scholarly journals Crystal structures of Ca2+–calmodulin bound to NaV C-terminal regions suggest role for EF-hand domain in binding and inactivation

2019 ◽  
Vol 116 (22) ◽  
pp. 10763-10772 ◽  
Author(s):  
Bernd R. Gardill ◽  
Ricardo E. Rivera-Acevedo ◽  
Ching-Chieh Tung ◽  
Filip Van Petegem
Keyword(s):  
Crystal Structure ◽  
Binding Sites ◽  
Binding Mode ◽  
Conformational Switch ◽  
Channel Inactivation ◽  
Dependent Inactivation ◽  
Ef Hand ◽  
Inherited Arrhythmia ◽  
A Site

Voltage-gated sodium (NaV) and calcium channels (CaV) form targets for calmodulin (CaM), which affects channel inactivation properties. A major interaction site for CaM resides in the C-terminal (CT) region, consisting of an IQ domain downstream of an EF-hand domain. We present a crystal structure of fully Ca2+-occupied CaM, bound to the CT of NaV1.5. The structure shows that the C-terminal lobe binds to a site ∼90° rotated relative to a previous site reported for an apoCaM complex with the NaV1.5 CT and for ternary complexes containing fibroblast growth factor homologous factors (FHF). We show that the binding of FHFs forces the EF-hand domain in a conformation that does not allow binding of the Ca2+-occupied C-lobe of CaM. These observations highlight the central role of the EF-hand domain in modulating the binding mode of CaM. The binding sites for Ca2+-free and Ca2+-occupied CaM contain targets for mutations linked to long-QT syndrome, a type of inherited arrhythmia. The related NaV1.4 channel has been shown to undergo Ca2+-dependent inactivation (CDI) akin to CaVs. We present a crystal structure of Ca2+/CaM bound to the NaV1.4 IQ domain, which shows a binding mode that would clash with the EF-hand domain. We postulate the relative reorientation of the EF-hand domain and the IQ domain as a possible conformational switch that underlies CDI.

Ca2+ Enhances U-Type Inactivation of N-Type (CaV2.2) Calcium Current in Rat Sympathetic Neurons

2006 ◽  
Vol 96 (3) ◽  
pp. 1075-1083 ◽  
Author(s):  
Yong Sook Goo ◽  
Wonil Lim ◽  
Keith S. Elmslie
Keyword(s):  
Slow Component ◽  
Divalent Cations ◽  
Sympathetic Neurons ◽  
Slow Inactivation ◽  
Fast Inactivation ◽  
Extracellular Solution ◽  
Channel Inactivation ◽  
Dependent Inactivation ◽  
Preferential Inactivation

Ca2+-dependent inactivation (CDI) has recently been shown in heterologously expressed N-type calcium channels (CaV2.2), but CDI has been inconsistently observed in native N-current. We examined the effect of Ca2+ on N-channel inactivation in rat sympathetic neurons to determine the role of CDI on mammalian N-channels. N-current inactivated with fast (τ ∼ 150 ms) and slow (τ ∼ 3 s) components in Ba2+. Ca2+ differentially affected these components by accelerating the slow component (slow inactivation) and enhancing the amplitude of the fast component (fast inactivation). Lowering intracellular BAPTA concentration from 20 to 0.1 mM accelerated slow inactivation, but only in Ca2+ as expected from CDI. However, low BAPTA accelerated fast inactivation in either Ca2+ or Ba2+, which was unexpected. Fast inactivation was abolished with monovalent cations as the charge carrier, but slow inactivation was similar to that in Ba2+. Increased Ca2+, but not Ba2+, concentration (5–30 mM) enhanced the amplitude of fast inactivation and accelerated slow inactivation. However, the enhancement of fast inactivation was independent of Ca2+ influx, which indicates the relevant site is exposed to the extracellular solution and is inconsistent with CDI. Fast inactivation showed U-shaped voltage dependence in both Ba2+ and Ca2+, which appears to result from preferential inactivation from intermediate closed states (U-type inactivation). Taken together, the data support a role for extracellular divalent cations in modulating U-type inactivation. CDI appears to play a role in N-channel inactivation, but on a slower (sec) time scale.

