Three-Dimensional Structure of the Fab′ Fragment of a Human Immunoglobulin at 2.8-Å Resolution

Poljak, Roberto J.; Amzel, L. Mario; Avey, H. P.; Chen, B. L.; Phizackerley, R. P.; Saul, F.A.

doi:10.1073/pnas.70.12.3305

Cited by 349 publications

(130 citation statements)

References 24 publications

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“…Therefore, the spacing of the binding of the Fabs along the PS calculated from these studies, about 3 nm per bound Fab at saturation, is relatively consistent with published estimates of the size of an antibody binding site (19) and with estimates of the dimensions of an Fab fragment (30,31,35,42), suggesting that anti-PS antibodies of moderately high affinities are capable of binding as densely along the PS chain as permitted by steric interactions between the IgG molecules and may therefore recognize structural or linear epitopes rather than conformational epitopes. Recognition of immunodominant and functionally important conformational epitopes by polyclonal antibodies and MAbs has been proposed for the group B streptococcal type III PS (7,46), the serotype 14 PS (20,21,45), and the Neisseria meningitidis serogroup B PS (6,11,23,24).…”

supporting

confidence: 72%

Thermodynamics and Density of Binding of a Panel of Antibodies to High-Molecular-Weight Capsular Polysaccharides

Harris

Fernsten

2009

Clin Vaccine Immunol

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Much detailed information regarding the thermodynamic parameters of monoclonal antibody (MAb) binding to oligosaccharides is available (8,9,11,15,26,29,38,39,43). However, very little thermodynamic information regarding binding of MAbs or Fab fragments to intact polysaccharides (PSs) is available. Of particular interest is understanding the density of MAb or Fab binding along high-molecular-weight PS chains.Isothermal titration microcalorimetry (ITC) can be used to investigate the thermodynamics of molecular interactions such as the binding of a MAb to its epitope (10). The thermodynamic binding constant (K), the change in the enthalpy of binding (⌬H), and the stoichiometry or density of binding (N) can all be determined directly from one ITC experiment. ITC directly measures the heat evolved (or consumed) in liquid samples upon the mixing of precise, known amounts of a ligand into a solution of a macromolecule. The heat released (or consumed) after each addition of ligand is directly proportional to the amount of binding that occurs. The data are analyzed by using fitting models to calculate ⌬H, K, and N. ⌬H is proportional to the magnitude of the inflection of the binding isotherm, K is derived from the slope at the midpoint of the binding isotherm, and N is derived from the midpoint of the rise or the inflection point of the binding isotherm. The change in free energy (⌬G) and the change in the entropy of binding (⌬S) are calculated values, since ⌬G is derived from K, that is, ⌬G ϭ ϪRTlnK, where R is the universal gas constant, T is the temperature of the interaction, and ⌬S is derived from ⌬G by the equation ⌬G ϭ ⌬H Ϫ T⌬S.We report here on the thermodynamic parameters of binding, K, ⌬H, and ⌬S, as well as N, for two MAbs with different specificities for Neisseria meningitidis serogroup C capsular PS (MnC PS) and five MAbs and two Fab fragments specific for Streptococcus pneumoniae serotype 4, 14, 6B, 9V, and 19F capsular PSs (Pn PSs). MATERIALS AND METHODSThe MnC PS and the Pn PSs were obtained from Wyeth Vaccines Research. The average molecular masses of the PSs were 360 kDa for MnC PS, 500 kDa for the serotype 4 PS, 850 kDa for the serotype 14 PS, 890 kDa for the serotype 6B PS, 900 kDa for the serotype 9V PS, and 940 kDa for the serotype 19F PS. The O-acetyl content of the MnC PS was 2.24 mol/mg, which is equivalent to approximately 0.7 O-acetyl groups per N-acetylneuraminic acid residue. The methods for the production and purification of anti-MnC PS MAbs Mn207-3 (immunoglobulin G1 [IgG1]), specific for O-acetylated epitopes of MnC PS, and Mn46-1 (IgG1), not specific for O-acetylated epitopes of MnC PS, were reported previously (16). Anti-Pn PS MAbs Pn31-1 (IgG1), Pn36-1 (IgG1), Pn45-1 (IgG1), and Pn42-1 (IgG2b), specific for serotype 4, 6B, 9V, and 14 capsular PSs, respectively, were produced in SJL mice immunized with a mixture of nine Pn PS-CRM 197 conjugates containing serotype 1,4,5, 6B, 9V,14, 18C, 19F,, specific for the serotype 19F PS, was produced in Swiss Webster mice immunized with the same nin...

