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Human antibody recognition of antigenic site IV on Pneumovirus fusion proteins.
Mousa JJ, Binshtein E, Human S, Fong RH, Alvarado G, Doranz BJ, Moore ML, Ohi MD, Crowe JE
(2018) PLoS Pathog 14: e1006837
MeSH Terms: Amino Acid Sequence, Amino Acid Substitution, Antibodies, Monoclonal, Antibodies, Neutralizing, Antibodies, Viral, Antibody Specificity, Binding Sites, Antibody, Binding, Competitive, Cross Reactions, Epitope Mapping, Epitopes, Humans, Kinetics, Metapneumovirus, Microscopy, Electron, Mutation, Recombinant Proteins, Respiratory Syncytial Virus, Human, Viral Fusion Proteins
Show Abstract · Added March 3, 2020
Respiratory syncytial virus (RSV) is a major human pathogen that infects the majority of children by two years of age. The RSV fusion (F) protein is a primary target of human antibodies, and it has several antigenic regions capable of inducing neutralizing antibodies. Antigenic site IV is preserved in both the pre-fusion and post-fusion conformations of RSV F. Antibodies to antigenic site IV have been described that bind and neutralize both RSV and human metapneumovirus (hMPV). To explore the diversity of binding modes at antigenic site IV, we generated a panel of four new human monoclonal antibodies (mAbs) and competition-binding suggested the mAbs bind at antigenic site IV. Mutagenesis experiments revealed that binding and neutralization of two mAbs (3M3 and 6F18) depended on arginine (R) residue R429. We discovered two R429-independent mAbs (17E10 and 2N6) at this site that neutralized an RSV R429A mutant strain, and one of these mAbs (17E10) neutralized both RSV and hMPV. To determine the mechanism of cross-reactivity, we performed competition-binding, recombinant protein mutagenesis, peptide binding, and electron microscopy experiments. It was determined that the human cross-reactive mAb 17E10 binds to RSV F with a binding pose similar to 101F, which may be indicative of cross-reactivity with hMPV F. The data presented provide new concepts in RSV immune recognition and vaccine design, as we describe the novel idea that binding pose may influence mAb cross-reactivity between RSV and hMPV. Characterization of the site IV epitope bound by human antibodies may inform the design of a pan-Pneumovirus vaccine.
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BRAF internal deletions and resistance to BRAF/MEK inhibitor therapy.
Johnson DB, Childress MA, Chalmers ZR, Frampton GM, Ali SM, Rubinstein SM, Fabrizio D, Ross JS, Balasubramanian S, Miller VA, Stephens PJ, Sosman JA, Lovly CM
(2018) Pigment Cell Melanoma Res 31: 432-436
MeSH Terms: Adult, Amino Acid Substitution, Base Sequence, Drug Resistance, Neoplasm, Female, Humans, Imidazoles, MAP Kinase Signaling System, Melanoma, Mutation, Missense, Oximes, Protein Kinase Inhibitors, Proto-Oncogene Proteins B-raf, Pyridones, Pyrimidinones, Sequence Deletion
Show Abstract · Added September 10, 2020
BRAF and MEK inhibitors have improved clinical outcomes in advanced, BRAF -mutated melanomas. Acquired resistance occurs in most patients, with numerous and diverse drivers. We obtained pretreatment and progression biopsies from a patient who progressed on dabrafenib and trametinib. In addition to a preserved BRAF mutation, an internal deletion (rearrangement) of BRAF was observed in the progression sample. This deletion involved exons 2-8, which includes the Ras-binding domain, and is analogous to previously documented BRAF fusions and splice variants known to reactivate RAS-RAF-MEK-ERK signaling. In a large cohort of melanomas, 10 additional internal deletions were identified (0.4% of all melanomas; nine of which had concurrent BRAF mutations), as well as sporadically in other tumor types. Thus, we describe a novel mechanism of resistance to BRAF and MEK inhibition.
© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
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Analysis of DNA binding by human factor xeroderma pigmentosum complementation group A (XPA) provides insight into its interactions with nucleotide excision repair substrates.