Critical Role of Calcium in the Regulation of the ER-to-Golgi Transport of FV and FVIII by the LMAN1-MCFD2 Cargo Receptor.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2140-2140
Author(s):  
Chunlei Zheng ◽  
Huihui Liu ◽  
David Ginsburg ◽  
Bin Zhang
Keyword(s):  
Factor Viii ◽  
Binding Sites ◽  
Critical Role ◽  
Carbohydrate Recognition Domain ◽  
Factor V ◽  
Receptor Complex ◽  
Carbohydrate Recognition ◽  
Golgi Transport ◽  
Ef Hand

Abstract Abstract 2140 Poster Board II-117 Coagulation factor V (FV) and factor VIII (FVIII) play key roles in hemostasis and thrombosis. The LMAN1 (ERGIC-53)-MCFD2 complex is a mammalian cargo receptor for efficient transport of FV and FVIII from the endoplasmic reticulum (ER) to the Golgi. Mutations in either LMAN1 or MCFD2 cause a bleeding disorder, combined deficiency of factor V and factor VIII. LMAN1 is a type-1 transmembrane protein with a Ca2+-dependent carbohydrate recognition domain homologous to leguminous lectins. MCFD2 is a small soluble protein with an N-terminal sequence of unknown structure and two Ca2+-binding EF-hand domains at the C terminus. LMAN1 and MCFD2 form a Ca2+-dependent protein complex in the ER-Golgi intermediate compartment (ERGIC), an organelle between the ER and Golgi that is unique to higher eukaryotic cells. FV and FVIII interact with the LMAN1-MCFD2 complex in a Ca2+ -dependent manner. To elucidate the role of Ca2+ in regulating the ER-to-Golgi transport of FV and FVIII, we determined the structural features important for the organization of the receptor complex and the interaction of this complex with its client cargo FV and FVIII. We show that the C-terminal Ca2+-binding EF hand domains of MCFD2 are both necessary and sufficient for interaction with LMAN1. The EF hand domains also mediate the interaction with FV and FVIII. All MCFD2 missense mutants identified in F5F8D patients are localized to the EF hand domains and fail to bind LMAN1. However, these mutants still retain the FV and FVIII binding activities. Circular dichroism spectroscopy studies on missense mutations localized to different structural elements of the EF hand domains suggest that Ca2+-induced folding of MCFD2 is important for LMAN1 interaction, but not essential for FV and FVIII binding. We also demonstrate that the carbohydrate recognition domain (CRD) of LMAN1 contains separate binding sites for MCFD2 and FV/FVIII. Mutations in the Ca2+ and sugar binding sites of CRD disrupt FV and FVIII interaction, without affecting MCFD2 binding, suggesting that the Ca2+ binding sites in LMAN1 are primarily required for the recognition of sugar residues in FV and FVIII. These results support a model in which Ca2+ plays a critical role in regulating the binding in the ER and the subsequent release in the ERGIC of FV and FVIII. Ca2+ concentration is higher in the ER than in the ERGIC and the Golgi. In the ER lumen, FV and FVIII loading is initiated by a flexible interaction with MCFD2 and stabilized by the follow-up interaction of sugar side chains of FV and FVIII with the carbohydrate binding site of LMAN1. The LMAN1-FV/FVIII interaction is more sensitive to Ca2+ concentration than the LMAN1-MCFD2 interaction, so that the lower Ca2+/pH in the ERGIC triggers the release of FV and FVIII but not the dissociation of the LMAN1-MCFD2 receptor complex. The empty receptor complex is subsequently recycled back to the ER for the next round of cargo loading. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Distinct effects of Q925 mutation on intracellular and extracellular Na+ and K+ binding to the Na+, K+-ATPase