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supporting

confidence: 72%

Thermodynamics and Density of Binding of a Panel of Antibodies to High-Molecular-Weight Capsular Polysaccharides

Harris

Fernsten

2009

Clin Vaccine Immunol

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“…In Igs, the Fv fragment is composed of a large conserved framework of &sheets and six hypervariable loops denoted L1, L2, and L3 for V, and HI, H2, and H3 for V, (Wu & Kabat, 1970;Poljak et al, 1973). Structural analysis of the binding site of Igs revealed that there are a small number of main-chain conformations found for five of the six hypervariable loops, termed canonical structures (Chothia & Lesk, 1987).…”

Section: The Atomic Model Of the Tcr Moleculementioning

confidence: 99%

Molecular modeling of a T-cell receptor bound to a major histocompatibility complex molecule: Implications for T-cell recognition

et al. 1995

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The main functions of the T-cell receptor (TCR) involve its specific interaction with short and linear antigenic peptides bound to the major histocompatibility complex (MHC) molecules. In the absence of a 3D structure for TCR and for the TCR/peptide/MHC complex, several attempts to characterize the structural components of the TCR/peptide/MHC interaction have been made. However, this subject is still troublesome.In this paper a computer-based 3D model for a TCR/peptide/MHC complex (5C.C7/moth cytochrome c [MCC] peptide 93-103/I-Ek) was obtained. The complex surface shows a high complementarity between the 5C.C7 structure and the peptide/I-Ek molecule. The mapping of residues involved in the TCR/peptide/MHC interaction shows close agreement with mutational experiments (Jorgensen JL, Reay PA, Ehrich EW, Davis MM, 1992b, Annu Rev Immunol10:835-873). Moreover, the results are consistent with a recent variability analysis of TCR sequences using three variability indexes (Almagro JC, Zenteno-Cuevas R, Vargas-Madrazo E, Lara-Ochoa F, 1995b, Int J Pept Protein Res 45:180-186). Accordingly, the 3D model of the 5C.C7/MCC peptide 93-103/I-Ek complex provides a framework to generate testable hypotheses about TCR recognition. Thus, starting from this model, the role played by each loop that forms the peptide/MHC binding site of the TCR is discussed. Keywords: TCR/peptide/MHC interaction complex; variability analysisThe specificity of the immune system is related to two protein families: Igs and TCRs. Igs directly recognize, via their V, and V, domains, the 3D structure of native antigenic proteins in solution (Davies et al., 1990), whereas TCRs recognize, via their V, and V, domains, short antigenic peptides bound to the MHC molecules (Davis, 1990). At present, significant advances have been made in the structural characterization of antigenic recognition mediated by Igs (Davies et al., 1990;Wilson & Stanfield, 1993;Padlan, 1994). In contrast, the nature of the interaction between TCRs and peptide-MHC complexes is poorly understood (Jorgensen et al., 1992b). This is partly due to the Reprint requests to: Juan C. Almagro, Instituto de Quimica, Universidad Nacional Autonoma de Mexico, Circuito Exterior, Ciudad Universitaria, C.P. lack of an experimental 3D structure for TCRs and for TCR/ peptide/MHC complexes.In the absence of 3D structures for TCRs, sequence analysis of their V domains shows that the V,/V, dimer has a framework structure that is very close to the Fv fragment of Igs (Novotny et al., 1986;Chothia et al., 1988). Further comparison has structurally assigned the six hypervariable loops that form the antigen-binding site of Igs to the binding site of TCRs (Chothia et al., 1988), termed a l , a2, and a3 for the V,, and 01, 02, and 03 for the V, in TCRs.Genetically, oll and a 2 are encoded by the V, gene, whereas a 3 is produced by the recombination of an additional gene segment: J a (Davis, 1990). In a similar way 01 and 02 are encoded by the V, gene, whereas 03 is a result of the recombination of two add...

