Mechanistic Insights into the Modulation of Voltage-Gated Ion Channels by Inhalational Anesthetics

General anesthesia is a relatively safe medical procedure, which for nearly 170 years has allowed life saving surgical interventions in animals and people. However, the molecular mechanism of general anesthesia continues to be a matter of importance and debate. A favored hypothesis proposes that general anesthesia results from direct multisite interactions with multiple and diverse ion channels in the brain. Neurotransmitter-gated ion channels and two-pore K⁺ channels are key players in the mechanism of anesthesia; however, new studies have also implicated voltage-gated ion channels. Recent biophysical and structural studies of Na⁺ and K⁺ channels strongly suggest that halogenated inhalational general anesthetics interact with gates and pore regions of these ion channels to modulate function. Here, we review these studies and provide a perspective to stimulate further advances.     

辅助亚基KChIPs对Kv4钾通道的“钳制”调节作用

快速失活电压门控型钾通道对于神经元兴奋性发挥重要的调节作用,从而影响神经元的功能,如疼痛的信号传导等.拥有四个钙结合位点"EF-hand"的胞浆蛋白KChIPs(Kv channel-interacting proteins)属于神经钙感受器(NCS)家族,与Kv4(Shal)的α亚基共组装成为天然复合体,在神经元和心肌分别编码瞬间低阈值A型K+电流ISA(transient subthreshold A-type K+ current)和瞬间外向K+电流ITO(transient outward K+ current).辅助亚基KChIPs与Kv4 N端的特异性结合有助于电压门控钾通道四聚体的形成和稳定,从而增加通道向细胞膜表面的转运,并调节通道的失活动力学和恢复速率等.本文基于近期解析出的Kv4 N末端与KChIP1的复合晶体结构,着重阐述Kv4钾通道与其辅助亚基KChIPs的相互作用机制.在Kv4 N端/KChIP1复合结晶体中,每一个KChIP1分子分别与两个邻近的Kv4 N末端相结合,即单个KChIP1同时与周围的两个Kv4相互作用,而形成分子数比为4:4的"钳制"结构.Kv4和KChIPs相互作用的结构机制为基于结构的化合物设计以及治疗膜兴奋性相关疾病提供了基础.     

Structural Insights into of the Allosteric Activation of the LicT Antiterminator by PTS-Mediated Phosphorylation

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Structural Basis of the Allosteric Inhibition of Human ABCG2 by Nanobodies

ABCG2 is an ATP-binding cassette transporter that exports a wide range of xenobiotic compounds and has been recognized as a contributing factor for multidrug resistance in cancer cells. Substrate and inhibitor interactions with ABCG2 have been extensively studied and small molecule inhibitors have been developed that prevent the export of anticancer drugs from tumor cells. Here, we explore the potential for inhibitors that target sites other than the substrate binding pocket of ABCG2. We developed novel nanobodies against ABCG2 and used functional analyses to select three inhibitory nanobodies (Nb8, Nb17 and Nb96) for structural studies by single particle cryo-electron microscopy. Our results showed that these nanobodies allosterically bind to different regions of the nucleotide binding domains. Two copies of Nb8 bind to the apex of the NBDs preventing them from fully closing. Nb17 binds near the two-fold axis of the transporter and interacts with both NBDs. Nb96 binds to the side of the NBD and immobilizes a region connected to key motifs involved in ATP binding and hydrolysis. All three nanobodies prevent the transporter from undergoing conformational changes required for substrate transport. These findings advance our understanding of the molecular basis of modulation of ABCG2 by external binders, which may contribute to the development of a new generation of inhibitors. Furthermore, this is the first example of modulation of human multidrug resistance transporters by nanobodies.     

Structural Insights into the Mechanisms and Pharmacology of K2P Potassium Channels