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Hang N. Nielsen ◽  
Kerri Spontarelli ◽  
Rikke Holm ◽  
Jens Peter Andersen ◽  
Anja P. Einholm ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Crystal Structures ◽  
Binding Sites ◽  
Functional Role ◽  
Transmembrane Helix ◽  
Binding Properties ◽  
Ion Translocation ◽  
Extracellular Side ◽  
Insight Into

Abstract Three Na+ sites are defined in the Na+-bound crystal structure of Na+, K+-ATPase. Sites I and II overlap with two K+ sites in the K+-bound structure, whereas site III is unique and Na+ specific. A glutamine in transmembrane helix M8 (Q925) appears from the crystal structures to coordinate Na+ at site III, but does not contribute to K+ coordination at sites I and II. Here we address the functional role of Q925 in the various conformational states of Na+, K+-ATPase by examining the mutants Q925A/G/E/N/L/I/Y. We characterized these mutants both enzymatically and electrophysiologically, thereby revealing their Na+ and K+ binding properties. Remarkably, Q925 substitutions had minor effects on Na+ binding from the intracellular side of the membrane – in fact, mutations Q925A and Q925G increased the apparent Na+ affinity – but caused dramatic reductions of the binding of K+ as well as Na+ from the extracellular side of the membrane. These results provide insight into the changes taking place in the Na+-binding sites, when they are transformed from intracellular- to extracellular-facing orientation in relation to the ion translocation process, and demonstrate the interaction between sites III and I and a possible gating function of Q925 in the release of Na+ at the extracellular side.

scholarly journals Pseudobactin Biogenesis in the Plant Growth-Promoting Rhizobacterium Pseudomonas Strain B10: Identification and Functional Analysis of the l-Ornithine N5-Oxygenase (psbA) Gene

2000 ◽  
Vol 182 (21) ◽  
pp. 6233-6238 ◽  
Author(s):  
C. Ambrosi ◽  
L. Leoni ◽  
L. Putignani ◽  
N. Orsi ◽  
P. Visca
Keyword(s):  
Binding Sites ◽  
Genomic Library ◽  
Erwinia Carotovora ◽  
Significant Similarity ◽  
Cofactor Binding ◽  
Plant Growth Promoting ◽  
Growth Promoting ◽  
A Site

ABSTRACT PseudobactinB10, the fluorescent siderophore produced by the rhizobacterium Pseudomonas strain B10, contains the hydroxamate ligandd-N5 -hydroxyornithine (d-N5 -OH-Orn). We cloned thel-Orn N5 -oxygenase (psbA) gene from a genomic library ofPseudomonas strain B10 and demonstrated that PsbA is involved in the conversion of l-Orn to itsN5 -OH derivative. PsbA shows significant similarity to microbial ω-amino acid hydroxylases containing flavin adenine dinucleotide and NADP cofactor-binding sites and the FATGY signature of the putative substrate recognition pocket. ThepsbA gene is monocistronic, and its transcription is negatively controlled by iron. A site-specific psbA mutant of Pseudomonas strain B10 was biochemically complemented with the precursor l-N5 -OH-Orn, suggesting that l-Orn is hydroxylated before conversion to the d isomer. The l-OrnN5 -hydroxylase-defective mutants ofPseudomonas strain B10 and Pseudomonas aeruginosa PAO1 were much less effective than the parental strains in suppressing the growth of the phytopathogen Erwinia carotovora in iron-poor medium. The extent of in vitro inhibition of E. carotovora was strictly iron dependent and directly correlated with the amount of released siderophores. These data strengthen the role of fluorescent siderophores in biocontrol of deleterious rhizomicroorganisms.

scholarly journals Modulation of CaV1.2 Channels by Mg2+ Acting at an EF-hand Motif in the COOH-terminal Domain

2005 ◽  
Vol 126 (4) ◽  
pp. 311-323 ◽  
Author(s):  
Sylvain Brunet ◽  
Todd Scheuer ◽  
Rachel Klevit ◽  
William A. Catterall
Keyword(s):  
Cardiac Myocytes ◽  
Voltage Dependence ◽  
Control Level ◽  
Site Model ◽  
Whole Cell Patch Clamp ◽  
Dependent Inactivation ◽  
Ef Hand ◽  
Voltage Dependent ◽  
Cav1.2 Channels

