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S100 Proteins in the Innate Immune Response to Pathogens.
Kozlyuk N, Monteith AJ, Garcia V, Damo SM, Skaar EP, Chazin WJ
(2019) Methods Mol Biol 1929: 275-290
MeSH Terms: Calcium, Host-Pathogen Interactions, Humans, Immunity, Innate, Inflammation, Manganese, Models, Molecular, Protein Conformation, S100 Proteins, Toll-Like Receptor 4
Show Abstract · Added March 26, 2019
S100 proteins are distinct dimeric EF-hand Ca-binding proteins that can bind Zn, Mn, and other transition metals with high affinity at two sites in the dimer interface. Certain S100 proteins, including S100A7, S100A12, S100A8, and S100A9, play key roles in the innate immune response to pathogens. These proteins function via a "nutritional immunity" mechanism by depleting essential transition metals in the infection that are required for the invading organism to grow and thrive. They also act as damage-associated molecular pattern ligands, which activate pattern recognition receptors (e.g., Toll-like receptor 4, RAGE) that mediate inflammation. Here we present protocols for these S100 proteins for high-level production of recombinant protein, measurement of binding affinities using isothermal titration calorimetry, and an assay of antimicrobial activity.
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10 MeSH Terms
An alternative N-terminal fold of the intestine-specific annexin A13a induces dimerization and regulates membrane-binding.
McCulloch KM, Yamakawa I, Shifrin DA, McConnell RE, Foegeding NJ, Singh PK, Mao S, Tyska MJ, Iverson TM
(2019) J Biol Chem 294: 3454-3463
MeSH Terms: Animals, Annexins, Cell Membrane, Epithelial Cells, Humans, Hydrogen-Ion Concentration, Intestinal Mucosa, Intestines, Liposomes, Mice, Models, Molecular, Organ Specificity, Protein Binding, Protein Conformation, alpha-Helical, Protein Multimerization, Protein Structure, Quaternary, Protein Transport
Show Abstract · Added April 1, 2019
Annexin proteins function as Ca-dependent regulators of membrane trafficking and repair that may also modulate membrane curvature. Here, using high-resolution confocal imaging, we report that the intestine-specific annexin A13 (ANX A13) localizes to the tips of intestinal microvilli and determined the crystal structure of the ANX A13a isoform to 2.6 Å resolution. The structure revealed that the N terminus exhibits an alternative fold that converts the first two helices and the associated helix-loop-helix motif into a continuous α-helix, as stabilized by a domain-swapped dimer. We also found that the dimer is present in solution and partially occludes the membrane-binding surfaces of annexin, suggesting that dimerization may function as a means for regulating membrane binding. Accordingly, as revealed by binding and cellular localization assays, ANX A13a variants that favor a monomeric state exhibited increased membrane association relative to variants that favor the dimeric form. Together, our findings support a mechanism for how the association of the ANX A13a isoform with the membrane is regulated.
© 2019 McCulloch et al.
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17 MeSH Terms
Yeast require redox switching in DNA primase.
O'Brien E, Salay LE, Epum EA, Friedman KL, Chazin WJ, Barton JK
(2018) Proc Natl Acad Sci U S A 115: 13186-13191
MeSH Terms: Crystallography, X-Ray, DNA Primase, Electron Transport, Iron-Sulfur Proteins, Models, Molecular, Mutation, Oxidation-Reduction, Protein Conformation, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins
Show Abstract · Added March 26, 2019
Eukaryotic DNA primases contain a [4Fe4S] cluster in the C-terminal domain of the p58 subunit (p58C) that affects substrate affinity but is not required for catalysis. We show that, in yeast primase, the cluster serves as a DNA-mediated redox switch governing DNA binding, just as in human primase. Despite a different structural arrangement of tyrosines to facilitate electron transfer between the DNA substrate and [4Fe4S] cluster, in yeast, mutation of tyrosines Y395 and Y397 alters the same electron transfer chemistry and redox switch. Mutation of conserved tyrosine 395 diminishes the extent of p58C participation in normal redox-switching reactions, whereas mutation of conserved tyrosine 397 causes oxidative cluster degradation to the [3Fe4S] species during p58C redox signaling. Switching between oxidized and reduced states in the presence of the Y397 mutations thus puts primase [4Fe4S] cluster integrity and function at risk. Consistent with these observations, we find that yeast tolerate mutations to Y395 in p58C, but the single-residue mutation Y397L in p58C is lethal. Our data thus show that a constellation of tyrosines for protein-DNA electron transfer mediates the redox switch in eukaryotic primases and is required for primase function in vivo.