Leak currents, defined as voltage and time independent flows of ions across cell membranes, are central to cellular electrical excitability control. The K_2P (KCNK) potassium channel class comprises an ion channel family that produces potassium leak currents that oppose excitation and stabilize the resting membrane potential in cells in the brain, cardiovascular system, immune system, and sensory organs. Due to their widespread tissue distribution, K_2Ps contribute to many physiological and pathophysiological processes including anesthesia, pain, arrythmias, ischemia, hypertension, migraine, intraocular pressure regulation, and lung injury responses. Structural studies of six homomeric K_2Ps have established the basic architecture of this channel family, revealed key moving parts involved in K_2P function, uncovered the importance of asymmetric pinching and dilation motions in the K_2P selectivity filter (SF) C-type gate, and defined two K_2P structural classes based on the absence or presence of an intracellular gate. Further, a series of structures characterizing K_2P:modulator interactions have revealed a striking polysite pharmacology housed within a relatively modestly sized (~70 kDa) channel. Binding sites for small molecules or lipids that control channel function are found at every layer of the channel structure, starting from its extracellular side through the portion that interacts with the membrane bilayer inner leaflet. This framework provides the basis for understanding how gating cues sensed by different channel parts control function and how small molecules and lipids modulate K_2P activity. Such knowledge should catalyze development of new K_2P modulators to probe function and treat a wide range of disorders.     

Structural Insights into the Assembly of CARMA1 and BCL10

The CBM complex (CARMA1, BCL10 and MALT1) plays a crucial role in B and T lymphocyte activation. CARMA1 serves as a scaffold for BCL10, MALT1 and other effector proteins and regulates various signaling pathways related to the immune response. The assembly of CARMA1 and BCL10 is mediated through a CARD-CARD interaction. Here, we report the crystal structure of the CARD domain of CARMA1 at a resolution of 1.75 Å. The structure consists of six helices, as previously determined for CARD domains. Structural and computational analysis identified the binding interface between CARMA1-CARD and BCL10-CARD, which consists of a basic patch in CARMA1 and an acidic patch in BCL10. Site-directed mutagenesis, co-immunoprecipitation and an NF-κB activation assay confirmed that the interface is necessary for association and downstream signaling. Our studies provide molecular insight into the assembly of CARMA1 and BCL10.     

Structural Insights into Functional Overlapping and Differentiation among Myosin V Motors

Myosin V (MyoV) motors have been implicated in the intracellular transport of diverse cargoes including vesicles, organelles, RNA-protein complexes, and regulatory proteins. Here, we have solved the cargo-binding domain (CBD) structures of the three human MyoV paralogs (Va, Vb, and Vc), revealing subtle structural changes that drive functional differentiation and a novel redox mechanism controlling the CBD dimerization process, which is unique for the MyoVc subclass. Moreover, the cargo- and motor-binding sites were structurally assigned, indicating the conservation of residues involved in the recognition of adaptors for peroxisome transport and providing high resolution insights into motor domain inhibition by CBD. These results contribute to understanding the structural requirements for cargo transport, autoinhibition, and regulatory mechanisms in myosin V motors.     

Structural and Mechanistic Insights into Lunatic Fringe from a Kinetic Analysis of Enzyme Mutants

