Structural insights into the ion selectivity of the MgtE channel for Mg2+ over Ca2+

MgtE is a Mg2+-selective ion channel whose orthologs are widely distributed from prokaryotes to eukaryotes, including humans, and play an important role in the maintenance of cellular Mg2+ homeostasis. Previous functional analyses showed that MgtE transports divalent cations with high selectivity for Mg2+ over Ca2+. Whereas the high-resolution structure determination of the MgtE transmembrane (TM) domain in complex with Mg2+ ions revealed a Mg2+ recognition mechanism of MgtE, the previous Ca2+-bound structure of the MgtE TM domain was determined only at moderate resolution (3.2 angstrom resolution), which was insufficient to visualize the water molecules coordinated to Ca2+ ions. Thus, the structural basis of the ion selectivity of MgtE for Mg2+ over Ca2+ has remained unclear. Here, we showed that the metal-binding site of the MgtE TM domain binds to Mg2+ ~500-fold more strongly than Ca2+. We then determined the crystal structure of the MgtE TM domain in complex with Ca2+ ions at a higher resolution (2.5 angstrom resolution), allowing us to reveal hexahydrated Ca2+, which is similarly observed in the previously determined Mg2+-bound structure but with extended metal-oxygen bond lengths. Our structural, biochemical, and computational analyses provide mechanistic insights into the ion selectivity of MgtE for Mg2+ over Ca2+.

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Tuning the ion selectivity of two-pore channels

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1616191114 ◽

2017 ◽

Vol 114 (5) ◽

pp. 1009-1014 ◽

Cited By ~ 39

Author(s):

Jiangtao Guo ◽

Weizhong Zeng ◽

Youxing Jiang

Keyword(s):

Sequence Similarity ◽

Ion Selectivity ◽

Structural Basis ◽

Monovalent Cations ◽

Nonselective Cation Channel ◽

High Sequence Similarity ◽

Selective Channel ◽

Tm Domain ◽

Key Residues

Organellar two-pore channels (TPCs) contain two copies of aShaker-like six-transmembrane (6-TM) domain in each subunit and are ubiquitously expressed in plants and animals. Interestingly, plant and animal TPCs share high sequence similarity in the filter region, yet exhibit drastically different ion selectivity. Plant TPC1 functions as a nonselective cation channel on the vacuole membrane, whereas mammalian TPC channels have been shown to be endo/lysosomal Na+-selective or Ca2+-release channels. In this study, we performed systematic characterization of the ion selectivity of TPC1 fromArabidopsis thaliana(AtTPC1) and compared its selectivity with the selectivity of human TPC2 (HsTPC2). We demonstrate that AtTPC1 is selective for Ca2+over Na+, but nonselective among monovalent cations (Li+, Na+, and K+). Our results also confirm that HsTPC2 is a Na+-selective channel activated by phosphatidylinositol 3,5-bisphosphate. Guided by our recent structure of AtTPC1, we converted AtTPC1 to a Na+-selective channel by mimicking the selectivity filter of HsTPC2 and identified key residues in the TPC filters that differentiate the selectivity between AtTPC1 and HsTPC2. Furthermore, the structure of the Na+-selective AtTPC1 mutant elucidates the structural basis for Na+selectivity in mammalian TPCs.

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Structural implications of weak Ca2+ block in Drosophila cyclic nucleotide–gated channels

The Journal of General Physiology ◽

10.1085/jgp.201511431 ◽

2015 ◽

Vol 146 (3) ◽

pp. 255-263 ◽

Cited By ~ 1

Author(s):

Yee Ling Lam ◽

Weizhong Zeng ◽

Mehabaw Getahun Derebe ◽

Youxing Jiang

Keyword(s):

