scholarly journals Structural basis of polyethylene glycol recognition by antibody

2020 ◽  
Vol 27 (1) ◽  
Author(s):  
Cheng-Chung Lee ◽  
Yu-Cheng Su ◽  
Tzu-Ping Ko ◽  
Li-Ling Lin ◽  
Chih-Ya Yang ◽  
...  
Keyword(s):  
Polyethylene Glycol ◽  
Crown Ether ◽  
Heavy Chain ◽  
Light Chain ◽  
Underlying Mechanism ◽  
Binding Mode ◽  
Structural Basis ◽  
Side Chains ◽  
Arginine Residues ◽  
Aromatic Side Chains

Abstract Background Polyethylene glycol (PEG) is widely used in industry and medicine. Anti-PEG antibodies have been developed for characterizing PEGylated drugs and other applications. However, the underlying mechanism for specific PEG binding has not been elucidated. Methods The Fab of two cognate anti-PEG antibodies 3.3 and 2B5 were each crystallized in complex with PEG, and their structures were determined by X-ray diffraction. The PEG-Fab interactions in these two crystals were analyzed and compared with those in a PEG-containing crystal of an unrelated anti-hemagglutinin 32D6-Fab. The PEG-binding stoichiometry was examined by using analytical ultracentrifuge (AUC). Results A common PEG-binding mode to 3.3 and 2B5 is seen with an S-shaped core PEG fragment bound to two dyad-related Fab molecules. A nearby satellite binding site may accommodate parts of a longer PEG molecule. The core PEG fragment mainly interacts with the heavy-chain residues D31, W33, L102, Y103 and Y104, making extensive contacts with the aromatic side chains. At the center of each half-circle of the S-shaped PEG, a water molecule makes alternating hydrogen bonds to the ether oxygen atoms, in a similar configuration to that of a crown ether-bound lysine. Each satellite fragment is clamped between two arginine residues, R52 from the heavy chain and R29 from the light chain, and also interacts with several aromatic side chains. In contrast, the non-specifically bound PEG fragments in the 32D6-Fab crystal are located in the elbow region or at lattice contacts. The AUC data suggest that 3.3-Fab exists as a monomer in PEG-free solution but forms a dimer in the presence of PEG-550-MME, which is about the size of the S-shaped core PEG fragment. Conclusions The differing amino acids in 3.3 and 2B5 are not involved in PEG binding but engaged in dimer formation. In particular, the light-chain residue K53 of 2B5-Fab makes significant contacts with the other Fab in a dimer, whereas the corresponding N53 of 3.3-Fab does not. This difference in the protein-protein interaction between two Fab molecules in a dimer may explain the temperature dependence of 2B5 in PEG binding, as well as its inhibition by crown ether.

scholarly journals Structural basis for amino acid exchange by a human heteromeric amino acid transporter

2020 ◽  
Vol 117 (35) ◽  
pp. 21281-21287 ◽  
Author(s):  
Di Wu ◽  
Tamara N. Grund ◽  
Sonja Welsch ◽  
Deryck J. Mills ◽  
Max Michel ◽  
...  
Keyword(s):  
Amino Acid ◽  
Heavy Chain ◽  
Light Chain ◽  
Basic Amino Acid ◽  
Binding Pocket ◽  
Structural Basis ◽  
Amino Acid Transporters ◽  
Wide Range ◽  
Functional Complex ◽  
Protein Components

Heteromeric amino acid transporters (HATs) comprise a group of membrane proteins that belong to the solute carrier (SLC) superfamily. They are formed by two different protein components: a light chain subunit from an SLC7 family member and a heavy chain subunit from the SLC3 family. The light chain constitutes the transport subunit whereas the heavy chain mediates trafficking to the plasma membrane and maturation of the functional complex. Mutation, malfunction, and dysregulation of HATs are associated with a wide range of pathologies or represent the direct cause of inherited and acquired disorders. Here we report the cryogenic electron microscopy structure of the neutral and basic amino acid transport complex (b[0,+]AT1-rBAT) which reveals a heterotetrameric protein assembly composed of two heavy and light chain subunits, respectively. The previously uncharacterized interaction between two HAT units is mediated via dimerization of the heavy chain subunits and does not include participation of the light chain subunits. The b(0,+)AT1 transporter adopts a LeuT fold and is captured in an inward-facing conformation. We identify an amino-acid–binding pocket that is formed by transmembrane helices 1, 6, and 10 and conserved among SLC7 transporters.