The Notch receptor is critical for proper development where it orchestrates numerous cell fate decisions. The Fringe family of β1,3-N-acetylglucosaminyltransferases are regulators of this pathway. Fringe enzymes add N-acetylglucosamine to O-linked fucose on the epidermal growth factor repeats of Notch. Here we have analyzed the reaction catalyzed by Lunatic Fringe (Lfng) in detail. A mutagenesis strategy for Lfng was guided by a multiple sequence alignment of Fringe proteins and solutions from docking an epidermal growth factor-like O-fucose acceptor substrate onto a homology model of Lfng. We targeted three main areas as follows: residues that could help resolve where the fucose binds, residues in two conserved loops not observed in the published structure of Manic Fringe, and residues predicted to be involved in UDP-N-acetylglucosamine (UDP-GlcNAc) donor specificity. We utilized a kinetic analysis of mutant enzyme activity toward the small molecule acceptor substrate 4-nitrophenyl-α-l-fucopyranoside to judge their effect on Lfng activity. Our results support the positioning of O-fucose in a specific orientation to the catalytic residue. We also found evidence that one loop closes off the active site coincident with, or subsequent to, substrate binding. We propose a mechanism whereby the ordering of this short loop may alter the conformation of the catalytic aspartate. Finally, we identify several residues near the UDP-GlcNAc-binding site, which are specifically permissive toward UDP-GlcNAc utilization.Defects in Notch signaling have been implicated in numerous human diseases, including multiple sclerosis (1), several forms of cancer (2-4), cerebral autosomal dominant arteriopathy with sub-cortical infarcts and leukoencephalopathy (5), and spondylocostal dysostosis (SCD)³ (6-8). The transmembrane Notch signaling receptor is activated by members of the DSL (Delta, Serrate, Lag2) family of ligands (9, 10). In the endoplasmic reticulum, O-linked fucose glycans are added to the epidermal growth factor-like (EGF) repeats of the Notch extracellular domain by protein O-fucosyltransferase 1 (11-13). These O-fucose monosaccharides can be elongated in the Golgi apparatus by three highly conserved β1,3-N-acetylglucosaminyltransferases of the Fringe family (Lunatic (Lfng), Manic (Mfng), and Radical Fringe (Rfng) in mammals) (14-16). The formation of this GlcNAc-β1,3-Fuc-α1, O-serine/threonine disaccharide is necessary and sufficient for subsequent elongation to a tetrasaccharide (15, 19), although elongation past the disaccharide in Drosophila is not yet clear (20, 21). Elongation of O-fucose by Fringe is known to potentiate Notch signaling from Delta ligands and inhibit signaling from Serrate ligands (22). Delta ligands are termed Delta-like (Delta-like1, -2, and -4) in mammals, and the homologs of Serrate are known as Jagged (Jagged1 and -2) in mammals. The effects of Fringe on Drosophila Notch can be recapitulated in Notch ligand in vitro binding assays using purified components, suggesting that the elongation of O-fucose by Fringe alters the binding of Notch to its ligands (21). Although Fringe also appears to alter Notch-ligand interactions in mammals, the effects of elongation of the glycan past the O-fucose monosaccharide is more complicated and appears to be cell type-, receptor-, and ligand-dependent (for a recent review see Ref. 23).The Fringe enzymes catalyze the transfer of GlcNAc from the donor substrate UDP-α-GlcNAc to the acceptor fucose, forming the GlcNAc-β1,3-Fuc disaccharide (14-16). They belong to the GT-A-fold of inverting glycosyltransferases, which includes N-acetylglucosaminyltransferase I and β1,4-galactosyltransferase I (17, 18). The mechanism is presumed to proceed through the abstraction of a proton from the acceptor substrate by a catalytic base (Asp or Glu) in the active site. This creates a nucleophile that attacks the anomeric carbon of the nucleotide-sugar donor, inverting its configuration from α (on the nucleotide sugar) to β (in the product) (24, 25). The enzyme then releases the acceptor substrate modified with a disaccharide and UDP. The Mfng structure (26) leaves little doubt as to the identity of the catalytic residue, which in all likelihood is aspartate 289 in mouse Lfng (we will use numbering for mouse Lunatic Fringe throughout, unless otherwise stated). The structure of Mfng with UDP-GlcNAc soaked into the crystals (26) showed density only for the UDP portion of the nucleotide-sugar donor and no density for two loops flanking either side of the active site. The presence of flexible loops that become ordered upon substrate binding is a common observation with glycosyltransferases in the GT-A fold family (18, 25). Density for the entire donor was observed in the structure of rabbit N-acetylglucosaminyltransferase I (27). In this case, ordering of a previously disordered loop upon UDP-GlcNAc binding may have contributed to increased stability of the donor. In the case of bovine β1,4-galactosyltransferase I, a section of flexible random coil from the apo-structure was observed to change its conformation to α-helical upon donor substrate binding (28). Both loops in Lfng are highly conserved, and we have mutated a number of residues in each to test the hypothesis that they interact with the substrates. The mutagenesis strategy was also guided by docking of an EGF-O-fucose acceptor substrate into the active site of the Lfng model as well as comparison of the Lfng model with a homology model of the β1,3-glucosyltransferase (β3GlcT) that modifies O-fucose on thrombospondin type 1 repeats (29, 30). The β3GlcT is predicted to be a GT-A fold enzyme related to the Fringe family (17, 18, 29).     

Depolarization Differentially Affects Allosteric Modulation by Neurotoxins of Scorpion α-Toxin Binding on Voltage-Gated Sodium Channels