Sugitani N, Voehler MW, Roh MS, Topolska-Woś AM, Chazin WJ
(2017) J Biol Chem 292: 16847-16857
MeSH Terms: Amino Acid Substitution, DNA Repair, DNA Repair Enzymes, DNA, Single-Stranded, Humans, Mutation, Missense, Nuclear Magnetic Resonance, Biomolecular, Protein Binding, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Structural Homology, Protein, Xeroderma Pigmentosum, Xeroderma Pigmentosum Group A Protein
Show Abstract · Added March 24, 2018
Xeroderma pigmentosum (XP) complementation group A (XPA) is an essential scaffolding protein in the multiprotein nucleotide excision repair (NER) machinery. The interaction of XPA with DNA is a core function of this protein; a number of mutations in the DNA-binding domain (DBD) are associated with XP disease. Although structures of the central globular domain of human XPA and data on binding of DNA substrates have been reported, the structural basis for XPA's DNA-binding activity remains unknown. X-ray crystal structures of the central globular domain of yeast XPA (Rad14) with lesion-containing DNA duplexes have provided valuable insights, but the DNA substrates used for this study do not correspond to the substrates of XPA as it functions within the NER machinery. To better understand the DNA-binding activity of human XPA in NER, we used NMR to investigate the interaction of its DBD with a range of DNA substrates. We found that XPA binds different single-stranded/double-stranded junction DNA substrates with a common surface. Comparisons of our NMR-based mapping of binding residues with the previously reported Rad14-DNA crystal structures revealed similarities and differences in substrate binding between XPA and Rad14. This includes direct evidence for DNA contacts to the residues extending C-terminally from the globular core, which are lacking in the Rad14 construct. Moreover, mutation of the XPA residue corresponding to Phe-262 in Rad14, previously reported as being critical for DNA binding, had only a moderate effect on the DNA-binding activity of XPA. The DNA-binding properties of several disease-associated mutations in the DBD were investigated. These results suggest that for XPA mutants exhibiting altered DNA-binding properties, a correlation exists between the extent of reduction in DNA-binding affinity and the severity of symptoms in XP patients.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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13 MeSH Terms
cytochrome P450 46A1 (CYP46A1) activation by neuroactive compounds.
Mast N, Anderson KW, Johnson KM, Phan TTN, Guengerich FP, Pikuleva IA
(2017) J Biol Chem 292: 12934-12946
MeSH Terms: Acetylcholine, Alkynes, Allosteric Regulation, Amino Acid Substitution, Anti-HIV Agents, Aspartic Acid, Benzoxazines, Binding Sites, Biocatalysis, Cholesterol 24-Hydroxylase, Cyclopropanes, Deuterium Exchange Measurement, Enzyme Activation, Glutamic Acid, Ligands, Models, Molecular, Molecular Docking Simulation, Mutagenesis, Site-Directed, Mutation, Nerve Tissue Proteins, Peptide Fragments, Protein Conformation, Recombinant Fusion Proteins, gamma-Aminobutyric Acid
Show Abstract · Added March 14, 2018
Cytochrome P450 46A1 (CYP46A1, cholesterol 24-hydroxylase) is the enzyme responsible for the majority of cholesterol elimination from the brain. Previously, we found that the anti-HIV drug efavirenz (EFV) can pharmacologically activate CYP46A1 in mice. Herein, we investigated whether CYP46A1 could also be activated by endogenous compounds, including major neurotransmitters. experiments with purified recombinant CYP46A1 indicated that CYP46A1 is activated by l-glutamate (l-Glu), l-aspartate, γ-aminobutyric acid, and acetylcholine, with l-Glu eliciting the highest increase (3-fold) in CYP46A1-mediated cholesterol 24-hydroxylation. We also found that l-Glu and other activating neurotransmitters bind to the same site on the CYP46A1 surface, which differs from the EFV-binding site. The other principal differences between EFV and l-Glu in CYP46A1 activation include an apparent lack of l-Glu binding to the P450 active site and different pathways of signal transduction from the allosteric site to the active site. EFV and l-Glu similarly increased the CYP46A1 , the rate of the "fast" phase of the enzyme reduction by the redox partner NADPH-cytochrome P450 oxidoreductase, and the amount of P450 reduced. Spectral titrations with cholesterol, in the presence of EFV or l-Glu, suggest that water displacement from the heme iron can be affected in activator-bound CYP46A1. Moreover, EFV and l-Glu synergistically activated CYP46A1. Collectively, our data, along with those from previous cell culture and studies by others, suggest that l-Glu-induced CYP46A1 activation is of physiological relevance.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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24 MeSH Terms
Structural and biochemical analyses reveal insights into covalent flavinylation of the Complex II homolog quinol:fumarate reductase.