Copyright © 2018 the Author(s). Published by PNAS.
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HCV Broadly Neutralizing Antibodies Use a CDRH3 Disulfide Motif to Recognize an E2 Glycoprotein Site that Can Be Targeted for Vaccine Design.
Flyak AI, Ruiz S, Colbert MD, Luong T, Crowe JE, Bailey JR, Bjorkman PJ
(2018) Cell Host Microbe 24: 703-716.e3
MeSH Terms: Antibodies, Neutralizing, Antibodies, Viral, Binding Sites, Disulfides, Drug Design, Epitopes, Hepacivirus, Hepatitis C, Hepatitis C Antibodies, Humans, Immunoglobulin G, Models, Molecular, Protein Conformation, Sequence Alignment, Viral Envelope Proteins, Viral Hepatitis Vaccines, X-Ray Diffraction
Show Abstract · Added March 31, 2019
Hepatitis C virus (HCV) vaccine efforts are hampered by the extensive genetic diversity of HCV envelope glycoproteins E1 and E2. Structures of broadly neutralizing antibodies (bNAbs) (e.g., HEPC3, HEPC74) isolated from individuals who spontaneously cleared HCV infection facilitate immunogen design to elicit antibodies against multiple HCV variants. However, challenges in expressing HCV glycoproteins previously limited bNAb-HCV structures to complexes with truncated E2 cores. Here we describe crystal structures of full-length E2 ectodomain complexes with HEPC3 and HEPC74, revealing lock-and-key antibody-antigen interactions, E2 regions (including a target of immunogen design) that were truncated or disordered in E2 cores, and an antibody CDRH3 disulfide motif that exhibits common interactions with a conserved epitope despite different bNAb-E2 binding orientations. The structures display unusual features relevant to common genetic signatures of HCV bNAbs and demonstrate extraordinary plasticity in antibody-antigen interactions. In addition, E2 variants that bind HEPC3/HEPC74-like germline precursors may represent candidate vaccine immunogens.
Copyright © 2018 Elsevier Inc. All rights reserved.
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17 MeSH Terms
Structural Biology of the HEAT-Like Repeat Family of DNA Glycosylases.
Shi R, Shen XX, Rokas A, Eichman BF
(2018) Bioessays 40: e1800133
MeSH Terms: Archaea, Bacteria, Crystallography, X-Ray, DNA, DNA Damage, DNA Glycosylases, DNA Repair, Eukaryota, Protein Conformation
Show Abstract · Added August 26, 2019
DNA glycosylases remove aberrant DNA nucleobases as the first enzymatic step of the base excision repair (BER) pathway. The alkyl-DNA glycosylases AlkC and AlkD adopt a unique structure based on α-helical HEAT repeats. Both enzymes identify and excise their substrates without a base-flipping mechanism used by other glycosylases and nucleic acid processing proteins to access nucleobases that are otherwise stacked inside the double-helix. Consequently, these glycosylases act on a variety of cationic nucleobase modifications, including bulky adducts, not previously associated with BER. The related non-enzymatic HEAT-like repeat (HLR) proteins, AlkD2, and AlkF, have unique nucleic acid binding properties that expand the functions of this relatively new protein superfamily beyond DNA repair. Here, we review the phylogeny, biochemistry, and structures of the HLR proteins, which have helped broaden our understanding of the mechanisms by which DNA glycosylases locate and excise chemically modified DNA nucleobases.
© 2018 WILEY Periodicals, Inc.
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Structure-function analyses of the ion channel TRPC3 reveal that its cytoplasmic domain allosterically modulates channel gating.