Abstract: Voltage-gated sodium channels serve as a target for many neurotoxins that bind to several distinct, allosterically interacting receptor sites. We examined the effect of membrane potentials (incited by increasing external K⁺ concentrations) on the binding modulation by veratridine, brevetoxin, and tetrodotoxin of the scorpion α-toxin AaH II to receptor site 3 on sodium channels of rat brain synaptosomes. Depolarization is shown to differentially modulate neurotoxin effects on AaH II binding: Veratridine increase is potentiated, brevetoxin's inhibitory effect is reduced, and tetrodotoxin enhancement is evident mainly at resting membrane potential (5 m M K⁺). Both tetrodotoxin and veratridine apparently reverse the inhibition of AaH II binding by brevetoxin at resting membrane potential, but only veratridine is able to partially restore AaH II binding at 0 mV (135 m M K⁺). Thus, the allosteric interactions are grouped into two categories, depending on the membrane potential. Under depolarized conditions, the cooperative effects among veratridine and brevetoxin on AaH II binding fit the previously described two-state conformational model. At resting membrane potential, additional interactions are revealed, which may be explained by assuming that toxin binding induces conformational changes on the channel structure, in addition to being state-dependent. Our results provide a new insight into neurotoxin action and the complex dynamic changes underlying allosteric coupling of neurotoxin receptor sites, which may be related to channel gating.     

Structural Insights into the Neutralization Mechanism of Monoclonal Antibody 6C2 against Ricin

Ricin belongs to the type II ribosome-inactivating proteins that depurinate the universally conserved α-sarcin loop of rRNA. The RNA N-glycosidase activity of ricin also largely depends on the ribosomal proteins that play an important role during the process of rRNA depurination. Therefore, the study of the interaction between ricin and the ribosomal elements will be better to understand the catalysis mechanism of ricin. The antibody 6C2 is a mouse monoclonal antibody exhibiting unusually potent neutralizing ability against ricin, but the neutralization mechanism remains unknown. Here, we report the 2.8 Å crystal structure of 6C2 Fab in complex with the A-chain of ricin (RTA), which reveals an extensive antigen-antibody interface that contains both hydrogen bonds and van der Waals contacts. The complementarity-determining region loops H1, H2, H3, and L3 form a pocket to accommodate the epitope on the RTA (residues Asp⁹⁶–Thr¹¹⁶). ELISA results show that Gln⁹⁸, Glu⁹⁹, Glu¹⁰², and Thr¹⁰⁵ (RTA) are the key residues that play an important role in recognizing 6C2. With the perturbation of the 6C2 Fab-RTA interface, 6C2 loses its neutralization ability, measured based on the inhibition of protein synthesis in a cell-free system. Finally, we propose that the neutralization mechanism of 6C2 against ricin is that the binding of 6C2 hinders the interaction between RTA and the ribosome and the surface plasmon resonance and pulldown results confirm our hypothesis. In short, our data explain the neutralization mechanism of mAb 6C2 against ricin and provide a structural basis for the development of improved antibody drugs with better specificity and higher affinity.     

Structural Insights into Ca2+-dependent Regulation of Inositol 1,4,5-Trisphosphate Receptors by CaBP1

