid membrane transporter that is a member of the vacuolar iron transporter (VIT) family and an orthologue of the yeast Ca2+-sensitive cross complementer (CCC1) protein plays a major role in cytosolic iron detoxification of Plasmodium species and functions in transport of ferrous iron ions into the endoplasmic reticulum for storage. While this transporter, termed PfVIT, is not critical for viability of the parasite evidence from studies of mice infected with VIT-deficient Plasmodium suggests it could still provide an efficient target for chemoprophylactic treatment of malaria. Individual amino acid residues that constitute the Fe2+ binding site of the protein were identified to better understand the structural basis of substrate recognition and binding by PfVIT. Methods Using the crystal structure of a recently published plant VIT as a template, a high-quality homology model of PfVIT was constructed to identify the amino acid composition of the transporter’s substrate binding site and to act as a guide for subsequent mutagenesis studies. To test the effect of mutation of the substrate binding-site residues on PfVIT function a yeast complementation assay assessed the ability of overexpressed, recombinant wild type and mutant PfVIT to rescue an iron-sensitive deletion strain (ccc1∆) of Saccharomyces cerevisiae yeast from the toxic effects of a high concentration of extracellular iron. Results The combined in silico and mutagenesis approach identified a methionine residue located within the cytoplasmic metal binding domain of the transporter as essential for PfVIT function and provided insight into the structural basis for the Fe2+-selectivity of the protein. Conclusion The structural model of the metal binding site of PfVIT opens the door for rational design of therapeutics to interfere with iron homeostasis within the malaria parasite.

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