Starbird CA, Maklashina E, Sharma P, Qualls-Histed S, Cecchini G, Iverson TM
(2017) J Biol Chem 292: 12921-12933
MeSH Terms: Amino Acid Substitution, Biocatalysis, Crystallography, X-Ray, Enzyme Stability, Escherichia coli, Escherichia coli Proteins, Flavin-Adenine Dinucleotide, Gene Deletion, Glutamic Acid, Hot Temperature, Models, Molecular, Molecular Docking Simulation, Mutagenesis, Site-Directed, Mutation, Oxidoreductases, Protein Conformation, Protein Denaturation, Protein Interaction Domains and Motifs, Protein Multimerization, Protein Processing, Post-Translational, Protein Subunits, Recombinant Proteins, Structural Homology, Protein, Succinate Dehydrogenase
Show Abstract · Added April 1, 2019
The Complex II homolog quinol:fumarate reductase (QFR, FrdABCD) catalyzes the interconversion of fumarate and succinate at a covalently attached FAD within the FrdA subunit. The SdhE assembly factor enhances covalent flavinylation of Complex II homologs, but the mechanisms underlying the covalent attachment of FAD remain to be fully elucidated. Here, we explored the mechanisms of covalent flavinylation of the QFR FrdA subunit. Using a Δ strain, we show that the requirement for the assembly factor depends on the cellular redox environment. We next identified residues important for the covalent attachment and selected the FrdA residue, which contributes to proton shuttling during fumarate reduction, for detailed biophysical and structural characterization. We found that QFR complexes containing FrdA have a structure similar to that of the WT flavoprotein, but lack detectable substrate binding and turnover. In the context of the isolated FrdA subunit, the anticipated assembly intermediate during covalent flavinylation, FrdA variants had stability similar to that of WT FrdA, contained noncovalent FAD, and displayed a reduced capacity to interact with SdhE. However, small-angle X-ray scattering (SAXS) analysis of WT FrdA cross-linked to SdhE suggested that the FrdA residue is unlikely to contribute directly to the FrdA-SdhE protein-protein interface. We also found that no auxiliary factor is absolutely required for flavinylation, indicating that the covalent flavinylation is autocatalytic. We propose that multiple factors, including the SdhE assembly factor and bound dicarboxylates, stimulate covalent flavinylation by preorganizing the active site to stabilize the quinone-methide intermediate.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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Implications of the differing roles of the β1 and β3 transmembrane and cytoplasmic domains for integrin function.
Lu Z, Mathew S, Chen J, Hadziselimovic A, Palamuttam R, Hudson BG, Fässler R, Pozzi A, Sanders CR, Zent R
(2016) Elife 5:
MeSH Terms: Amino Acid Substitution, Cell Adhesion, Cells, Cultured, Epithelial Cells, Humans, Integrin alpha1, Integrin beta1, Integrin beta3, Mutagenesis, Site-Directed, Mutant Proteins, Platelet Membrane Glycoprotein IIb, Protein Binding, Protein Multimerization
Show Abstract · Added March 26, 2017
Integrins are transmembrane receptors composed of α and β subunits. Although most integrins contain β1, canonical activation mechanisms are based on studies of the platelet integrin, αIIbβ3. Its inactive conformation is characterized by the association of the αIIb transmembrane and cytosolic domain (TM/CT) with a tilted β3 TM/CT that leads to activation when disrupted. We show significant structural differences between β1 and β3 TM/CT in bicelles. Moreover, the 'snorkeling' lysine at the TM/CT interface of β subunits, previously proposed to regulate αIIbβ3 activation by ion pairing with nearby lipids, plays opposite roles in β1 and β3 integrin function and in neither case is responsible for TM tilt. A range of affinities from almost no interaction to the relatively high avidity that characterizes αIIbβ3 is seen between various α subunits and β1 TM/CTs. The αIIbβ3-based canonical model for the roles of the TM/CT in integrin activation and function clearly does not extend to all mammalian integrins.
