Faculty Opinions recommendation of Structural basis for a pH-sensitive calcium leak across membranes.

ABSTRACTHuman noroviruses (NoVs) are the main cause of epidemic and sporadic gastroenteritis. Phylogenetically, noroviruses are divided into seven genogroups, with each divided into multiple genotypes. NoVs belonging to genogroup II and genotype 4 (GII.4) are globally most prevalent. Genetic diversity among the NoVs and the periodic emergence of novel strains present a challenge for the development of vaccines and antivirals to treat NoV infection. NoV protease is essential for viral replication and is an attractive target for the development of antivirals. The available structure of GI.1 protease provided a basis for the design of inhibitors targeting the active site of the protease. These inhibitors, although potent against the GI proteases, poorly inhibit the GII proteases, for which structural information is lacking. To elucidate the structural basis for this difference in the inhibitor efficiency, we determined the crystal structure of a GII.4 protease. The structure revealed significant changes in the S2 substrate-binding pocket, making it noticeably smaller, and in the active site, with the catalytic triad residues showing conformational changes. Furthermore, a conserved arginine is found inserted into the active site, interacting with the catalytic histidine and restricting substrate/inhibitor access to the S2 pocket. This interaction alters the relationships between the catalytic residues and may allow for a pH-dependent regulation of protease activity. The changes we observed in the GII.4 protease structure may explain the reduced potency of the GI-specific inhibitors against the GII protease and therefore must be taken into account when designing broadly cross-reactive antivirals against NoVs.IMPORTANCEHuman noroviruses (NoVs) cause sporadic and epidemic gastroenteritis worldwide. They are divided into seven genogroups (GI to GVII), with each genogroup further divided into several genotypes. Human NoVs belonging to genogroup II and genotype 4 (GII.4) are the most prevalent. Currently, there are no vaccines or antiviral drugs available for NoV infection. The protease encoded by NoV is considered a valuable target because of its essential role in replication. NoV protease structures have only been determined for the GI genogroup. We show here that the structure of the GII.4 protease exhibits several significant changes from GI proteases, including a unique pairing of an arginine with the catalytic histidine that makes the proteolytic activity of GII.4 protease pH sensitive. A comparative analysis of NoV protease structures may provide a rational framework for structure-based drug design of broadly cross-reactive inhibitors targeting NoVs.

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Structural basis for a pH-sensitive calcium leak across membranes

Science ◽

10.1126/science.1252043 ◽

2014 ◽

Vol 344 (6188) ◽

pp. 1131-1135 ◽

Cited By ~ 51

Author(s):

Y. Chang ◽

R. Bruni ◽

B. Kloss ◽

Z. Assur ◽

E. Kloppmann ◽

...

Keyword(s):

Ph Sensitive ◽

Structural Basis

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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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