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PubMed2003

Electrophysiological analysis of the yeast V-type proton pump: variable coupling ratio and proton shunt.

Isolated vacuoles from the yeast Saccharomyces cerevisiae were examined in the whole-vacuole mode of patch recording, to get a detailed functional description of the vacuolar proton pump, the V-ATPase. Functioning of the V-ATPase was characterized by its current-voltage (I-V) relationship, obtained for various levels of vacuolar and cytosolic pH. I-V curves for the V-ATPase were computed as the difference between I-V curves obtained with the pump switched on (ATP, ADP, and Pi present) or off (no ATP). These difference current-voltage relationships usually crossed the voltage axis within the experimental range (from -80 to +80 mV), thus measuring the reversal voltage (ER) for the V-ATPase, which could be compared with the standing ion gradients and free energy of ATP hydrolysis, to calculate the apparent pump stoichiometry or coupling ratio: the number of protons transported for each ATP molecule hydrolyzed. This ratio was found to depend strongly upon the pH difference (DeltapH) across the vacuolar membrane, being approximately 2H+/ATP at high DeltapH (4 pH units) and increasing to >4H+/ATP for small or zero DeltapH. That result is in quantitative agreement with previous determinations on plant vacuoles. Considerations of purely electrical behavior, together with the physical properties of a recent detailed structural model for V-ATPases, led to a linear equivalent circuit--which quantitatively accounts for all observations of variable coupling ratios in fungal and plant V-ATPases by variations of the conductance for bona fide proton pumping (GP) through the ATPase relative to independent proton shunting (GS) through the same protein.

Adenosine Triphosphate↗

PubMed2001

Characterization of the murine gammaherpesvirus 68 ORF74 product: a novel oncogenic G protein-coupled receptor.

Murine gammaherpesvirus (MHV-68) is well established as a small animal model for the study of gammaherpesviruses. The MHV-68 genome contains an open reading frame (ORF74) that has significant sequence homology with mammalian G-protein coupled receptors (GPCRs) and the GPCR from the related Kaposi's sarcoma-associated herpesvirus (KSHV). Here we show that the MHV-68 ORF74 is predicted to encode a GPCR since it has seven potential transmembrane helices and that it has other sequence motifs in common with GPCRS: Of interest is the observation that the sequence around a conserved arginine at the start of the second intracellular loop suggests that the ORF74 product may signal constitutively (agonist independent). Given that the ORF74 product is predicted to encode a GPCR we named it MHV-GPCR. In studies on the transcription of the MHV-GPCR, we determined that it was encoded on multiple early transcripts of 3.4, 4.4, 6.6 and 8.7 kb in size. At least one of these transcripts was bicistronic, containing the ORF encoding the Bcl-2 homologue also. In vivo, we found that MHV GPCR was expressed during acute infection but also during persistence, particularly in the lungs of infected mice. Immunofluorescence studies indicated that the MHV GPCR protein was expressed on the surface of cells in patches. Finally, like the KSHV GPCR, expression of the MHV GPCR resulted in transformation of NIH 3T3 cells. We surmise, therefore, that the MHV GPCR may act in concert with genes with which it is expressed such as vBcl-2 to enhance the growth and survival of MHV-68-infected cells.

3T3 Cells↗

PubMed2003

Functional network integration of embryonic stem cell-derived astrocytes in hippocampal slice cultures.

Embryonic stem (ES) cells provide attractive prospects for neural transplantation. So far, grafting strategies in the CNS have focused mainly on neuronal replacement. Employing a slice culture model, we found that ES cell-derived glial precursors (ESGPs) possess a remarkable capacity to integrate into the host glial network. Following deposition on the surface of hippocampal slices, ESGPs actively migrate into the recipient tissue and establish extensive cell-cell contacts with recipient glia. Gap junction-mediated coupling between donor and host astrocytes permits widespread delivery of dye from single donor cells. During maturation, engrafted donor cells display morphological, immunochemical and electrophysiological properties that are characteristic of differentiating native glia. Our findings provide the first evidence of functional integration of grafted astrocytes, and depict glial network integration as a potential route for widespread transcellular delivery of small molecules to the CNS.

Animals↗

PubMed2005

Effect of local anaesthesia on neuronal c-fos expression in the spinal dorsal horn and hypothalamic paraventricular nucleus after surgery in rats.