Structural basis for DNA-mediated allosteric regulation facilitated by the AAA+module of Lon protease

2014 ◽  
Vol 70 (2) ◽  
pp. 218-230 ◽  
Author(s):  
Alan Yueh-Luen Lee ◽  
Yu-Da Chen ◽  
Yu-Yung Chang ◽  
Yu-Ching Lin ◽  
Chi-Fon Chang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Dna Binding ◽  
Electrostatic Interactions ◽  
Allosteric Regulation ◽  
Binding Mode ◽  
Negative Regulation ◽  
Enzymatic Activities ◽  
Structural Basis ◽  
Lon Protease ◽  
Arginine Residues

Lon belongs to a unique group of AAA+proteases that bind DNA. However, the DNA-mediated regulation of Lon remains elusive. Here, the crystal structure of the α subdomain of the Lon protease fromBrevibacillus thermoruber(Bt-Lon) is presented, together with biochemical data, and the DNA-binding mode is delineated, showing that Arg518, Arg557 and Arg566 play a crucial role in DNA binding. Electrostatic interactions contributed by arginine residues in the AAA+module are suggested to be important to DNA binding and allosteric regulation of enzymatic activities. Intriguingly, Arg557, which directly binds DNA in the α subdomain, has a dual role in the negative regulation of ATPase stimulation by DNA and in the domain–domain communication in allosteric regulation of Bt-Lon by substrate. In conclusion, structural and biochemical evidence is provided to show that electrostatic interaction in the AAA+module is important for DNA binding by Lon and allosteric regulation of its enzymatic activities by DNA and substrate.

scholarly journals The binding of 2,4,6-trinitrophenyl derivatives to the mouse myeloma immunoglobulin A protein MOPC 315

1978 ◽  
Vol 169 (1) ◽  
pp. 179-188 ◽  
Author(s):  
Steven K. Dower ◽  
Peter Gettins ◽  
Roland Jackson ◽  
Raymond A. Dwek ◽  
David Givol
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Chemical Shifts ◽  
Variable Region ◽  
Proton Nuclear Magnetic Resonance ◽  
Structural Basis ◽  
Tryptophan Residue ◽  
Difference Spectra ◽  
Fab Fragment ◽  
Mouse Myeloma

The binding of Tnp (2,4,6-trinitrophenyl) derivatives to the Fv fragment (variable region of heavy and light chains) of the mouse myeloma IgA protein MOPC 315 was investigated by 270MHz proton nuclear magnetic resonance. Two of the haptens, Tnp-glycine and Tnp-l-aspartate, are in fast exchange with the Fv fragment, and the changes in chemical shifts for both protein and hapten resonances were determined by titrations. For the tightly binding hapten ε-N-Tnp-α-N-acetyl-l-lysine, which is in slow exchange with the Fv fragment, the changes in chemical shifts for the hapten H3+H5 resonances were determined by cross-saturation. By using these data and the known structure of the combining site of protein MOPC 315 [Dwek, Wain-Hobson, Dower, Gettins, Sutton, Perkins & Givol (1977), Nature (London) 266, 31–37] the mode of binding of Tnp derivatives is deduced by ring-current calculations. The trinitrophenyl ring stacks with tryptophan-93L (light chain) in the ‘aromatic box’ formed by tryptophan-93L, tyrosine-34L and phenyl-alanine-34H (heavy chain). Further evidence for the stacking interaction with a tryptophan residue is provided by the similarity of the optical-difference spectra observed with Tnp-aminomethylphosphonate in the presence of either the Fab fragment (light chain and N-terminal half of heavy chain) of protein MOPC 315 or tryptophan. These data show that the modes of binding of all the Tnp derivatives are very similar, despite a 100-fold range in their affinities. It is also concluded that the modes of binding of Dnp (2,4-dinitrophenyl) and Tnp derivatives to protein MOPC 315 are very similar, and that the structural basis for this is that the aromatic box is large enought to allow the trinitrophenyl ring to stack with tryptophan-93L while still forming hydrogen bonds to asparagine-36L and tyrosine-34L.