Calcium-binding protein 1 (CaBP1), a neuron-specific member of the calmodulin (CaM) superfamily, modulates Ca²⁺-dependent activity of inositol 1,4,5-trisphosphate receptors (InsP₃Rs). Here we present NMR structures of CaBP1 in both Mg²⁺-bound and Ca²⁺-bound states and their structural interaction with InsP₃Rs. CaBP1 contains four EF-hands in two separate domains. The N-domain consists of EF1 and EF2 in a closed conformation with Mg²⁺ bound at EF1. The C-domain binds Ca²⁺ at EF3 and EF4, and exhibits a Ca²⁺-induced closed to open transition like that of CaM. The Ca²⁺-bound C-domain contains exposed hydrophobic residues (Leu¹³², His¹³⁴, Ile¹⁴¹, Ile¹⁴⁴, and Val¹⁴⁸) that may account for selective binding to InsP₃Rs. Isothermal titration calorimetry analysis reveals a Ca²⁺-induced binding of the CaBP1 C-domain to the N-terminal region of InsP₃R (residues 1-587), whereas CaM and the CaBP1 N-domain did not show appreciable binding. CaBP1 binding to InsP₃Rs requires both the suppressor and ligand-binding core domains, but has no effect on InsP₃ binding to the receptor. We propose that CaBP1 may regulate Ca²⁺-dependent activity of InsP₃Rs by promoting structural contacts between the suppressor and core domains.Calcium ion (Ca²⁺) in the cell functions as an important messenger that controls neurotransmitter release, gene expression, muscle contraction, apoptosis, and disease processes (1). Receptor stimulation in neurons promotes large increases in intracellular Ca²⁺ levels controlled by Ca²⁺ release from intracellular stores through InsP₃Rs (2). The neuronal type-1 receptor (InsP₃R1)² is positively and negatively regulated by cytosolic Ca²⁺ (3-6), important for the generation of repetitive Ca²⁺ transients known as Ca²⁺ spikes and waves (1). Ca²⁺-dependent activation of InsP₃R1 contributes to the fast rising phase of Ca²⁺ signaling known as Ca²⁺-induced Ca²⁺ release (7). Ca²⁺-induced inhibition of InsP₃R1, triggered at higher cytosolic Ca²⁺ levels, coordinates the temporal decay of Ca²⁺ transients (6). The mechanism of Ca²⁺-dependent regulation of InsP₃Rs is complex (8, 9), and involves direct Ca²⁺ binding sites (5, 10) as well as remote sensing by extrinsic Ca²⁺-binding proteins such as CaM (11, 12), CaBP1 (13, 14), CIB1 (15), and NCS-1 (16).Neuronal Ca²⁺-binding proteins (CaBP1-5 (17)) represent a new sub-branch of the CaM superfamily (18) that regulate various Ca²⁺ channel targets. Multiple splice variants and isoforms of CaBPs are localized in different neuronal cell types (19-21) and perform specialized roles in signal transduction. CaBP1, also termed caldendrin (22), has been shown to modulate the Ca²⁺-sensitive activity of InsP₃Rs (13, 14). CaBP1 also regulates P/Q-type voltage-gated Ca²⁺ channels (23), L-type channels (24), and the transient receptor potential channel, TRPC5 (25). CaBP4 regulates Ca²⁺-dependent inhibition of L-type channels in the retina and may be genetically linked to retinal degeneration (26). Thus, the CaBP proteins are receiving increased attention as a family of Ca²⁺ sensors that control a variety of Ca²⁺ channel targets implicated in neuronal degenerative diseases.CaBP proteins contain four EF-hands, similar in sequence to those found in CaM and troponin C (18) (Fig. 1). By analogy to CaM (27), the four EF-hands are grouped into two domains connected by a central linker that is four residues longer in CaBPs than in CaM. In contrast to CaM, the CaBPs contain non-conserved amino acids within the N-terminal region that may confer target specificity. Another distinguishing property of CaBPs is that the second EF-hand lacks critical residues required for high affinity Ca²⁺ binding (17). CaBP1 binds Ca²⁺ only at EF3 and EF4, whereas it binds Mg²⁺ at EF1 that may serve a functional role (28). Indeed, changes in cytosolic Mg²⁺ levels have been detected in cortical neurons after treatment with neurotransmitter (29). Other neuronal Ca²⁺-binding proteins such as DREAM (30), CIB1 (31), and NCS-1 (32) also bind Mg²⁺ and exhibit Mg²⁺-induced physiological effects. Mg²⁺ binding in each of these proteins helps stabilize their Ca²⁺-free state to interact with signaling targets.Open in a separate windowFIGURE 1.Amino acid sequence alignment of human CaBP1 with CaM. Secondary structural elements (α-helices and β-strands) were derived from NMR analysis. The four EF-hands (EF1, EF2, EF3, and EF4) are highlighted green, red, cyan, and yellow. Residues in the 12-residue Ca²⁺-binding loops are underlined and chelating residues are highlighted bold. Non-conserved residues in the hydrophobic patch are colored red.Despite extensive studies on CaBP1, little is known about its structure and target binding properties, and regulation of InsP₃Rs by CaBP1 is somewhat controversial and not well understood. Here, we present the NMR solution structures of both Mg²⁺-bound and Ca²⁺-bound conformational states of CaBP1 and their structural interactions with InsP₃R1. These CaBP1 structures reveal important Ca²⁺-induced structural changes that control its binding to InsP₃R1. Our target binding analysis demonstrates that the C-domain of CaBP1 exhibits Ca²⁺-induced binding to the N-terminal cytosolic region of InsP₃R1. We propose that CaBP1 may regulate Ca²⁺-dependent channel activity in InsP₃Rs by promoting a structural interaction between the N-terminal suppressor and ligand-binding core domains that modulates Ca²⁺-dependent channel gating (8, 33, 34).     

