Structural basis for the gating modulation of Kv4.3 by auxiliary subunits

AbstractAcid-sensing ion channels (ASICs) are proton-gated cation channels critical for neuronal functions. Studies of ASIC1, a major ASIC isoform and proton sensor, have identified acidic pocket, an extracellular region enriched in acidic residues, as a key participant in channel gating. While binding to this region by the venom peptide psalmotoxin modulates channel gating, molecular and structural mechanisms of ASIC gating modulation by small molecules are poorly understood. Here, combining functional, crystallographic, computational and mutational approaches, we show that two structurally distinct small molecules potently and allosterically inhibit channel activation and desensitization by binding at the acidic pocket and stabilizing the closed state of rat/chicken ASIC1. Our work identifies a previously unidentified binding site, elucidates a molecular mechanism of small molecule modulation of ASIC gating, and demonstrates directly the structural basis of such modulation, providing mechanistic and structural insight into ASIC gating, modulation and therapeutic targeting.

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Structural basis of gating modulation of Kv4 channel complexes

Nature ◽

10.1038/s41586-021-03935-z ◽

2021 ◽

Author(s):

Yoshiaki Kise ◽

Go Kasuya ◽

Hiroyuki H. Okamoto ◽

Daichi Yamanouchi ◽

Kan Kobayashi ◽

...

Keyword(s):

Structural Basis ◽

Macromolecular Complex ◽

Auxiliary Subunits ◽

Voltage Gated ◽

Kv Channels ◽

C Terminus ◽

Voltage Dependent ◽

Gated Channel ◽

Related Proteins ◽

Voltage Sensing Domain

AbstractModulation of voltage-gated potassium (Kv) channels by auxiliary subunits is central to the physiological function of channels in the brain and heart1,2. Native Kv4 tetrameric channels form macromolecular ternary complexes with two auxiliary β-subunits—intracellular Kv channel-interacting proteins (KChIPs) and transmembrane dipeptidyl peptidase-related proteins (DPPs)—to evoke rapidly activating and inactivating A-type currents, which prevent the backpropagation of action potentials1–5. However, the modulatory mechanisms of Kv4 channel complexes remain largely unknown. Here we report cryo-electron microscopy structures of the Kv4.2–DPP6S–KChIP1 dodecamer complex, the Kv4.2–KChIP1 and Kv4.2–DPP6S octamer complexes, and Kv4.2 alone. The structure of the Kv4.2–KChIP1 complex reveals that the intracellular N terminus of Kv4.2 interacts with its C terminus that extends from the S6 gating helix of the neighbouring Kv4.2 subunit. KChIP1 captures both the N and the C terminus of Kv4.2. In consequence, KChIP1 would prevent N-type inactivation and stabilize the S6 conformation to modulate gating of the S6 helices within the tetramer. By contrast, unlike the reported auxiliary subunits of voltage-gated channel complexes, DPP6S interacts with the S1 and S2 helices of the Kv4.2 voltage-sensing domain, which suggests that DPP6S stabilizes the conformation of the S1–S2 helices. DPP6S may therefore accelerate the voltage-dependent movement of the S4 helices. KChIP1 and DPP6S do not directly interact with each other in the Kv4.2–KChIP1–DPP6S ternary complex. Thus, our data suggest that two distinct modes of modulation contribute in an additive manner to evoke A-type currents from the native Kv4 macromolecular complex.

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Organization of tight junctions in the choroid plexus epithelium

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100082546 ◽

1978 ◽

Vol 36 (2) ◽

pp. 336-337

Author(s):

B. Van Deurs ◽

J. K. Koehler

Keyword(s):