Sierra-Valdez F, Azumaya CM, Romero LO, Nakagawa T, Cordero-Morales JF
(2018) J Biol Chem 293: 16102-16114
MeSH Terms: Allosteric Regulation, Ankyrin Repeat, HEK293 Cells, Humans, Ion Channel Gating, Mutation, Protein Conformation, alpha-Helical, Protein Domains, TRPC Cation Channels
Show Abstract · Added April 10, 2019
The transient receptor potential ion channels support Ca permeation in many organs, including the heart, brain, and kidney. Genetic mutations in transient receptor potential cation channel subfamily C member 3 (TRPC3) are associated with neurodegenerative diseases, memory loss, and hypertension. To better understand the conformational changes that regulate TRPC3 function, we solved the cryo-EM structures for the full-length human TRPC3 and its cytoplasmic domain (CPD) in the apo state at 5.8- and 4.0-Å resolution, respectively. These structures revealed that the TRPC3 transmembrane domain resembles those of other TRP channels and that the CPD is a stable module involved in channel assembly and gating. We observed the presence of a C-terminal domain swap at the center of the CPD where horizontal helices (HHs) transition into a coiled-coil bundle. Comparison of TRPC3 structures revealed that the HHs can reside in two distinct positions. Electrophysiological analyses disclosed that shortening the length of the C-terminal loop connecting the HH with the TRP helices increases TRPC3 activity and that elongating the length of the loop has the opposite effect. Our findings indicate that the C-terminal loop affects channel gating by altering the allosteric coupling between the cytoplasmic and transmembrane domains. We propose that molecules that target the HH may represent a promising strategy for controlling TRPC3-associated neurological disorders and hypertension.
© 2018 Sierra-Valdez et al.
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9 MeSH Terms
De novo designed transmembrane peptides activating the α5β1 integrin.
Mravic M, Hu H, Lu Z, Bennett JS, Sanders CR, Orr AW, DeGrado WF
(2018) Protein Eng Des Sel 31: 181-190
MeSH Terms: Amino Acid Sequence, Cell Membrane, Computer-Aided Design, Drug Design, Humans, Integrin alpha5beta1, Micelles, Peptides, Protein Conformation, alpha-Helical, Protein Domains
Show Abstract · Added November 21, 2018
Computationally designed transmembrane α-helical peptides (CHAMP) have been used to compete for helix-helix interactions within the membrane, enabling the ability to probe the activation of the integrins αIIbβ3 and αvβ3. Here, this method is extended towards the design of CHAMP peptides that inhibit the association of the α5β1 transmembrane (TM) domains, targeting the Ala-X3-Gly motif within α5. Our previous design algorithm was performed alongside a new workflow implemented within the widely used Rosetta molecular modeling suite. Peptides from each computational approach activated integrin α5β1 but not αVβ3 in human endothelial cells. Two CHAMP peptides were shown to directly associate with an α5 TM domain peptide in detergent micelles to a similar degree as a β1 TM peptide does. By solution-state nuclear magnetic resonance, one of these CHAMP peptides was shown to bind primarily the integrin β1 TM domain, which itself has a Gly-X3-Gly motif. The second peptide associated modestly with both α5 and β1 constructs, with slight preference for α5. Although the design goal was not fully realized, this work characterizes novel CHAMP peptides activating α5β1 that can serve as useful reagents for probing integrin biology.
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10 MeSH Terms
Structural and Functional Features of the Reovirus σ1 Tail.
Dietrich MH, Ogden KM, Long JM, Ebenhoch R, Thor A, Dermody TS, Stehle T
(2018) J Virol 92:
MeSH Terms: Amino Acid Sequence, Capsid Proteins, Cells, Cultured, Crystallography, X-Ray, Protein Binding, Protein Conformation, Receptors, Virus, Reoviridae, Reoviridae Infections, Sequence Homology, Virus Attachment, Virus Internalization, Virus Replication
Show Abstract · Added April 3, 2019
Mammalian orthoreovirus attachment to target cells is mediated by the outer capsid protein σ1, which projects from the virion surface. The σ1 protein is a homotrimer consisting of a filamentous tail, which is partly inserted into the virion; a body domain constructed from β-spiral repeats; and a globular head with receptor-binding properties. The σ1 tail is predicted to form an α-helical coiled coil. Although σ1 undergoes a conformational change during cell entry, the nature of this change and its contributions to viral replication are unknown. Electron micrographs of σ1 molecules released from virions identified three regions of flexibility, including one at the midpoint of the molecule, that may be involved in its structural rearrangement. To enable a detailed understanding of essential σ1 tail organization and properties, we determined high-resolution structures of the reovirus type 1 Lang (T1L) and type 3 Dearing (T3D) σ1 tail domains. Both molecules feature extended α-helical coiled coils, with T1L σ1 harboring central chloride ions. Each molecule displays a discontinuity (stutter) within the coiled coil and an unexpectedly seamless transition to the body domain. The transition region features conserved interdomain interactions and appears rigid rather than highly flexible. Functional analyses of reoviruses containing engineered σ1 mutations suggest that conserved residues predicted to stabilize the coiled-coil-to-body junction are essential for σ1 folding and encapsidation, whereas central chloride ion coordination and the stutter are dispensable for efficient replication. Together, these findings enable modeling of full-length reovirus σ1 and provide insight into the stabilization of a multidomain virus attachment protein. While it is established that different conformational states of attachment proteins of enveloped viruses mediate receptor binding and membrane fusion, less is understood about how such proteins mediate attachment and entry of nonenveloped viruses. The filamentous reovirus attachment protein σ1 binds cellular receptors; contains regions of predicted flexibility, including one at the fiber midpoint; and undergoes a conformational change during cell entry. Neither the nature of the structural change nor its contribution to viral infection is understood. We determined crystal structures of large σ1 fragments for two different reovirus serotypes. We observed an unexpectedly tight transition between two domains spanning the fiber midpoint, which allows for little flexibility. Studies of reoviruses with engineered changes near the σ1 midpoint suggest that the stabilization of this region is critical for function. Together with a previously determined structure, we now have a complete model of the full-length, elongated reovirus σ1 attachment protein.