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“…S211 is a myeloma K chain from the LOU rat (11), LEW and DA are sequences from pooled light chains, and M321 is a mouse myeloma K chain (17). The numbering of peptides is given below the sequences.…”

mentioning

confidence: 99%

Structure and regulation of immunoglobulins: kappa allotypes in the rat have multiple amino-acid differences in the constant region.

Gutman¹,

Loh²,

Hood³

1975

Proc. Natl. Acad. Sci. U.S.A.

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Immunoglobulin kappa chains from various inbred strains of rats have two serologically detectable forms that segregate in a Mendelian fashion (allotypes a and b of the RI-1 locus). Partial amino-acid sequences from the constant regions of these two forms have been compared. Of the 81 residues of the constant region studied, 10 amino-acid substitutions as well as one size difference (sequence gap) were found. This large number of sequence differences among alternative forms of the K allotype raises provocative questions as to the genetic and evolutionary implications of these light chain allotypes. We designate allotypes whose alternative forms differ at multiple residue positions as complex allotypes. There are basically two genetic models that might explain complex allotypes. First, these allotypes are alleles of a single structural gene with an unusual evolutionary history. Second, all rats have genes that code for each of the light chain allotypes and a control mechanism that permits them to be expressed so that they mimic a Mendelian pattern of segregation. We discuss evidence from other immunoglobulin systems that is compatible with this second model.The immune system is one of the most complex physiological systems that has been studied at the molecular, genetic, and cellular levels. Fig. 1 with the previously published CK sequences of myeloma (Bence-Jones) K chains from the LOU strain of rat (S211) (9) and the BALB/c strain of mouse (M321) (10). The LEW and LOU strains are of the b serotype, whereas DA belongs to the a serotype. The LEW and DA CK regions differ by 10 residues plus one sequence gap, whereas the LEW and S211 CK regions differ by only two residues. This is a minimum estimate of the total number of differences for several reasons: (i) only 81 of the 108 residues of the C region were compared; (ii) the acid and amide forms of aspartic and glutamic acid were not distinguished; and (iii) the V regions were not examined.These allotype-associated differences are distributed in a nonrandom manner. Ten of the 11 differences occur at positions where the LEW sequence differs from that of the mouse, indicating that certain positions are more likely to accumulate changes than others. The fact that the DA sequence is identical to that of the mouse at five of these positions is a puzzling point which may indicate a more rapid accumulation of changes in the LEW gene. Further, the distribution of the substitutions in the tertiary structure of the light chain is not random. Most of the substitutions lie on the external portion of the polypeptide chain (only position 136 is internal) (ref. 11; R. Poljak, personal communication). Two clusters of differences (one including 153 and 155, the other 184, 185, and 188) are external, and both lie very close to one another in a region already known to encompass the serological markers Oz and Inv, and the sequence marker Kern. Since it is known the RI-1 determinants lie exclusively in the C-region (12), it seems likely that one or more of these external subst...

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Three-Dimensional Structure of the Fab′ Fragment of a Human Immunoglobulin at 2.8-Å Resolution

Cited by 349 publications

References 24 publications

Thermodynamics and Density of Binding of a Panel of Antibodies to High-Molecular-Weight Capsular Polysaccharides

Thermodynamics and Density of Binding of a Panel of Antibodies to High-Molecular-Weight Capsular Polysaccharides

Molecular modeling of a T-cell receptor bound to a major histocompatibility complex molecule: Implications for T-cell recognition

Structure and regulation of immunoglobulins: kappa allotypes in the rat have multiple amino-acid differences in the constant region.

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