Choroid Plexus ◽

Present Report ◽

Freeze Fracture ◽

Barrier Properties ◽

Cell Junctions ◽

Structural Basis ◽

Apical Surface ◽

Choroid Plexus Epithelium ◽

Solid Nitrogen ◽

Special Composition

The choroid plexus epithelium constitutes a blood-cerebrospinal fluid (CSF) barrier, and is involved in regulation of the special composition of the CSF. The epithelium is provided with an ouabain-sensitive Na/K-pump located at the apical surface, actively pumping ions into the CSF. The choroid plexus epithelium has been described as “leaky” with a low transepithelial resistance, and a passive transepithelial flux following a paracellular route (intercellular spaces and cell junctions) also takes place. The present report describes the structural basis for these “barrier” properties of the choroid plexus epithelium as revealed by freeze fracture.Choroid plexus from the lateral, third and fourth ventricles of rats were used. The tissue was fixed in glutaraldehyde and stored in 30% glycerol. Freezing was performed either in liquid nitrogen-cooled Freon 22, or directly in a mixture of liquid and solid nitrogen prepared in a special vacuum chamber. The latter method was always used, and considered necessary, when preparations of complementary (double) replicas were made.

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High resolution spot scan imaging of frozen, hydrated actin bundles with 400kV electrons

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100122964 ◽

1992 ◽

Vol 50 (1) ◽

pp. 512-513

Author(s):

J. Jakana ◽

M.F. Schmid ◽

P. Matsudaira ◽

W. Chiu

Keyword(s):

High Resolution ◽

Binding Proteins ◽

Actin Binding ◽

Eukaryotic Cells ◽

Dimensional Structure ◽

Structural Basis ◽

Actin Bundle ◽

Actin Bundles ◽

3 Dimensional ◽

Macromolecular Assembly

Actin is a protein found in all eukaryotic cells. In its polymerized form, the cells use it for motility, cytokinesis and for cytoskeletal support. An example of this latter class is the actin bundle in the acrosomal process from the Limulus sperm. The different functions actin performs seem to arise from its interaction with the actin binding proteins. A 3-dimensional structure of this macromolecular assembly is essential to provide a structural basis for understanding this interaction in relationship to its development and functions.

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Electron Microscopy and image reconstruction reveal the structural basis for spectrin's elastic properties

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100122952 ◽

1992 ◽

Vol 50 (1) ◽

pp. 510-511

Author(s):

Amy M. McGough ◽

Robert Josephs

Keyword(s):

Electron Microscopy ◽

Elastic Properties ◽

Conformational Changes ◽

Quaternary Structure ◽

Three Dimensional ◽

Membrane Skeleton ◽

Projection Algorithm ◽

Structural Basis ◽

Iterative Approximation ◽

Membrane Skeletons

The remarkable deformability of the erythrocyte derives in large part from the elastic properties of spectrin, the major component of the membrane skeleton. It is generally accepted that spectrin's elasticity arises from marked conformational changes which include variations in its overall length (1). In this work the structure of spectrin in partially expanded membrane skeletons was studied by electron microscopy to determine the molecular basis for spectrin's elastic properties. Spectrin molecules were analysed with respect to three features: length, conformation, and quaternary structure. The results of these studies lead to a model of how spectrin mediates the elastic deformation of the erythrocyte.Membrane skeletons were isolated from erythrocyte membrane ghosts, negatively stained, and examined by transmission electron microscopy (2). Particle lengths and end-to-end distances were measured from enlarged prints using the computer program MACMEASURE. Spectrin conformation (straightness) was assessed by calculating the particles’ correlation length by iterative approximation (3). Digitised spectrin images were correlation averaged or Fourier filtered to improve their signal-to-noise ratios. Three-dimensional reconstructions were performed using a suite of programs which were based on the filtered back-projection algorithm and executed on a cluster of Microvax 3200 workstations (4).