Cyclic Nucleotide ◽

Ion Selectivity ◽

Structural Difference ◽

Selectivity Filter ◽

Structural Basis ◽

Functional Studies ◽

Cyclic Nucleotide Gated Channels ◽

Cng Channel ◽

High Resolution Structure ◽

Calcium Permeability

Calcium permeability and the concomitant calcium block of monovalent ion current (“Ca2+ block”) are properties of cyclic nucleotide–gated (CNG) channel fundamental to visual and olfactory signal transduction. Although most CNG channels bear a conserved glutamate residue crucial for Ca2+ block, the degree of block displayed by different CNG channels varies greatly. For instance, the Drosophila melanogaster CNG channel shows only weak Ca2+ block despite the presence of this glutamate. We previously constructed a series of chimeric channels in which we replaced the selectivity filter of the bacterial nonselective cation channel NaK with a set of CNG channel filter sequences and determined that the resulting NaK2CNG chimeras displayed the ion selectivity and Ca2+ block properties of the parent CNG channels. Here, we used the same strategy to determine the structural basis of the weak Ca2+ block observed in the Drosophila CNG channel. The selectivity filter of the Drosophila CNG channel is similar to that of most other CNG channels except that it has a threonine at residue 318 instead of a proline. We constructed a NaK chimera, which we called NaK2CNG-Dm, which contained the Drosophila selectivity filter sequence. The high resolution structure of NaK2CNG-Dm revealed a filter structure different from those of NaK and all other previously investigated NaK2CNG chimeric channels. Consistent with this structural difference, functional studies of the NaK2CNG-Dm chimeric channel demonstrated a loss of Ca2+ block compared with other NaK2CNG chimeras. Moreover, mutating the corresponding threonine (T318) to proline in Drosophila CNG channels increased Ca2+ block by 16 times. These results imply that a simple replacement of a threonine for a proline in Drosophila CNG channels has likely given rise to a distinct selectivity filter conformation that results in weak Ca2+ block.

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Structural basis for the Mg2+ recognition and regulation of the CorC Mg2+ transporter

Science Advances ◽

10.1126/sciadv.abe6140 ◽

2021 ◽

Vol 7 (7) ◽

pp. eabe6140

Author(s):

Yichen Huang ◽

Fei Jin ◽

Yosuke Funato ◽

Zhijian Xu ◽

Weiliang Zhu ◽

...

Keyword(s):

Binding Site ◽

Genetic Disorders ◽

Genetic Diseases ◽

Structural Basis ◽

Atp Binding ◽

Pathogenic Microorganisms ◽

Domains Of Life ◽

Body Absorption ◽

Tm Domain ◽

Functional Analyses

The CNNM/CorC family proteins are Mg2+ transporters that are widely distributed in all domains of life. In bacteria, CorC has been implicated in the survival of pathogenic microorganisms. In humans, CNNM proteins are involved in various biological events, such as body absorption/reabsorption of Mg2+ and genetic disorders. Here, we determined the crystal structure of the Mg2+-bound CorC TM domain dimer. Each protomer has a single Mg2+ binding site with a fully dehydrated Mg2+ ion. The residues at the Mg2+ binding site are strictly conserved in both human CNNM2 and CNNM4, and many of these residues are associated with genetic diseases. Furthermore, we determined the structures of the CorC cytoplasmic region containing its regulatory ATP-binding domain. A combination of structural and functional analyses not only revealed the potential interface between the TM and cytoplasmic domains but also showed that ATP binding is important for the Mg2+ export activity of CorC.

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Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

Biochemical Journal ◽

10.1042/bcj20190399 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3333-3353 ◽

Cited By ~ 3

Author(s):

Malti Yadav ◽

Kamalendu Pal ◽

Udayaditya Sen

Keyword(s):

Active Site ◽

Metal Binding ◽

Metal Ion ◽

Triosephosphate Isomerase ◽

Catalytic Mechanism ◽

Degradation Mechanism ◽

Structural Basis ◽

Conserved Residues ◽

Phosphodiester Bond ◽

Cyclic Dinucleotides

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

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A Model for Predicting Cation Selectivity and Permeability in AMPA and NMDA Receptors Based on Receptor Subunit Composition

Frontiers in Synaptic Neuroscience ◽

10.3389/fnsyn.2021.779759 ◽

2021 ◽

Vol 13 ◽

Author(s):

Sampath Kumar ◽

Sanjay S. Kumar

Keyword(s):

Experimental Data ◽

Divalent Cations ◽

Ion Selectivity ◽

Gene Families ◽

Subunit Composition ◽

Receptor Subunit ◽

Cation Selectivity ◽

Subunit Stoichiometry ◽

Ampa And Nmda Receptors

Glutamatergic AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) and NMDA (N-methyl-D-aspartate) receptors are implicated in diverse functions ranging from synaptic plasticity to cell death. They are heterotetrameric proteins whose subunits are derived from multiple distinct gene families. The subunit composition of these receptors determines their permeability to monovalent and/or divalent cations, but it is not entirely clear how this selectivity arises in native and recombinantly-expressed receptor populations. By analyzing the sequence of amino acids lining the selectivity filters within the pore forming membrane helices (M2) of these subunits and by correlating subunit stoichiometry of these receptors with their ability to permeate Na+ and/or Ca2+, we propose here a mathematical model for predicting cation selectivity and permeability in these receptors. The model proposed is based on principles of charge attractivity and charge neutralization within the pore forming region of these receptors; it accurately predicts and reconciles experimental data across various platforms including Ca2+ permeability of GluA2-lacking AMPARs and ion selectivity within GluN3-containing di- and tri-heteromeric NMDARs. Additionally, the model provides insights into biophysical mechanisms regulating cation selectivity and permeability of these receptors and the role of various subunits in these processes.