Structural Basis of Focal Adhesion Localization of LIM-only Adaptor PINCH by Integrin-linked Kinase

The LIM-only adaptor PINCH (the particularly interesting cysteine- and histidine-rich protein) plays a pivotal role in the assembly of focal adhesions (FAs), supramolecular complexes that transmit mechanical and biochemical information between extracellular matrix and actin cytoskeleton, regulating diverse cell adhesive processes such as cell migration, cell spreading, and survival. A key step for the PINCH function is its localization to FAs, which depends critically on the tight binding of PINCH to integrin-linked kinase (ILK). Here we report the solution NMR structure of the core ILK·PINCH complex (28 kDa, K_D ∼ 68 nm) involving the N-terminal ankyrin repeat domain (ARD) of ILK and the first LIM domain (LIM1) of PINCH. We show that the ILK ARD exhibits five sequentially stacked ankyrin repeat units, which provide a large concave surface to grip the two contiguous zinc fingers of the PINCH LIM1. The highly electrostatic interface is evolutionally conserved but differs drastically from those of known ARD and LIM bound to other types of protein domains. Consistently mutation of a hot spot in LIM1, which is not conserved in other LIM domains, disrupted the PINCH binding to ILK and abolished the PINCH targeting to FAs. These data provide atomic insight into a novel modular recognition and demonstrate how PINCH is specifically recruited by ILK to mediate the FA assembly and cell-extracellular matrix communication.Cell-extracellular matrix (ECM)³ adhesion, migration, and survival are essential for the development and maintenance of tissues and organs in living organisms. They are mediated by integrin transmembrane receptors, which function by adhering to ECM proteins via their large extracellular domains while connecting to the actin cytoskeleton via their small cytoplasmic tails (20-70 residues) (1). The integrin-actin connection supports strong cell-ECM adhesion, and its alteration leads to dynamic cell shape change, migration, and survival (2). The molecular details of such connection, however, are highly complex, involving a large protein complex network called focal adhesions (FAs) (3, 4).Integrin-linked kinase (ILK) is a 50-kDa FA protein that contains an N-terminal ankyrin repeat domain (ARD), a middle pleckstrin homology domain, and a C-terminal kinase domain. Originally discovered as an integrin β cytoplasmic tail-binding protein (5), ILK has been established as a major regulator that controls the complex FA assembly and transmits many cell adhesive signals between integrins and actin (6-8). Soon after the discovery of ILK, Tu et al. (9) identified an ILK binding partner called PINCH that contains five LIM domains. Extensive studies have shown that the PINCH binding to ILK is essential for triggering the FA assembly and for relaying diverse mechanical and biochemical signals between ECM and the actin cytoskeleton (9-11). Consistent with the importance of the ILK/PINCH association in almost all cellular behavior and fate, ablation of either ILK (12) or PINCH in mice is embryonically lethal (13, 14). PINCH also has a highly homologous isoform called PINCH-2. However, although complementary to PINCH in many cellular behaviors (for reviews, see Refs. 8 and 15), PINCH-2 appears to be involved at the later stage of development (16), and thus its ablation in mice is not embryonically lethal (17). At the clinical level, dysregulation of the ILK/PINCH interaction has been implicated in the development of numerous human disorders such as cancer (6, 18) and heart diseases (19, 20). A Phase I clinical trial is ongoing on a drug called thymosin β-4 (RegeneRx) that appears to specifically target ILK/PINCH for treating myocardial infarction, a major heart failure disorder (19).Despite the cellular, physiological, and pathological importance of the ILK/PINCH interaction, the structural basis for how exactly PINCH binds to ILK has not been well understood. Previous biochemical/structural analyses have indicated that ILK utilizes its N-terminal ARD to recognize the LIM1 domain of PINCH, and such binding may promote the targeting of PINCH to FAs (9, 21). However, the precise atomic basis for such targeting process is elusive. No structure of any ARD·LIM complex has been reported. Using a combination of NMR-based techniques, we have solved the solution structure of the ILK ARD·PINCH LIM1 complex that revealed an interface that is distinct from other ARD and LIM bound to non-ARD/LIM domains. Structure-based mutation of a hot spot in PINCH LIM1, which is not conserved in other LIM domains, abolished the PINCH binding to ILK and its localization to FAs. These results not only reveal a unique LIM/ARD recognition mode but also provide a definitive functional basis for how PINCH is recruited by ILK to focal adhesion site, a major step toward the dynamic cell adhesion and migration processes.     

