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Manuel Covarrubias Annika?F. Barber Vincenzo Carnevale Werner Treptow Roderic?G. Eckenhoff 《Biophysical journal》2015,109(10):2003-2011
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. 相似文献
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辅助亚基KChIPs对Kv4钾通道的“钳制”调节作用 总被引:1,自引:0,他引:1
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. 相似文献
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《Structure (London, England : 1993)》2020,28(2):244-251.e3
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《Journal of molecular biology》2023,435(19):168234
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. 相似文献
7.
《Journal of molecular biology》2021,433(17):166995
Leak currents, defined as voltage and time independent flows of ions across cell membranes, are central to cellular electrical excitability control. The K2P (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, K2Ps 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 K2Ps have established the basic architecture of this channel family, revealed key moving parts involved in K2P function, uncovered the importance of asymmetric pinching and dilation motions in the K2P selectivity filter (SF) C-type gate, and defined two K2P structural classes based on the absence or presence of an intracellular gate. Further, a series of structures characterizing K2P: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 K2P activity. Such knowledge should catalyze development of new K2P modulators to probe function and treat a wide range of disorders. 相似文献
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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. 相似文献
9.
Andrey F. Z. Nascimento Daniel M. Trindade Celisa C. C. Tonoli Priscila O. de Giuseppe Leandro H. P. Assis Rodrigo V. Honorato Paulo S. L. de Oliveira Pravin Mahajan Nicola A. Burgess-Brown Frank von Delft Roy E. Larson Mario T. Murakami 《The Journal of biological chemistry》2013,288(47):34131-34145
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. 相似文献
10.
Kelvin B. Luther Hermann Schindelin Robert S. Haltiwanger 《The Journal of biological chemistry》2009,284(5):3294-3305
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). 相似文献
11.
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. 相似文献
12.
Yuwei Zhu Jianxin Dai Tiancheng Zhang Xu Li Pengfei Fang Huajing Wang Yongliang Jiang Xiaojie Yu Tian Xia Liwen Niu Yajun Guo Maikun Teng 《The Journal of biological chemistry》2013,288(35):25165-25172
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. 相似文献
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Congmin Li Jenny Chan Franciose Haeseleer Katsuhiko Mikoshiba Krzysztof Palczewski Mitsuhiko Ikura James B. Ames 《The Journal of biological chemistry》2009,284(4):2472-2481
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). 相似文献
15.
16.
Yanwu Yang Xiaoxia Wang Cheryl A. Hawkins Kan Chen Julia Vaynberg Xian Mao Yizeng Tu Xiaobing Zuo Jinbu Wang Yun-xing Wang Chuanyue Wu Nico Tjandra Jun Qin 《The Journal of biological chemistry》2009,284(9):5836-5844
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, KD ∼ 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. 相似文献
17.
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. 相似文献
18.
Shun Hirota Naoki Tanaka Ivan Mi?eti? Paolo Di Muro Satoshi Nagao Hiroaki Kitagishi Koji Kano Richard S. Magliozzo Jack Peisach Mariano Beltramini Luigi Bubacco 《The Journal of biological chemistry》2010,285(25):19338-19345
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 kobs 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. 相似文献
19.
Anders B. Sorensen Jesper J. Madsen L. Anders Svensson Anette A. Pedersen Henrik ?stergaard Michael T. Overgaard Ole H. Olsen Prafull S. Gandhi 《The Journal of biological chemistry》2016,291(9):4671-4683
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. 相似文献