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13 MeSH Terms
Kinetic and Structural Impact of Metal Ions and Genetic Variations on Human DNA Polymerase ι.
Choi JY, Patra A, Yeom M, Lee YS, Zhang Q, Egli M, Guengerich FP
(2016) J Biol Chem 291: 21063-21073
MeSH Terms: Amino Acid Substitution, Crystallography, X-Ray, DNA-Directed DNA Polymerase, Deoxycytosine Nucleotides, Humans, Hydrogen Bonding, Kinetics, Magnesium, Manganese, Mutation, Missense, Protein Domains
Show Abstract · Added March 14, 2018
DNA polymerase (pol) ι is a Y-family polymerase involved in translesion synthesis, exhibiting higher catalytic activity with Mn than Mg The human germline R96G variant impairs both Mn-dependent and Mg-dependent activities of pol ι, whereas the Δ1-25 variant selectively enhances its Mg-dependent activity. We analyzed pre-steady-state kinetic and structural effects of these two metal ions and genetic variations on pol ι using pol ι core (residues 1-445) proteins. The presence of Mn (0.15 mm) instead of Mg (2 mm) caused a 770-fold increase in efficiency (k/K) of pol ι for dCTP insertion opposite G, mainly due to a 450-fold decrease in K The R96G and Δ1-25 variants displayed a 53-fold decrease and a 3-fold increase, respectively, in k/K for dCTP insertion opposite G with Mg when compared with wild type, substantially attenuated by substitution with Mn Crystal structures of pol ι ternary complexes, including the primer terminus 3'-OH and a non-hydrolyzable dCTP analogue opposite G with the active-site Mg or Mn, revealed that Mn achieves more optimal octahedral coordination geometry than Mg, with lower values in average coordination distance geometry in the catalytic metal A-site. Crystal structures of R96G revealed the loss of three H-bonds of residues Gly-96 and Tyr-93 with an incoming dNTP, due to the lack of an arginine, as well as a destabilized Tyr-93 side chain secondary to the loss of a cation-π interaction between both side chains. These results provide a mechanistic basis for alteration in pol ι catalytic function with coordinating metals and genetic variation.
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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11 MeSH Terms
A Derived Allosteric Switch Underlies the Evolution of Conditional Cooperativity between HOXA11 and FOXO1.
Nnamani MC, Ganguly S, Erkenbrack EM, Lynch VJ, Mizoue LS, Tong Y, Darling HL, Fuxreiter M, Meiler J, Wagner GP
(2016) Cell Rep 15: 2097-2108
MeSH Terms: Allosteric Regulation, Amino Acid Sequence, Amino Acid Substitution, Animals, Biological Evolution, CREB-Binding Protein, DNA-Activated Protein Kinase, Forkhead Box Protein O1, HeLa Cells, Homeodomain Proteins, Humans, Intrinsically Disordered Proteins, Mice, Models, Biological, Models, Molecular, Phosphorylation, Protein Binding, Protein Domains, Protein Structure, Secondary, Transcriptional Activation
Show Abstract · Added April 8, 2017
Transcription factors (TFs) play multiple roles in development. Given this multifunctionality, it has been assumed that TFs are evolutionarily highly constrained. Here, we investigate the molecular mechanisms for the origin of a derived functional interaction between two TFs, HOXA11 and FOXO1. We have previously shown that the regulatory role of HOXA11 in mammalian endometrial stromal cells requires interaction with FOXO1, and that the physical interaction between these proteins evolved before their functional cooperativity. Here, we demonstrate that the derived functional cooperativity between HOXA11 and FOXO1 is due to derived allosteric regulation of HOXA11 by FOXO1. This study shows that TF function can evolve through changes affecting the functional output of a pre-existing protein complex.
Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.
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20 MeSH Terms
Long antibody HCDR3s from HIV-naïve donors presented on a PG9 neutralizing antibody background mediate HIV neutralization.