The surgical stress response is the neurophysiologic reflex response to surgery, which involves activation of the hypothalamic-pituitary-adrenal axis and is regulated by the hypothalamic paraventricular nucleus. The effect of pre-operative use of local anaesthetics on activation of neurones in the paraventricular nucleus during surgery was studied by quantification of the neuronal expression of the c-fos-gene after a standardized plantar incision in rats. Furthermore, c-fos expression in the spinal dorsal horn was used as a measure of spinal nociception. Six halothane-anaesthetized animals underwent surgery following infiltration with lidocaine and bupivacaine, six animals were operated without local anaesthetics, and six control animals were subjected to the anaesthetic procedures. After two hours, the animals were perfused with 4% formaldehyde and the spinal cords and brains were collected and processed by immunohistochemistry for stereological quantification of the number of neurones with Fos-like immunoreactivity. Furthermore, brain and spinal cord were sampled from nine control animals right after induction of halothane anaesthesia. Surgery without local anaesthetics caused a significant increased number of neurones with Fos-like immunoreactivity in the spinal cord (4258+/-1710; mean+/-S.D.; P<0.01) compared to the anaesthesia control group (1204+/-436). Local anaesthetics reduced this number to 2029+/-919 (P<0.05), which was not significantly different from the anaesthesia control group. After surgery, the number of neurones with Fos-like immunoreactivity in paraventricular nucleus increased from 2948+/-1365 in the anaesthetized control group to 5550+/-3875 and 5191+/-1558 in the surgery and local anaesthetics plus surgery group, respectively, although significance was only reached for the group receiving local anaesthetics (P<0.05). In conclusion, preoperative local anaesthetic infiltration did not reduce the surgery-induced c-fos expression in paraventricular nucleus after paw surgery in rats, although spinal nociception was reduced.

Anesthetics, Local↗

PubMed1998

Coupling protein stability and protein function in Escherichia coli CspA.

BACKGROUND: CspA is a small protein that binds single-stranded RNA and DNA. The binding site of CspA consists of a cluster of aromatic amino acids, which form an unusually large nonpolar patch on the surface of the protein. Because nonpolar residues are generally found in the interiors of proteins, this cluster may have evolved to bind nucleic acids at the expense of protein stability. RESULTS: Three neighboring phenylalanines have been mutated singly and in combination to leucine and to serine. All mutations adversely affect DNA binding. Surprisingly, all mutations, and especially those to serine, are destabilizing. CONCLUSIONS: The aromatic cluster in CspA is required not only for protein function but also for protein stability. This result is pertinent to the design of beta-sheet proteins and single-stranded nucleic acid binding proteins, whose binding mode is proposed to be of aromatic-aromatic intercalation.

Bacterial Proteins↗

PubMed2000

Spatial correlation analysis of atrial activation patterns during sustained atrial fibrillation in conscious goats.

In this study we applied both linear and nonlinear spatial correlation measures to characterize epicardial activation patterns of sustained atrial fibrillation in instrumented conscious goats. It was investigated if nonlinearity was involved in the spatial coupling of atrial regions and to what extent fibrillation was organized in the experimental model of sustained atrial fibrillation (AF) in instrumented goats. Data were collected in five goats during experiments to convert AF by continuous infusion of cibenzoline. Spatial organization during AF was quantified with the linear spatial cross correlation function and the nonlinear spatial cross redundancy which was calculated using the Grassberger-Procaccia correlation integral. Two different types of correlation were evaluated to distinguish simultaneous interaction from non-simultaneous interaction, for instance resulting from propagation of fibrillation waves. The nonlinear association length and the linear correlation length were estimated along the principal axes of iso-correlation contours in two-dimensional correlation maps of the nonlinear spatial redundancy and the linear spatial correlation function, respectively. To quantitatively assess the degree of nonlinearity, the association length was also estimated from the linearized spatial redundancy using multivariate surrogate data. The differences between the nonlinear and linearized association lengths indicated that a nonlinear component in the spatial organization of AF predominantly existed in the right atrium. The degree of organization characterized by association length along the short principal axis was higher in the right atrium (15 +/- 7 mm) than in the left atrium (8 +/- 4 mm). The spatial extension of coherent atrial patches was estimated from a surface of association equal to the area spanned by the principal axes of iso-correlation contours from the redundancy, including the effects from non-simultaneous interaction. Interpreting this area as the spatial domain of a fibrillation wavelet, the results suggest that the mapped region was activated on average by two wavelets in the left atrium and by one wavelet in the right atrium. Therefore, the activation pattern of sustained AF in goats was relatively organized, consistent with type II of AF. It is suggested that the surface of association is a measure of the number of independent wavelets present in the atria during sustained AF, and that larger association lengths result from fewer and larger reentrant circuits.