Magnesium levels in cardiac myocytes change in cardiovascular diseases. Intracellular free magnesium (Mgi) inhibits L-type Ca2+ currents through CaV1.2 channels in cardiac myocytes, but the mechanism of this effect is unknown. We hypothesized that Mgi acts through the COOH-terminal EF-hand of CaV1.2. EF-hand mutants were engineered to have either decreased (D1546A/N/S/K) or increased (K1543D and K1539D) Mg2+ affinity. In whole-cell patch clamp experiments, increased Mgi reduced both Ba2+ and Ca2+ currents conducted by wild type (WT) CaV1.2 channels expressed in tsA-201 cells with similar affinity. Exposure of WT CaV1.2 to lower Mgi (0.26 mM) increased the amplitudes of Ba2+ currents 2.6 ± 0.4–fold without effects on the voltage dependence of activation and inactivation. In contrast, increasing Mgi to 2.4 or 7.2 mM reduced current amplitude to 0.5 ± 0.1 and 0.26 ± 0.05 of the control level at 0.8 mM Mgi. The effects of Mgi on peak Ba2+ currents were approximately fit by a single binding site model with an apparent Kd of 0.65 mM. The apparent Kd for this effect of Mgi was shifted ∼3.3- to 16.5-fold to higher concentration in D1546A/N/S mutants, with only small effects on the voltage dependence of activation and inactivation. Moreover, mutant D1546K was insensitive to Mgi up to 7.2 mM. In contrast to these results, peak Ba2+ currents through the K1543D mutant were inhibited by lower concentrations of Mgi compared with WT, consistent with approximately fourfold reduction in apparent Kd for Mgi, and inhibition of mutant K1539D by Mgi was also increased comparably. In addition to these effects, voltage-dependent inactivation of K1543D and K1539D was incomplete at positive membrane potentials when Mgi was reduced to 0.26 or 0.1 mM, respectively. These results support a novel mechanism linking the COOH-terminal EF-hand with modulation of CaV1.2 channels by Mgi. Our findings expand the repertoire of modulatory interactions taking place at the COOH terminus of CaV1.2 channels, and reveal a potentially important role of Mgi binding to the COOH-terminal EF-hand in regulating Ca2+ influx in physiological and pathophysiological states.

scholarly journals Binding of Neomycin-Class Aminoglycoside Antibiotics to Mutant Ribosomes with Alterations in the A Site of 16S rRNA

2006 ◽  
Vol 50 (4) ◽  
pp. 1489-1496 ◽  
Author(s):  
Sven N. Hobbie ◽  
Peter Pfister ◽  
Christian Bruell ◽  
Peter Sander ◽  
Boris François ◽  
...  
Keyword(s):  
Crystal Structure ◽  
16S Rrna ◽  
Base Pair ◽  
Mycobacterium Smegmatis ◽  
Drug Binding ◽  
Aminoglycoside Antibiotics ◽  
Wild Type ◽  
The Core ◽  
A Site

ABSTRACT Aminoglycoside antibiotics that bind to the aminoacyl-tRNA site (A site) of the ribosome are composed of a common neamine core in which a glycopyranosyl ring is attached to position 4 of a 2-deoxystreptamine moiety. The core is further substituted by one (ribostamycin), two (neomycin and paromomycin), or three (lividomycin A) additional sugars attached to position 5 of the 2-deoxystreptamine. To study the role of rings III, IV, and V in aminoglycoside binding, we used isogenic Mycobacterium smegmatis ΔrrnB mutants carrying homogeneous populations of mutant ribosomes with alterations in the 16S rRNA A site. MICs were determined to investigate drug-ribosome interactions, and the results were compared with that of the previously published crystal structure of paromomycin bound to the ribosomal A site. Our analysis demonstrates that the stacking interaction between ring I and G1491 is largely sequence independent, that rings III and IV each increase the strength of drug binding to the ribosome, that ring IV of the 6′-NH3 + aminoglycosides compensates for loss of interactions between ring II and U1495 and between ring III and G1491, that the aminoglycosides rely on pseudo-base pairing between ring I and A1408 for binding independently of the number of sugar rings attached to the neamine core, that addition of ring V to the 6′-OH 4,5-aminoglycoside paromomycin does not alter the mode of binding, and that alteration of the U1406 · U1495 wobble base pair to the Watson-Crick interaction pair 1406C-1495G yields ribosomal drug susceptibilities to 4,5-aminoglycosides comparable to those seen with the wild-type A site.