Level of galactosylation determines cryoglobulin activity of murine IgG3 monoclonal rheumatoid factor

Blood ◽  
2002 ◽  
Vol 99 (8) ◽  
pp. 2922-2928 ◽  
Author(s):  
Aki Kuroki ◽  
Yasuhiro Kuroda ◽  
Shuichi Kikuchi ◽  
Frédéric Lajaunias ◽  
Thierry Fulpius ◽  
...  
Keyword(s):  
Rheumatoid Factor ◽  
Heavy Chain ◽  
Glycosylation Site ◽  
Structural Basis ◽  
Side Chains ◽  
Murine Lupus ◽  
Marked Contrast ◽  
Autoimmune Syndrome ◽  
Molecular Variants ◽  
Charged Residues

Abstract Autoantibodies of the cryoprecipitating IgG3 isotype have been shown to play a significant role in the development of murine lupus–like autoimmune syndrome. At present, the structural basis of IgG3 cryoprecipitation and its role in autoantibody pathogenicity remain to be defined. Using molecular variants of an IgG3 monoclonal rheumatoid factor, 6-19, derived from an autoimmune MRL-Faslpr mouse, we have investigated the implication of charged residues in the heavy-chain variable (VH) region, potential CH3-linked oligosaccharides, and galactosylation of CH2-linked oligosaccharides in its cryoglobulin activity. The cryoglobulin activity of the IgG3 6-19 mutant bearing more negatively charged residues at VH 6 and 23 was found to be reduced but still highly significant, whereas that of the mutant lacking a potential CH3 glycosylation site remained unchanged. In marked contrast, IgG3 6-19 variants obtained from 6-19 heavy-chain transgenic mice displayed barely detectable cryoglobulin activity associated with an increased level of galactosylation in the CH2 oligosaccharide side chains. Thus, our data strongly suggest that the cryoglobulin activity of IgG3 6-19 autoantibody is critically determined by levels of galactosylation in the CH2 oligosaccharide side chains, whereas VH residues play a secondary role in 6-19 IgG3 cryoglobulin activity.

scholarly journals Structural basis for heme-dependent NCoR binding to the transcriptional repressor REV-ERBβ

2021 ◽  
Vol 7 (5) ◽  
pp. eabc6479
Author(s):  
Sarah A. Mosure ◽  
Timothy S. Strutzenberg ◽  
Jinsai Shang ◽  
Paola Munoz-Tello ◽  
Laura A. Solt ◽  
...  
Keyword(s):  
Nuclear Receptors ◽  
Binding Mode ◽  
Structural Basis ◽  
Receptor Interaction ◽  
Heme Binding ◽  
Endogenous Ligand ◽  
Interaction Domain ◽  
Response Elements ◽  
Biochemical Assays ◽  
Chemical Cross Linking

Heme is the endogenous ligand for the constitutively repressive REV-ERB nuclear receptors, REV-ERBα (NR1D1) and REV-ERBβ (NR1D2), but how heme regulates REV-ERB activity remains unclear. Cellular studies indicate that heme is required for the REV-ERBs to bind the corepressor NCoR and repress transcription. However, fluorescence-based biochemical assays suggest that heme displaces NCoR; here, we show that this is due to a heme-dependent artifact. Using ITC and NMR spectroscopy, we show that heme binding remodels the thermodynamic interaction profile of NCoR receptor interaction domain (RID) binding to REV-ERBβ ligand-binding domain (LBD). We solved two crystal structures of REV-ERBβ LBD cobound to heme and NCoR peptides, revealing the heme-dependent NCoR binding mode. ITC and chemical cross-linking mass spectrometry reveals a 2:1 LBD:RID stoichiometry, consistent with cellular studies showing that NCoR-dependent repression of REV-ERB transcription occurs on dimeric DNA response elements. Our findings should facilitate renewed progress toward understanding heme-dependent REV-ERB activity.