Animals↗

PubMed2004

The structural basis of ClC chloride channel function.

ClC channels constitute a large family of Cl(-)-selective ion channels that are present in diverse organisms. The channels have a complex gating behavior, in which channel opening and closing is tightly coupled to ion permeation. Recent success in the structure determination of bacterial channels has revealed the overall architecture of this family and provided important insight into ion selectivity and gating. The ClC channels are homodimers, with each subunit containing an ion conduction pore. In the narrow selectivity filter of each pore, Cl(-) are bound to several sites through electrostatic interactions with helix dipoles and the partial charges of protein residues. In the closed conformation of the channel, a conserved glutamate residue occupies one of the ion-binding sites; on channel opening, this glutamate residue moves out of the binding site, thereby making room for an additional Cl(-) ion.

Amino Acid Sequence↗

PubMed1998

Excitability of the squid giant axon revisited.

The electrical properties of the giant axon from the common squid Loligo pealei have been reexamined. The primary motivation for this work was the observation that the refractoriness of the axon was significantly greater than the predictions of the standard model of nerve excitability. In particular, the axon fired only once in response to a sustained, suprathreshold stimulus. Similarly, only a single action potential was observed in response to the first pulse of a train of 1-ms duration current pulses, when the pulses were separated in time by approximately 10 ms. The axon was refractory to all subsequent pulses in the train. The underlying mechanisms for these results concern both the sodium and potassium ion currents INa and IK. Specifically, Na+ channel activation has long been known to be coupled to inactivation during a depolarizing voltage-clamp step. This feature appears to be required to simulate the pulse train results in a revised model of nerve excitability. Moreover, the activation curve for IK has a significantly steeper voltage dependence, especially near its threshold (approximately -60 mV), than in the standard model, which contributes to reduced excitability, and the fully activated current-voltage relation for IK has a nonlinear, rather than a linear, dependence on driving force. An additional aspect of the revised model is accumulation/depeletion of K+ in the space between the axon and the glial cells surrounding the axon, which is significant even during a single action potential and which can account for the 15-20 mV difference between the potassium equilibrium potential EK and the maximum afterhyperpolarization of the action potential. The modifications in IK can also account for the shape of voltage changes near the foot of the action potential.

Action Potentials↗

PubMed1991

Phase resetting and entrainment of pacemaker activity in single sinus nodal cells.

The phase-resetting and entrainment properties of single pacemaker cells were studied using computer simulations in a model of the rabbit sinus nodal cell, as well as using the whole-cell patch-clamp (current-clamp) technique in isolated rabbit sinus nodal cells. Spontaneous electrical activity in the cell model was reconstructed using Hodgkin-Huxley-type equations describing time- and voltage-dependent membrane currents. In both simulations and experiments, single subthreshold current pulses (depolarizing or hyperpolarizing) were used to scan the spontaneous cycle of the cells. Such pulses perturbed the subsequent discharge, producing temporary phasic changes in pacemaker period, and enabled the construction of phase response curves. On the basis of these results, we studied entrainment characteristics of the cells. For example, application of repetitive pulses allowed for phasic changes in the spontaneous cycle and resulted in stable 1:1 entrainment at a range of basic cycle length around the spontaneous cycle, or a 2:1 pattern at basic cycle length values about half the spontaneous cycle length. Between the two entrainment zones, complex Wenckebach-like patterns (e.g., 5:4, 4:3, and 3:2) were observed. The experimental data from the isolated cell were further analyzed from a theoretical perspective, and the results showed that the topological characteristics of the phase-resetting behavior accounts for the experimentally observed patterns during repetitive stimulation of the cell. This first demonstration of phase resetting in single cells provides the basis for phenomena such as mutual entrainment between electrically coupled pacemaker cells, apparent intranodal conduction, and reflex vagal control of heart rate.

Animals↗

PubMed2002

Changes in excitation-contraction coupling in an isolated ventricular myocyte model of cardiac stunning.