Willis JR, Finn JA, Briney B, Sapparapu G, Singh V, King H, LaBranche CC, Montefiori DC, Meiler J, Crowe JE
(2016) Proc Natl Acad Sci U S A 113: 4446-51
MeSH Terms: Amino Acid Substitution, Antibodies, Neutralizing, Blood Donors, Complementarity Determining Regions, Female, HIV Antibodies, HIV Envelope Protein gp120, HIV-1, Humans, Immunoglobulin Heavy Chains, Male, Mutation, Missense
Show Abstract · Added May 4, 2016
Development of broadly neutralizing antibodies (bnAbs) against HIV-1 usually requires prolonged infection and induction of Abs with unusual features, such as long heavy-chain complementarity-determining region 3 (HCDR3) loops. Here we sought to determine whether the repertoires of HIV-1-naïve individuals contain Abs with long HCDR3 loops that could mediate HIV-1 neutralization. We interrogated at massive scale the structural properties of long Ab HCDR3 loops in HIV-1-naïve donors, searching for structured HCDR3s similar to those of the HIV-1 bnAb PG9. We determined the nucleotide sequences encoding 2.3 × 10(7)unique HCDR3 amino acid regions from 70 different HIV-1-naïve donors. Of the 26,917 HCDR3 loops with 30-amino acid length identified, we tested 30 for further study that were predicted to have PG9-like structure when chimerized onto PG9. Three of these 30 PG9 chimeras bound to the HIV-1 gp120 monomer, and two were neutralizing. In addition, we found 14 naturally occurring HCDR3 sequences that acquired the ability to bind to the HIV-1 gp120 monomer when adding 2- to 7-amino acid mutations via computational design. Of those 14 designed Abs, 8 neutralized HIV-1, with IC50values ranging from 0.7 to 98 µg/mL. These data suggest that the repertoire of HIV-1-naïve individuals contains rare B cells that encode HCDR3 loops that bind or neutralize HIV-1 when presented on a PG9 background with relatively few or no additional mutations. Long HCDR3 sequences are present in the HIV-naïve B-cell repertoire, suggesting that this class of bnAbs is a favorable target for rationally designed preventative vaccine efforts.
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LMO2 Oncoprotein Stability in T-Cell Leukemia Requires Direct LDB1 Binding.
Layer JH, Alford CE, McDonald WH, Davé UP
(2016) Mol Cell Biol 36: 488-506
MeSH Terms: Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Amino Acid Substitution, Cell Line, DNA-Binding Proteins, Humans, Jurkat Cells, LIM Domain Proteins, Leukemia, T-Cell, Molecular Sequence Data, Mutation, Protein Interaction Domains and Motifs, Protein Interaction Maps, Protein Stability, Proto-Oncogene Proteins, Transcription Factors, Transcriptional Activation
Show Abstract · Added January 26, 2016
LMO2 is a component of multisubunit DNA-binding transcription factor complexes that regulate gene expression in hematopoietic stem and progenitor cell development. Enforced expression of LMO2 causes leukemia by inducing hematopoietic stem cell-like features in T-cell progenitor cells, but the biochemical mechanisms of LMO2 function have not been fully elucidated. In this study, we systematically dissected the LMO2/LDB1-binding interface to investigate the role of this interaction in T-cell leukemia. Alanine scanning mutagenesis of the LIM interaction domain of LDB1 revealed a discrete motif, R(320)LITR, required for LMO2 binding. Most strikingly, coexpression of full-length, wild-type LDB1 increased LMO2 steady-state abundance, whereas coexpression of mutant proteins deficient in LMO2 binding compromised LMO2 stability. These mutant LDB1 proteins also exerted dominant negative effects on growth and transcription in diverse leukemic cell lines. Mass spectrometric analysis of LDB1 binding partners in leukemic lines supports the notion that LMO2/LDB1 function in leukemia occurs in the context of multisubunit complexes, which also protect the LMO2 oncoprotein from degradation. Collectively, these data suggest that the assembly of LMO2 into complexes, via direct LDB1 interaction, is a potential molecular target that could be exploited in LMO2-driven leukemias resistant to existing chemotherapy regimens.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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