Modulation of Kv4.2 channel expression and gating by dipeptidyl peptidase 10 (DPP10)

The dipeptidyl aminopeptidase-like protein DPPX (DPP6) associates with Kv4 potassium channels, increasing surface trafficking and reconstituting native neuronal ISA-like properties. Dipeptidyl peptidase 10 (DPP10) shares with DPP6 a high amino acid identity, lack of enzymatic activity, and expression predominantly in the brain. We used a two-electrode voltage-clamp and oocyte expression system to determine if DPP10 also interacts with Kv4 channels and modulates their expression and function. Kv4.2 coimmunoprecipitated with HA/DPP10 from extracts of oocytes heterologously expressing both proteins. Coexpression with DPP10 and HA/DPP10 enhanced Kv4.2 current by approximately fivefold without increasing protein level. DPP10 also remodeled Kv4.2 kinetic and steady-state properties by accelerating time courses of inactivation and recovery (taurec: WT = 200 ms, +DPP10 = 78 ms). Furthermore, DPP10 introduced hyperpolarizing shifts in the conductance-voltage relationship (approximately 19 mV) as well as steady-state inactivation (approximately 7 mV). The effects of DPP10 on Kv4.1 were similar to Kv4.2; however, distinct biophysical differences were observed. Additional experiments suggested that the cytoplasmic N-terminal domain of DPP10 determines the acceleration of inactivation. In summary, DPP10 is a potent modulator of Kv4 expression and biophysical properties and may be a critical component of somatodendritic ISA channels in the brain.     

Structural Basis of the Lactate-dependent Allosteric Regulation of Oxygen Binding in Arthropod Hemocyanin

Hemocyanin (Hc) is an oxygen carrier protein in which oxygen binding is regulated by allosteric effectors such as H⁺ and l-lactate. Isothermal titration calorimetric measurements showed that l-lactate binds to dodecameric and heterohexameric Hc and to the CaeSS3 homohexamer but not to the CaeSS2 monomer. The binding of lactate caused no change in the optical absorption and x-ray absorption spectra of either oxy- or deoxy-Hc, suggesting that no structural rearrangement of the active site occurred. At pH 6.5, the oxygen binding rate constant k_obs obtained by flash photolysis showed a significant increase upon addition of l-lactate, whereas l-lactate addition had little effect at pH 8.3. Lactate binding caused a concentration-dependent shift in the interhexameric distances at pH 6.5 based on small angle x-ray scattering measurements. These results show that l-lactate affects oxygen affinity at pH 6.5 by modulating the global structure of Hc without affecting its binuclear copper center (the active site). In contrast to this, the active site structure of deoxy-Hc is affected by changes in pH (Hirota, S., Kawahara, T., Beltramini, M., Di Muro, P., Magliozzo, R. S., Peisach, J., Powers, L. S., Tanaka, N., Nagao, S., and Bubacco, L. (2008) J. Biol. Chem. 283, 31941–31948). Upon addiction of lactate, the kinetic behavior of oxygen rebinding for Hc was heterogeneous under low oxygen concentrations at pH 6.5 due to changes in the T and R state populations, and the equilibrium was found to shift from the T toward the R state with addition of lactate.     

Molecular Basis of Enhanced Activity in Factor VIIa-Trypsin Variants Conveys Insights into Tissue Factor-mediated Allosteric Regulation of Factor VIIa Activity

The complex of coagulation factor VIIa (FVIIa), a trypsin-like serine protease, and membrane-bound tissue factor (TF) initiates blood coagulation upon vascular injury. Binding of TF to FVIIa promotes allosteric conformational changes in the FVIIa protease domain and improves its catalytic properties. Extensive studies have revealed two putative pathways for this allosteric communication. Here we provide further details of this allosteric communication by investigating FVIIa loop swap variants containing the 170 loop of trypsin that display TF-independent enhanced activity. Using x-ray crystallography, we show that the introduced 170 loop from trypsin directly interacts with the FVIIa active site, stabilizing segment 215–217 and activation loop 3, leading to enhanced activity. Molecular dynamics simulations and novel fluorescence quenching studies support that segment 215–217 conformation is pivotal to the enhanced activity of the FVIIa variants. We speculate that the allosteric regulation of FVIIa activity by TF binding follows a similar path in conjunction with protease domain N terminus insertion, suggesting a more complete molecular basis of TF-mediated allosteric enhancement of FVIIa activity.