To investigate cardiac stunning, we recorded intracellular [Ca(2+)], contractions, and electrical activity in isolated guinea pig ventricular myocytes exposed to simulated ischemia and reperfusion. After equilibration, ischemia was simulated by exposing myocytes to hypoxia, acidosis, hyperkalemia, hypercapnia, lactate accumulation, and substrate deprivation for 30 min at 37 degrees C. Reperfusion was simulated by exposure to Tyrode solution. Field-stimulated myocytes exhibited stunning upon reperfusion. By 10 min of reperfusion, contraction amplitude decreased to 43.0 +/- 5.5% of preischemic values (n = 15, P < 0.05), although action potential configuration and sarcoplasmic reticulum Ca(2+) stores, assessed with caffeine, were normal. Diastolic [Ca(2+)] and Ca(2+) transients (fura 2) were also normal in stunned myocytes. In voltage-clamped cells, peak L-type Ca(2+) current was reduced to 47.4 +/- 4.5% of preischemic values at 10 min of reperfusion (n = 21, P < 0.05). Contractions elicited by Ca(2+)-induced Ca(2+) release and the voltage-sensitive release mechanism were both depressed in reperfusion. Our observations suggest that stunning is associated with reduced L-type Ca(2+) current but that alterations in Ca(2+) homeostasis and release are not directly responsible for stunning.

Action Potentials↗

PubMed2003

The human D2 dopamine receptor synergizes with the A2A adenosine receptor to stimulate adenylyl cyclase in PC12 cells.

The adenosine A(2A) receptor and the dopamine D(2) receptor are prototypically coupled to G(s) and G(i)/G(o), respectively. In striatal intermediate spiny neurons, these receptors are colocalized in dendritic spines and act as mutual antagonists. This antagonism has been proposed to occur at the level of the receptors or of receptor-G protein coupling. We tested this model in PC12 cells which endogenously express A(2A) receptors. The human D(2) receptor was introduced into PC12 cells by stable transfection. A(2A)-agonist-mediated inhibition of D(2) agonist binding was absent in PC12 cell membranes but present in HEK293 cells transfected as a control. However, in the resulting PC12 cell lines, the action of the D(2) agonist quinpirole depended on the expression level of the D(2) receptor: at low and high receptor levels, the A(2A)-agonist-induced elevation of cAMP was enhanced and inhibited, respectively. Forskolin-stimulated cAMP formation was invariably inhibited by quinpirole. The effects of quinpirole were abolished by pretreatment with pertussis toxin. A(2A)-receptor-mediated cAMP formation was inhibited by other G(i)/G(o)-coupled receptors that were either endogenously present (P(2y12)-like receptor for ADP) or stably expressed after transfection (A(1) adenosine, metabotropic glutamate receptor-7A). Similarly, voltage activated Ca(2+) channels were inhibited by the endogenous P(2Y) receptor and by the heterologously expressed A(1) receptor but not by the D(2) receptor. These data indicate functional segregation of signaling components. Our observations are thus compatible with the proposed model that D(2) and A(2A) receptors are closely associated, but they highlight the fact that this interaction can also support synergism.

4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone↗

PubMed2006

The role of the persistent Na(+) current during cardiac ischemia and hypoxia.

INTRODUCTION: Inadequate or zero cardiac perfusion, if prolonged beyond about 10 minutes, can result in irreversible cell damage. Paradoxically, much of this damage occurs when perfusion is restored, and this appears to be linked to an uncontrolled rise in intracellular calcium. This article reviews the causes of this rise in calcium. METHODS AND RESULTS: Data that have arisen from a variety of techniques to measure intracellular ion concentrations in cardiac cells are reviewed. Fluorescence measurements in intact hearts and isolated cells show that the rise in [Ca(2+)](i) is preceded by a rise in [Na(+)](i), a finding that has led to the coupled exchanger theory, which postulates that the high activity of the Na-H exchanger, as a result of intracellular acidification, increases [Na(+)](i) and that this slows or reverses the Na-Ca exchanger. However, the [Na(+)](i) appears to come from several sources: the Na-H exchanger, the Na-HCO(3) symporter, and the persistent Na(+) current, I(Na(p)). The latter appears to be important because blockers of Na-H exchange (e.g., Cariporide) have been shown to be only partially protective against reperfusion damage, whereas TTX and other Na(+) channel blockers offer equal or better protection. Patch clamp experiments in isolated cells have shown that I(Na(p)) is increased by hypoxia, although the mechanisms are not known. CONCLUSION: Blockers of I(Na(p)) may provide an alternative strategy for preventing reperfusion damage in myocardium, either alone or in combination with Na-H exchange blockers.