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Structural basis of AdoMet-dependent aminocarboxypropyl transfer reaction catalyzed by tRNA-wybutosine synthesizing enzyme, TYW2

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0905270106 ◽

2009 ◽

Vol 106 (37) ◽

pp. 15616-15621 ◽

Cited By ~ 30

Author(s):

Masataka Umitsu ◽

Hiroshi Nishimasu ◽

Akiko Noma ◽

Tsutomu Suzuki ◽

Ryuichiro Ishitani ◽

...

Keyword(s):

Crystal Structures ◽

Binding Pocket ◽

Structural Basis ◽

Binding Modes ◽

Substrate Binding Pocket ◽

Methyl Group ◽

Group Transfer ◽

Canonical Class ◽

Functional Analyses

S-adenosylmethionine (AdoMet) is a methyl donor used by a wide variety of methyltransferases, and it is also used as the source of an α-amino-α-carboxypropyl (“acp”) group by several enzymes. tRNA-yW synthesizing enzyme-2 (TYW2) is involved in the biogenesis of a hypermodified nucleotide, wybutosine (yW), and it catalyzes the transfer of the “acp” group from AdoMet to the C7 position of the imG-14 base, a yW precursor. This modified nucleoside yW is exclusively located at position 37 of eukaryotic tRNAPhe, and it ensures the anticodon-codon pairing on the ribosomal decoding site. Although this “acp” group has a significant role in preventing decoding frame shifts, the mechanism of the “acp” group transfer by TYW2 remains unresolved. Here we report the crystal structures and functional analyses of two archaeal homologs of TYW2 from Pyrococcus horikoshii and Methanococcus jannaschii. The in vitro mass spectrometric and radioisotope-labeling analyses confirmed that these archaeal TYW2 homologues have the same activity as yeast TYW2. The crystal structures verified that the archaeal TYW2 contains a canonical class-I methyltransferase (MTase) fold. However, their AdoMet-bound structures revealed distinctive AdoMet-binding modes, in which the “acp” group, instead of the methyl group, of AdoMet is directed to the substrate binding pocket. Our findings, which were confirmed by extensive mutagenesis studies, explain why TYW2 transfers the “acp” group, and not the methyl group, from AdoMet to the nucleobase.

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Structural basis for auxiliary subunit KCTD16 regulation of the GABABreceptor

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1903024116 ◽

2019 ◽

Vol 116 (17) ◽

pp. 8370-8379 ◽

Cited By ~ 11

Author(s):

Hao Zuo ◽

Ian Glaaser ◽

Yulin Zhao ◽

Igor Kurinov ◽

Lidia Mosyak ◽

...

Keyword(s):

Structural Basis ◽

Functional Coupling ◽

Girk Channels ◽

Receptor Complexes ◽

Binding Interface ◽

Tetramerization Domain ◽

High Resolution Structure ◽

Inwardly Rectifying ◽

The Brain ◽

Oligomerization Domain

Metabotropic GABABreceptors mediate a significant fraction of inhibitory neurotransmission in the brain. Native GABABreceptor complexes contain the principal subunits GABAB1and GABAB2, which form an obligate heterodimer, and auxiliary subunits, known as potassium channel tetramerization domain-containing proteins (KCTDs). KCTDs interact with GABABreceptors and modify the kinetics of GABABreceptor signaling. Little is known about the molecular mechanism governing the direct association and functional coupling of GABABreceptors with these auxiliary proteins. Here, we describe the high-resolution structure of the KCTD16 oligomerization domain in complex with part of the GABAB2receptor. A single GABAB2C-terminal peptide is bound to the interior of an open pentamer formed by the oligomerization domain of five KCTD16 subunits. Mutation of specific amino acids identified in the structure of the GABAB2–KCTD16 interface disrupted both the biochemical association and functional modulation of GABABreceptors and G protein-activated inwardly rectifying K+channel (GIRK) channels. These interfacial residues are conserved among KCTDs, suggesting a common mode of KCTD interaction with GABABreceptors. Defining the binding interface of GABABreceptor and KCTD reveals a potential regulatory site for modulating GABAB-receptor function in the brain.

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Emergence of a novel immune-evasion strategy from an ancestral protein fold in bacteriophage Mu

Nucleic Acids Research ◽

10.1093/nar/gkaa319 ◽

2020 ◽

Vol 48 (10) ◽

pp. 5294-5305

Author(s):

Shweta Karambelkar ◽

Shubha Udupa ◽

Vykuntham Naga Gowthami ◽

Sharmila Giliyaru Ramachandra ◽

Ganduri Swapna ◽

...

