Mechanism of calmodulin inactivation of the calcium-selective TRP channel TRPV6

Calcium (Ca2+) plays a major role in numerous physiological processes. Ca2+ homeostasis is tightly controlled by ion channels, the aberrant regulation of which results in various diseases including cancers. Calmodulin (CaM)–mediated Ca2+-induced inactivation is an ion channel regulatory mechanism that protects cells against the toxic effects of Ca2+ overload. We used cryo-electron microscopy to capture the epithelial calcium channel TRPV6 (transient receptor potential vanilloid subfamily member 6) inactivated by CaM. The TRPV6-CaM complex exhibits 1:1 stoichiometry; one TRPV6 tetramer binds both CaM lobes, which adopt a distinct head-to-tail arrangement. The CaM carboxyl-terminal lobe plugs the channel through a unique cation-π interaction by inserting the side chain of lysine K115 into a tetra-tryptophan cage at the pore’s intracellular entrance. We propose a mechanism of CaM-mediated Ca2+-induced inactivation that can be explored for therapeutic design

Cardiovascular involvement in severe malaria: A prospective study in Ranchi, Jharkhand

Background & objectives: Malaria is considered as the most important parasitic disease of humans, causing seri- ous illness that can be fatal, if not diagnosed and treated immediately. It is a multisystem disorder affecting nearly every system of the body. The aim of the present study was to evaluate the involvement of cardiovascular system in severe malaria using non-invasive methods. Methods: This prospective study was conducted on patients of severe malaria who were admitted between June and November 2015 in the Department of Medicine, Rajendra Institute of Medical Sciences and Hospital, Ranchi, Jharkhand, India. A total of 27 cases (18 males and 9 females; age ranging between 15 and 70 yr) of severe malaria (P. falciparum 24; P. vivax 1; mixed 2) were diagnosed by microscopic examination of peripheral blood smear and bivalent rapid diagnostic test (RDT) kit. The assessment of cardiovascular system was done by clinical examination, chest X-ray, ECG and transthoracic echocardiography. Results: In all, 7 (26%) patients were found to be suffering from circulatory failure, out of which one was P. vivax case and rest were cases of P. falciparum infection with high parasite density. One patient died due to cardiovascular collapse. ECG revealed sinus bradycardia [Heart rate (HR): 40-60] in 7% of the cases, extreme tachycardia (HR: 120-150) in 3.7% of cases and premature arterial ectopic with tachycardia in 3.7% of patients (p <0.05). The echo- cardiographic findings were global hypokinesia with decreased left ventricular ejection fraction (<55%) in 11.1%, grade 1 left ventricular diastolic dysfunction in 3.7%, mild tricuspid regurgitation (TR) with mild pulmonary artery hypertension (PAH) in 3.7% and mild pericardial effusion in 3.7% of the cases. The ECG and echocardiography changes indicated myocardial involvement in severe malaria. Interpretation & conclusion: The present study indicated involvement of cardiovascular system in severe malaria as evidenced from ECG and echocardiography. The study also revealed that cardiovascular instabilities are common in falciparum malaria, but can also be observed in vivax malaria

Co-Factor Binding Confers Substrate Specificity to Xylose Reductase from <em>Debaryomyces hansenii</em>

<div><p>Binding of substrates into the active site, often through complementarity of shapes and charges, is central to the specificity of an enzyme. In many cases, substrate binding induces conformational changes in the active site, promoting specific interactions between them. In contrast, non-substrates either fail to bind or do not induce the requisite conformational changes upon binding and thus no catalysis occurs. In principle, both lock and key and induced-fit binding can provide specific interactions between the substrate and the enzyme. In this study, we present an interesting case where cofactor binding pre-tunes the active site geometry to recognize only the cognate substrates. We illustrate this principle by studying the substrate binding and kinetic properties of Xylose Reductase from <em>Debaryomyces hansenii</em> (<em>Dh</em>XR), an AKR family enzyme which catalyzes the reduction of carbonyl substrates using NADPH as co-factor. <em>Dh</em>XR reduces D-xylose with increased specificity and shows no activity towards “non-substrate” sugars like L-rhamnose. Interestingly, apo-<em>Dh</em>XR binds to D-xylose and L-rhamnose with similar affinity (K_d∼5.0–10.0 mM). Crystal structure of apo-<em>Dh</em>XR-rhamnose complex shows that L-rhamnose is bound to the active site cavity. L-rhamnose does not bind to holo-<em>Dh</em>XR complex and thus, it cannot competitively inhibit D-xylose binding and catalysis even at 4–5 fold molar excess. Comparison of K_d values with K_m values reveals that increased specificity for D-xylose is achieved at the cost of moderately reduced affinity. The present work reveals a latent regulatory role for cofactor binding which was previously unknown and suggests that cofactor induced conformational changes may increase the complimentarity between D-xylose and active site similar to specificity achieved through induced-fit mechanism.</p> </div

Steady state kinetic parameters for different carbonyl substrates catalyzed by <i>Dh</i>XR and kinetic analyses of D-xylose reduction by <i>Dh</i>XR in the presence of non-xylose sugars.

<p>Note: G, Galactose; A, Arabinose; R, Rhamnose.</p

Steady-state kinetic characterization of <i>Dh</i>XR.

<p>Substrate specificity of <i>Dh</i>XR checked and kinetic data were fit to Michaelis-Menten model as described in methods; enzyme concentration is same for all experiments (0.18 µM). A) Kinetic study using D-xylose as substrate; B) Comparative kinetic study using different carbonyl substrates; C) Examination of <i>Dh</i>XR kinetic properties towards D-xylose in the presence of fixed amounts (40 mM) of non-xylose substrates.</p

Structural analyses of L-rhamnose interaction with apoenzyme.

<p>A) F_o−F_c omit-electron density map (2.5 σ level) shows L-rhamnose backbones and hydroxyl groups and 2 F_o−F_c electron density map (1.0 σ level) also shows rhamnose is bound to active site cavity. B) Interactions of bound L-rhamnose with side chains of residues lining the active site cavity. The aldehyde part of ligand is aligned towards side chain of Y217 with O1 forming strong hydrogen bond with OH of Y217 and also interacting with near by aromatic side chains. C) Superposition of NADPH bound structure to apoenzyme-rhamnose complex. The plane of side chain of Y217 tilted nearly perpendicularly in cofactor bound structure, causing to be atop of NADPH. Apoenzyme-rhamnose complex is shown in green and NADPH bound complex is shown in blue.</p

Specific activities of <i>Dh</i>XR with xylose in presence of different carbonyl substrates.

*<p>amount of substrate added in 100 mM of D-xylose reaction.</p

Determination of equilibrium binding constants for ligands binding to active site mutants.

*<p>all units are in mM.</p