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Electron diffraction detection of ordliking in annealed PbTe thin film

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100178227 ◽

1990 ◽

Vol 48 (4) ◽

pp. 1018-1019

Author(s):

Karimat El-Sayed

Keyword(s):

Thin Film ◽

Electron Diffraction ◽

Lead Telluride ◽

Structural Basis ◽

Face Centered Cubic ◽

X Ray ◽

Polycrystalline Thin Films ◽

Stage Process ◽

Diffraction Patterns ◽

Face Centered

Lead telluride is an important semiconductor of many applications. Many Investigators showed that there are anamolous descripancies in most of the electrophysical properties of PbTe polycrystalline thin films on annealing. X-Ray and electron diffraction studies are being undertaken in the present work in order to explain the cause of this anamolous behaviour.Figures 1-3 show the electron diffraction of the unheated, heated in air at 100°C and heated in air at 250°C respectively of a 300°A polycrystalline PbTe thin film. It can be seen that Fig. 1 is a typical [100] projection of a face centered cubic with unmixed (hkl) indices. Fig. 2 shows the appearance of faint superlattice reflections having mixed (hkl) indices. Fig. 3 shows the disappearance of thf superlattice reflections and the appearance of polycrystalline PbO phase superimposed on the [l00] PbTe diffraction patterns. The mechanism of this three stage process can be explained on structural basis as follows :

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Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

Biochemical Journal ◽

10.1042/bcj20190399 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3333-3353 ◽

Cited By ~ 3

Author(s):

Malti Yadav ◽

Kamalendu Pal ◽

Udayaditya Sen

Keyword(s):

Active Site ◽

Metal Binding ◽

Metal Ion ◽

Triosephosphate Isomerase ◽

Catalytic Mechanism ◽

Degradation Mechanism ◽

Structural Basis ◽

Conserved Residues ◽

Phosphodiester Bond ◽

Cyclic Dinucleotides

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

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Reading the phosphorylation code: binding of the 14-3-3 protein to multivalent client phosphoproteins

Biochemical Journal ◽

10.1042/bcj20200084 ◽

2020 ◽

Vol 477 (7) ◽

pp. 1219-1225 ◽

Cited By ~ 4

Author(s):

Nikolai N. Sluchanko

Keyword(s):

Protein Function ◽

Intrinsically Disordered Protein ◽

Structural Basis ◽

Major Protein ◽

Post Translational Modifications ◽

Protein Protein Interaction ◽

Intrinsically Disordered ◽

Biochemical Journal ◽

Multiple Binding ◽

And Control

Many major protein–protein interaction networks are maintained by ‘hub’ proteins with multiple binding partners, where interactions are often facilitated by intrinsically disordered protein regions that undergo post-translational modifications, such as phosphorylation. Phosphorylation can directly affect protein function and control recognition by proteins that ‘read’ the phosphorylation code, re-wiring the interactome. The eukaryotic 14-3-3 proteins recognizing multiple phosphoproteins nicely exemplify these concepts. Although recent studies established the biochemical and structural basis for the interaction of the 14-3-3 dimers with several phosphorylated clients, understanding their assembly with partners phosphorylated at multiple sites represents a challenge. Suboptimal sequence context around the phosphorylated residue may reduce binding affinity, resulting in quantitative differences for distinct phosphorylation sites, making hierarchy and priority in their binding rather uncertain. Recently, Stevers et al. [Biochemical Journal (2017) 474: 1273–1287] undertook a remarkable attempt to untangle the mechanism of 14-3-3 dimer binding to leucine-rich repeat kinase 2 (LRRK2) that contains multiple candidate 14-3-3-binding sites and is mutated in Parkinson's disease. By using the protein-peptide binding approach, the authors systematically analyzed affinities for a set of LRRK2 phosphopeptides, alone or in combination, to a 14-3-3 protein and determined crystal structures for 14-3-3 complexes with selected phosphopeptides. This study addresses a long-standing question in the 14-3-3 biology, unearthing a range of important details that are relevant for understanding binding mechanisms of other polyvalent proteins.

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Crystal structures of Magnaporthe oryzae trehalose-6-phosphate synthase (MoTps1) suggest a model for catalytic process of Tps1

Biochemical Journal ◽

10.1042/bcj20190289 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3227-3240 ◽

Cited By ~ 1

Author(s):

Shanshan Wang ◽

Yanxiang Zhao ◽

Long Yi ◽

Minghe Shen ◽

Chao Wang ◽

...