Action Potentials↗

PubMed2000

Electrotonic suppression of early afterdepolarizations in isolated rabbit Purkinje myocytes.

Many studies suggest that early afterdepolarizations (EADs) arising from Purkinje fibers initiate triggered arrhythmias under pathological conditions. However, electrotonic interactions between Purkinje and ventricular myocytes may either facilitate or suppress EAD formation at the Purkinje-ventricular interface. To determine conditions that facilitated or suppressed EADs during Purkinje-ventricular interactions, we coupled single Purkinje myocytes and aggregates isolated from rabbit hearts to a passive model cell via an electronic circuit with junctional resistance (R(j)). The model cell had input resistance (R(m,v)) of 50 M Omega, capacitance of 39 pF, and a variable rest potential (V(rest,v)). EADs were induced in Purkinje myocytes during superfusion with 1 microM isoproterenol. Coupling at high R(j) to normally polarized V(rest,v) established a repolarizing coupling current during all phases of the Purkinje action potential. This coupling current preferentially suppressed EADs in single cells with mean membrane resistance (R(m,p)) of 297 M Omega, whereas EAD suppression in larger aggregates with mean R(m,p) of 80 M Omega required larger coupling currents. In contrast, coupling to elevated V(rest,v) established a depolarizing coupling current during late phase 2, phase 3, and phase 4 that facilitated EAD formation and induced spontaneous activity in single Purkinje myocytes and aggregates. These results have important implications for arrhythmogenesis in the infarcted heart when reduction of the ventricular mass due to scarring alters the R(m,p)-to-R(m,v) ratio and in the ischemic heart when injury currents are established during coupling between polarized Purkinje myocytes and depolarized ventricular myocytes.

Animals↗

PubMed2004

AMG 9810 [(E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b][1,4] dioxin-6-yl)acrylamide], a novel vanilloid receptor 1 (TRPV1) antagonist with antihyperalgesic properties.

The vanilloid receptor 1 (VR1 or TRPV1) is a membrane-bound, nonselective cation channel expressed by peripheral sensory neurons. TRPV1 antagonists produce antihyperalgesic effects in animal models of inflammatory and neuropathic pain. Here, we describe the in vitro and in vivo pharmacology of a novel TRPV1 antagonist, AMG 9810, (E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)acrylamide. AMG 9810 is a competitive antagonist of capsaicin activation (IC50 value for human TRPV1, 24.5 +/- 15.7 nM; rat TRPV1, 85.6 +/- 39.4 nM) and blocks all known modes of TRPV1 activation, including protons (IC50 value for rat TRPV1, 294 +/- 192 nM; human TRPV1, 92.7 +/- 72.8 nM), heat (IC50 value for rat TRPV1, 21 +/- 17 nM; human TRPV1, 15.8 +/- 10.8 nM), and endogenous ligands, such as anandamide, N-arachidonyl dopamine, and oleoyldopamine. AMG 9810 blocks capsaicin-evoked depolarization and calcitonin gene-related peptide release in cultures of rat dorsal root ganglion primary neurons. Screening of AMG 9810 against a panel of G protein-coupled receptors and ion channels indicated selectivity toward TRPV1. In vivo, AMG 9810 is effective at preventing capsaicin-induced eye wiping in a dose-dependent manner, and it reverses thermal and mechanical hyperalgesia in a model of inflammatory pain induced by intraplantar injection of complete Freund's adjuvant. At effective doses, AMG 9810 did not show any significant effects on motor function, as measured by open field locomotor activity and motor coordination tests. AMG 9810 is the first cinnamide TRPV1 antagonist reported to block capsaicin-induced eye wiping behavior and reverse hyperalgesia in an animal model of inflammatory pain.

Acrylamides↗

PubMed1991

The action of luteinizing hormone on the testis.