scholarly journals Insight into the dimer dissociation process of the Chromobacterium violaceum (S)-selective amine transaminase

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Federica Ruggieri ◽  
Jonatan C. Campillo-Brocal ◽  
Shan Chen ◽  
Maria S. Humble ◽  
Björn Walse ◽  
...  
Keyword(s):  
Enzyme Engineering ◽  
Chromobacterium Violaceum ◽  
Wild Type ◽  
Conformational Switch ◽  
Dependent Inactivation ◽  
Catalytically Active ◽  
The Stability ◽  
Computational Mutagenesis ◽  
Insight Into

AbstractOne of the main factors hampering the implementation in industry of transaminase-based processes for the synthesis of enantiopure amines is their often low storage and operational stability. Our still limited understanding of the inactivation processes undermining the stability of wild-type transaminases represents an obstacle to improving their stability through enzyme engineering. In this paper we present a model describing the inactivation process of the well-characterized (S)-selective amine transaminase from Chromobacterium violaceum. The cornerstone of the model, supported by structural, computational, mutagenesis and biophysical data, is the central role of the catalytic lysine as a conformational switch. Upon breakage of the lysine-PLP Schiff base, the strain associated with the catalytically active lysine conformation is dissipated in a slow relaxation process capable of triggering the known structural rearrangements occurring in the holo-to-apo transition and ultimately promoting dimer dissociation. Due to the occurrence in the literature of similar PLP-dependent inactivation models valid for other non-transaminase enzymes belonging to the same fold-class, the role of the catalytic lysine as conformational switch might extend beyond the transaminase enzyme group and offer new insight to drive future non-trivial engineering strategies.

Ruthenium(II) Complexes Bearing Thioether-Appended α- Iminopyridine Ligands: Arene Precursors Permit Access to K2-N,N and K3-N,N,S Complexes

2019 ◽  
Author(s):  
Victoria A. Ternes ◽  
Hannah A. Morgan ◽  
Austin P. Lanquist ◽  
Michael P. Murray ◽  
Bradley Wile
Keyword(s):  
Crystal Structure ◽  
Solid State ◽  
Strong Field ◽  
Active Role ◽  
Binding Mode ◽  
Phenethyl Alcohol ◽  
Ru Complexes

Herein we report the preparation of a series of Ru(II) complexes featuring alpha-iminopyridine ligands bearing thioether functionality (NNS<sup>R</sup>, where R = Me, CH<sub>2</sub>Ph, Ph). Metallation using (<i>p</i> cymene)RuCl dimer permits access to (k<sup>2</sup>-N,N)Ru complexes in which the thioether moiety remains uncoordinated. In the presence of a strong field ligand such as acetonitrile or triphenylphosphine, the p-cymene moiety is displaced, and the ligand adopts a k<sup>3</sup>-N,N,S binding mode. These complexes are characterized using a combination of solution and solid state methods, including the crystal structure of [(NNS<sup>Me</sup>)Ru(NCMe)<sub>2</sub>Cl]Cl. The k<sup>2</sup>-N,N Ru(II) complexes are shown to serve as efficient precatalysts for the oxidation of sec-phenethyl alcohol at 5 mol% loadings, using a variety of external oxidants and solvents. The complex bearing an S-Ph donor was found to be the most active of those surveyed, suggesting that the thioether donor plays an active role in catalyst speciation for this transformation.

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