Keyword(s):

Active Site ◽

Metal Binding ◽

Broad Host Range ◽

Dna Modification ◽

Bacteriophage Mu ◽

Acetyl Coa ◽

Protein Architecture ◽

Iron Binding Protein ◽

Free Radical Chemistry ◽

Computational Analyses

Abstract The broad host range bacteriophage Mu employs a novel ‘methylcarbamoyl’ modification to protect its DNA from diverse restriction systems of its hosts. The DNA modification is catalyzed by a phage-encoded protein Mom, whose mechanism of action is a mystery. Here, we characterized the co-factor and metal-binding properties of Mom and provide a molecular mechanism to explain ‘methylcarbamoyl’ation of DNA by Mom. Computational analyses revealed a conserved GNAT (GCN5-related N-acetyltransferase) fold in Mom. We demonstrate that Mom binds to acetyl CoA and identify the active site. We discovered that Mom is an iron-binding protein, with loss of Fe2+/3+-binding associated with loss of DNA modification activity. The importance of Fe2+/3+ is highlighted by the colocalization of Fe2+/3+ with acetyl CoA within the Mom active site. Puzzlingly, acid-base mechanisms employed by >309,000 GNAT members identified so far, fail to support methylcarbamoylation of adenine using acetyl CoA. In contrast, free-radical chemistry catalyzed by transition metals like Fe2+/3+ can explain the seemingly challenging reaction, accomplished by collaboration between acetyl CoA and Fe2+/3+. Thus, binding to Fe2+/3+, a small but unprecedented step in the evolution of Mom, allows a giant chemical leap from ordinary acetylation to a novel methylcarbamoylation function, while conserving the overall protein architecture.

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Structures of ribosome-bound initiation factor 2 reveal the mechanism of subunit association

Science Advances ◽

10.1126/sciadv.1501502 ◽

2016 ◽

Vol 2 (3) ◽

pp. e1501502 ◽

Cited By ~ 36

Author(s):

Thiemo Sprink ◽

David J. F. Ramrath ◽

Hiroshi Yamamoto ◽

Kaori Yamamoto ◽

Justus Loerke ◽

...

Keyword(s):

Translation Initiation ◽

Protein Biosynthesis ◽

Initiation Factor ◽

Structural Basis ◽

Initiation Complex ◽

Cryo Electron Microscopy ◽

Initiation Factor 2 ◽

High Resolution Structure ◽

Guanosine Triphosphatase ◽

Resolution Structure

Throughout the four phases of protein biosynthesis—initiation, elongation, termination, and recycling—the ribosome is controlled and regulated by at least one specified translational guanosine triphosphatase (trGTPase). Although the structural basis for trGTPase interaction with the ribosome has been solved for the last three steps of translation, the high-resolution structure for the key initiation trGTPase, initiation factor 2 (IF2), complexed with the ribosome, remains elusive. We determine the structure of IF2 complexed with a nonhydrolyzable guanosine triphosphate analog and initiator fMet-tRNAiMet in the context of the Escherichia coli ribosome to 3.7-Å resolution using cryo-electron microscopy. The structural analysis reveals previously unseen intrinsic conformational modes of the 70S initiation complex, establishing the mutual interplay of IF2 and initator transfer RNA (tRNA) with the ribsosome and providing the structural foundation for a mechanistic understanding of the final steps of translation initiation.

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The high-resolution structure of DNA-binding protein HU from Bacillus stearothermophilus

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444999000578 ◽

1999 ◽

Vol 55 (4) ◽

pp. 801-809 ◽

Cited By ~ 31

Author(s):

Stephen W. White ◽

Keith S. Wilson ◽

Krzysztof Appelt ◽

Isao Tanaka

Keyword(s):

High Resolution ◽

Protein Interactions ◽

Bacillus Stearothermophilus ◽

Helical Structure ◽

Structural Basis ◽

Hydrophobic Core ◽

Salt Bridges ◽

Medium Resolution ◽

High Resolution Structure ◽

Resolution Structure

Protein HU is a ubiquitous prokaryotic protein which controls the architecture of genomic DNA. It binds DNA non-specifically and promotes the bending and supercoiling of the double helical structure. HU is involved in many DNA-associated cellular processes, including replication, transcription and the packaging of DNA into chromosome-like structures. Originally determined at medium resolution, the crystal structure of HU has now been refined at 2.0 Å resolution. The high-resolution structure shows that the dimeric molecule is essentially a compact platform for two flexible and basic arms which wrap around the DNA molecule. To maximize the protein's stability, non-secondary structural regions are reduced to a minimum, there is an extensive aromatic hydrophobic core and several salt bridges and hydrogen-bonded water molecules knit together crucial regions. Based on the original medium-resolution structure of HU, several proposals were made concerning the structural basis of HU's ability to bind, bend and supercoil DNA. Each of these proposals is fully supported by the high-resolution structure. Most notably, the surfaces of the molecule which appear to mediate protein–DNA and protein–protein interactions have the ideal shapes and physicochemical properties to perform these functions.

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