Copyright © 2018 American Society for Microbiology.
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The unassembled flavoprotein subunits of human and bacterial complex II have impaired catalytic activity and generate only minor amounts of ROS.
Maklashina E, Rajagukguk S, Iverson TM, Cecchini G
(2018) J Biol Chem 293: 7754-7765
MeSH Terms: Bacterial Proteins, Catalysis, Crystallography, X-Ray, Electron Transport Complex II, Escherichia coli, Flavoproteins, Humans, Models, Molecular, Oxidation-Reduction, Protein Conformation, Protein Subunits, Reactive Oxygen Species
Show Abstract · Added April 1, 2019
Complex II (SdhABCD) is a membrane-bound component of mitochondrial and bacterial electron transport chains, as well as of the TCA cycle. In this capacity, it catalyzes the reversible oxidation of succinate. SdhABCD contains the SDHA protein harboring a covalently bound FAD redox center and the iron-sulfur protein SDHB, containing three distinct iron-sulfur centers. When assembly of this complex is compromised, the flavoprotein SDHA may accumulate in the mitochondrial matrix or bacterial cytoplasm. Whether the unassembled SDHA has any catalytic activity, for example in succinate oxidation, fumarate reduction, reactive oxygen species (ROS) generation, or other off-pathway reactions, is not known. Therefore, here we investigated whether unassembled SdhA flavoprotein, its homolog fumarate reductase (FrdA), and the human SDHA protein have succinate oxidase or fumarate reductase activity and can produce ROS. Using recombinant expression in , we found that the free flavoproteins from these divergent biological sources have inherently low catalytic activity and generate little ROS. These results suggest that the iron-sulfur protein SDHB in complex II is necessary for robust catalytic activity. Our findings are consistent with those reported for single-subunit flavoprotein homologs that are not associated with iron-sulfur or heme partner proteins.
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Integrated Structural Biology for α-Helical Membrane Protein Structure Determination.
Xia Y, Fischer AW, Teixeira P, Weiner B, Meiler J
(2018) Structure 26: 657-666.e2
MeSH Terms: Algorithms, Binding Sites, Electron Spin Resonance Spectroscopy, Humans, Membrane Proteins, Microscopy, Electron, Models, Molecular, Monte Carlo Method, Nuclear Magnetic Resonance, Biomolecular, Protein Binding, Protein Conformation, alpha-Helical, Protein Folding, Protein Interaction Domains and Motifs, Rhodopsin, Thermodynamics
Show Abstract · Added March 17, 2018
While great progress has been made, only 10% of the nearly 1,000 integral, α-helical, multi-span membrane protein families are represented by at least one experimentally determined structure in the PDB. Previously, we developed the algorithm BCL::MP-Fold, which samples the large conformational space of membrane proteins de novo by assembling predicted secondary structure elements guided by knowledge-based potentials. Here, we present a case study of rhodopsin fold determination by integrating sparse and/or low-resolution restraints from multiple experimental techniques including electron microscopy, electron paramagnetic resonance spectroscopy, and nuclear magnetic resonance spectroscopy. Simultaneous incorporation of orthogonal experimental restraints not only significantly improved the sampling accuracy but also allowed identification of the correct fold, which is demonstrated by a protein size-normalized transmembrane root-mean-square deviation as low as 1.2 Å. The protocol developed in this case study can be used for the determination of unknown membrane protein folds when limited experimental restraints are available.
Copyright © 2018 Elsevier Ltd. All rights reserved.
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15 MeSH Terms