LIPOPOLYSACCHARIDE-SENSITIVE SERINE-PROTEASE ZYMOGEN (FACTOR C) OF LIMULUS HEMOCYTES: IDENTIFICATION AND ALIGNMENT OF PROTEOLYTIC FRAGMENTS PRODUCED DURING THE ACTIVATION SHOW THAT IT IS A NOVEL TYPE OF SERINE-PROTEASE

1987 ◽  
Author(s):  
F Tokunaga ◽  
T Miyata ◽  
T Nakamura ◽  
T Morita ◽  
S Iwanaga
Keyword(s):  
Serine Protease ◽  
Heavy Chain ◽  
Light Chain ◽  
Interleukin 2 ◽  
Coagulation Factor ◽  
Clotting Factor ◽  
Single Chain ◽  
Terminal Sequence ◽  
A Chain ◽  
Factor C

Limulus clotting factor, factor C, is a lipopolysaccharide (LPS)-sensitive serine-protease zymogen present in the hemocytes. It is a two-chain glycoprotein (M.W. = 123,000) composed of a heavy chain (M.W. = 80,000) and a light chain (M.W. = 43,000) T. Nakamura et al. (1986) Eur. J. Biochem. 154, 511-521 .On further studies of this zymogen, a single-chain factor C (M.W. = 123,000) was identified by Western blotting technique. The heavy chain had an NH2-terminal sequence of Ser-Gly-Val-Asp-, which was consistent with the NH2-terminal sequence of the single-chain factor C, indicating that the heavy chain is located in the NH2-terminal part of the zymogen. The light chain had an NH22-terminal sequence of Ser-Ser-Gln-Pro-. Incubation of the two-chain zymogen with LPS resulted in the cleavage of a Phe-Ile bond between residues 72 and 73 of the light chain. Concomitant with this cleavage, the A (72.amino acids) and B chains derived from the light chain was formed. The complete amino acid sequence of the A chain was determined by automated Edman degradation. The A chain contained a typical segment which is similar structuraly to those a family of repeats in human β2 -glycoprotein I, complement factors B, Clr, Cls, H, C4b-binding protein, 02, coagulation factor XIII b subunit, haptoglobin a chain, and interleukin 2 receptor. The NH2-terminal sequence of the B chain was Ile-Trp-Asn-Gly-. This chain contained the serine-active site sequence of -ASP-Ala-Cys-Ser-Gly-Asp-SER-Gly-Gly-Pro-.These results indicate that limulus factor C exists in the hemocytes in a single-chain zymogen form and is converted to an active serine-protease by hydrolysis of a specific Phe-Ile peptide bond. The correlation of limulus factor C and mammalian complement proteins was also suggested.

scholarly journals Variance analysis of immunoglobulin alleles in natural populations of rabbit (Oryctolagus cuniculus): the extensive interallelic divergence at the b locus could be the outcome of overdominance-type selection.

Genetics ◽  
1993 ◽  
Vol 135 (1) ◽  
pp. 171-187 ◽  
Author(s):  
W van der Loo
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Gene Diversity ◽  
Natural Populations ◽  
Constant Region ◽  
Immunoglobulin Light Chain ◽  
Evolutionary Patterns ◽  
B Locus ◽  
Type Selection ◽  
Predominant Allele

Abstract Population genetic data are presented which should contribute to evaluation of the hypothesis that the extraordinary evolutionary patterns observed at the b locus of the rabbit immunoglobulin light chain constant region can be the outcome of overdominance-type selection. The analysis of allele correlations in natural populations revealed an excess of heterozygotes of about 10% at the b locus while heterozygote excess was not observed at loci determining the immunoglobulin heavy chain. Data from the published literature, where homozygote advantage was suggested, were reevaluated and found in agreement with data here presented. Gene diversity was evenly distributed among populations and showed similarities with patterns reported for histocompatibility loci. Analysis of genotypic disequilibria revealed strong digenic associations between the leading alleles of heavy and light chain constant region loci in conjunction with trigenic disequilibria corresponding to a preferential association of b locus heterozygosity with the predominant allele of the heavy chain e locus. It is argued that this may indicate compensatory or nonadditive aspects of a putative heterozygosity enhancing mechanism, implying that effects at the light chain might be more pronounced in populations fixed for the heavy chain polymorphism.

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