Luteinizing hormone (LH) and human chorionic gonadotrophin (hCG) receptors are coupled to intracellular effector systems, most notably adenylate cyclase, through guanyl nucleotide-binding proteins or G-proteins. The molecular mechanism involved in the dynamic coupling of the LH/hCG receptor however, are not known. It has been postulated that receptor aggregation at the molecular level plays a critical role in this process. There have been attempts to understand the receptor association and dissociation phenomena at the molecular level. One of them involves the participation of the major histocompatibility complex (MHC) class I antigen in the mechanism of receptor activation and/or expression. One molecular basis for these mechanisms consists of a physical interaction between MHC proteins and receptors to form "compound receptors" able to transfer a hormonal signal to the cell. Using a photo-reactive probe we demonstrated that the LH/hCG receptors and the class I antigens are closely associated in the membrane. Thus, it is possible to form covalent complexes of hCG and class I antigens through the binding of the hormone to specific receptors. These findings imply that LH/hCG receptors and the MHC class I antigens may interact at the level of the plasma membrane in the mechanism of LH action. We also performed experiments using a single cell and limiting stimulation to a patch of membrane. The results stimulating the cell in a localized area suggested that even if all components are entirely free to float there is a constraint in the localization of the receptor, G-protein, and/or the effector, supporting the constraint dissociation model. Within a limited area subunits could dissociate, but they would not be free to diffuse throughout the membrane. Moreover the concept of compartmentalization that has been utilized to explain some inconsistencies in second-messenger action now can be proved by experimental design.

Animals↗

PubMed1994

Glucose induces oscillatory Ca2+ signalling and insulin release in human pancreatic beta cells.

Mechanisms of pulsatile insulin release in man were explored by studying the induction of oscillatory Ca2+ signals in individual beta cells and islets isolated from the human pancreas. Evidence was provided for a glucose-induced closure of ATP-regulated K+ channels, resulting in voltage-dependent entry of Ca2+. The observation of step-wise increases of capacitance in response to depolarizing pulses suggests that an enhanced influx of Ca2+ is an effective means of stimulating the secretory activity of the isolated human beta cell. Activation of muscarinic receptors (1-10 mumol/l carbachol) and of purinergic P2 receptors (0.01-1 mumol/l ATP) resulted in repetitive transients followed by sustained elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i). Periodic mobilisation of intracellular calcium was seen also when injecting 100 mumol/l GTP-gamma-S into beta cells hyperpolarized to -70 mV. Individual beta cells responded to glucose and tolbutamide with increases of [Ca2+]i, manifested either as large amplitude oscillations (frequency 0.1-0.5/min) or as a sustained elevation. Glucose regulation was based on sudden transitions between the basal and the two alternative states of raised [Ca2+]i at threshold concentrations of the sugar characteristic for the individual beta cells. The oscillatory characteristics of coupled cells were determined collectively rather than by particular pacemaker cells. In intact pancreatic islets the glucose induction of well-synchronized [Ca2+]i oscillations had its counterpart in 2-5 min pulses of insulin. Each of these pulses could be resolved into regularly occurring short insulin transients. It is concluded that glucose stimulation of insulin release in man is determined by the number of beta cells entering into a state with Ca(2+)-induced secretory pulses.

Adenosine Triphosphate↗

PubMed2002

Dissecting intersubunit contacts in cyclic nucleotide-gated ion channels.

In cyclic nucleotide-gated (CNG) ion channels, binding of cGMP or cAMP drives a conformational change that leads to opening of an ion-conducting pore. One region implicated in the coupling of ligand binding to opening of the pore is the C linker region. Here, we used crosslinking of endogenous cysteines to study interregion proximity. We demonstrate that an individual amino acid--C481--in the C linker region of each of two neighboring subunits can form a disulfide bond. Further, using tandem dimers, we show that a disulfide bond between C35 in the N-terminal region and C481 in the C linker region can form either within a subunit or between subunits. From our data on proximity between individual amino acids and previous studies, a picture emerges of the C linker as a potential dimerization interface.

Animals↗

PubMed2000

Structure of the human anti-apoptotic protein survivin reveals a dimeric arrangement.

Survivin is a 16.5 kDa protein that is expressed during the G2/M phase of the cell cycle and is hypothesized to inhibit a default apoptotic cascade initiated in mitosis. This inhibitory function is coupled to survivin's localization to the mitotic spindle. To begin to address the structural basis of survivin's function, we report the X-ray crystal structure of a recombinant form of full length survivin to 2.58 A resolution. Survivin consists of two defined domains including an N-terminal Zn2+-binding BIR domain linked to a 65 A amphipathic C-terminal alpha-helix. The crystal structure reveals an extensive dimerization interface along a hydrophobic surface on the BIR domain of each survivin monomer. A basic patch acting as a sulfate/phosphate-binding module, an acidic cluster projecting off the BIR domain, and a solvent-accessible hydrophobic surface residing on the C-terminal amphipathic helix, are suggestive of functional protein-protein interaction surfaces.

Amino Acid Sequence↗