Keyword(s):

Crystal Structures ◽

Conformational Changes ◽

Magnaporthe Oryzae ◽

Structural Information ◽

Complex Structure ◽

Critical Role ◽

Catalytic Process ◽

Structural Basis ◽

Salt Bridges ◽

Terminal Domain

Trehalose-6-phosphate (T6P) synthase (Tps1) catalyzes the formation of T6P from UDP-glucose (UDPG) (or GDPG, etc.) and glucose-6-phosphate (G6P), and structural basis of this process has not been well studied. MoTps1 (Magnaporthe oryzae Tps1) plays a critical role in carbon and nitrogen metabolism, but its structural information is unknown. Here we present the crystal structures of MoTps1 apo, binary (with UDPG) and ternary (with UDPG/G6P or UDP/T6P) complexes. MoTps1 consists of two modified Rossmann-fold domains and a catalytic center in-between. Unlike Escherichia coli OtsA (EcOtsA, the Tps1 of E. coli), MoTps1 exists as a mixture of monomer, dimer, and oligomer in solution. Inter-chain salt bridges, which are not fully conserved in EcOtsA, play primary roles in MoTps1 oligomerization. Binding of UDPG by MoTps1 C-terminal domain modifies the substrate pocket of MoTps1. In the MoTps1 ternary complex structure, UDP and T6P, the products of UDPG and G6P, are detected, and substantial conformational rearrangements of N-terminal domain, including structural reshuffling (β3–β4 loop to α0 helix) and movement of a ‘shift region' towards the catalytic centre, are observed. These conformational changes render MoTps1 to a ‘closed' state compared with its ‘open' state in apo or UDPG complex structures. By solving the EcOtsA apo structure, we confirmed that similar ligand binding induced conformational changes also exist in EcOtsA, although no structural reshuffling involved. Based on our research and previous studies, we present a model for the catalytic process of Tps1. Our research provides novel information on MoTps1, Tps1 family, and structure-based antifungal drug design.

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Designing TIMP (tissue inhibitor of metalloproteinases) variants that are selective metalloproteinase inhibitors

Biochemical Society Symposium ◽

10.1042/bss0700201 ◽

2003 ◽

Vol 70 ◽

pp. 201-212 ◽

Cited By ~ 51

Author(s):

Hideaki Nagase ◽

Keith Brew

Keyword(s):

Three Dimensional ◽

Therapeutic Interventions ◽

Tissue Inhibitors Of Metalloproteinases ◽

Structural Basis ◽

Structural Elements ◽

Ridge Structure ◽

Extracellular Matrix Components ◽

Metalloproteinase Inhibitors ◽

The Matrix ◽

A Disintegrin And Metalloproteinase

The tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of the matrix metalloproteinases (MMPs), enzymes that play central roles in the degradation of extracellular matrix components. The balance between MMPs and TIMPs is important in the maintenance of tissues, and its disruption affects tissue homoeostasis. Four related TIMPs (TIMP-1 to TIMP-4) can each form a complex with MMPs in a 1:1 stoichiometry with high affinity, but their inhibitory activities towards different MMPs are not particularly selective. The three-dimensional structures of TIMP-MMP complexes reveal that TIMPs have an extended ridge structure that slots into the active site of MMPs. Mutation of three separate residues in the ridge, at positions 2, 4 and 68 in the amino acid sequence of the N-terminal inhibitory domain of TIMP-1 (N-TIMP-1), separately and in combination has produced N-TIMP-1 variants with higher binding affinity and specificity for individual MMPs. TIMP-3 is unique in that it inhibits not only MMPs, but also several ADAM (a disintegrin and metalloproteinase) and ADAMTS (ADAM with thrombospondin motifs) metalloproteinases. Inhibition of the latter groups of metalloproteinases, as exemplified with ADAMTS-4 (aggrecanase 1), requires additional structural elements in TIMP-3 that have not yet been identified. Knowledge of the structural basis of the inhibitory action of TIMPs will facilitate the design of selective TIMP variants for investigating the biological roles of specific MMPs and for developing therapeutic interventions for MMP-associated diseases.

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