The three-dimensional structures of alpha-helices can be represented by two-dimensional projections which we call helical wheels. Initially, the wheels were employed as graphical restatements of the known structures determined by Kendrew, Perutz, Watson, and their colleagues at the University of Cambridge and by Phillips and his coworkers at The Royal Institution. The characteristics of the helices, discussed by Perutz et al. (1965), and Blake et al. (1965), can be readily visualized by examination of these wheels. For example, the projections for most helical segments of myoglobin, hemoglobin, and lysozyme have distinctive hydrophobic arcs. Moreover, the hydrophobic residues tend to be clustered in the n +/- 3, n, n +/- 4 positions of adjacent helical turns. Such hydrophobic arcs are not observed when the sequences of nonhelical segments are plotted on the wheels. Since the features of these projections are also distinctive, however, the wheels can be used to divide sequences into segments with either helical or nonhelical potential. The sequences of insulin, cytochrome c, ribonuclease A, chymotrypsinogen A, tobacco mosaic virus protein, and human growth hormone were chosen for application of the wheels for this purpose.

Crystal structures of the Fabs from an autoantibody (BV04-01) with specificity for single-stranded DNA have been determined in the presence and absence of a trinucleotide of deoxythymidylic acid, d(pT)3. Formation of the ligand-protein complex was accompanied by small adjustments in the orientations of the variable (VL and VH) domains. In addition, there were local conformational changes in the first hypervariable loop of the light chain and the third hypervariable loop of the heavy chain, which together with the domain shifts led to an improvement in the complementarity of nucleotide and Fab. The sugar-phosphate chain adopted an extended and "open" conformation, with the base, sugar, and phosphate components available for interactions with the protein. Nucleotide 1 (5'-end) was associated exclusively with the heavy chain, nucleotide 2 was shared by both heavy and light chains, and nucleotide 3 was bound by the light chain. The orientation of phosphate 1 was stabilized by hydrogen bonds with serine H52a and asparagine H53. Phosphate 2 formed an ion pair with arginine H52, but no other charge-charge interactions were observed. Insertion of the side chain of histidine L27d between nucleotides 2 and 3 resulted in a bend in the sugar-phosphate chain. The most dominant contacts with the protein involved the central thymine base, which was immobilized by cooperative stacking and hydrogen bonding interactions. This base was intercalated between a tryptophan ring (no. H100a) from the heavy chain and a tyrosine ring (no. L32) from the light chain. The resulting orientation of thymine was favorable for the simultaneous formation of two hydrogen bonds with the backbone carbonyl oxygen and the side chain hydroxyl group of serine L91 (the thymine atoms were the hydrogen on nitrogen 3 and keto oxygen 4).

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The crystal structure of a fluorescein-Fab (4-4-20) complex was determined at 2.7 A resolution by molecular replacement methods. The starting model was the refined 2.7 A structure of unliganded Fab from an autoantibody (BV04-01) with specificity for single-stranded DNA. In the 4-4-20 complex fluorescein fits tightly into a relatively deep slot formed by a network of tryptophan and tyrosine side chains. The planar xanthonyl ring of the hapten is accommodated at the bottom of the slot while the phenylcarboxyl group interfaces with solvent. Tyrosine 37 (light chain) and tryptophan 33 (heavy chain) flank the xanthonyl group and tryptophan 101 (light chain) provides the floor of the combining site. Tyrosine 103 (heavy chain) is situated near the phenyl ring of the hapten and tyrosine 102 (heavy chain) forms part of the boundary of the slot. Histidine 31 and arginine 39 of the light chain are located in positions adjacent to the two enolic groups at opposite ends of the xanthonyl ring, and thus account for neutralization of one of two negative charges in the haptenic dianion. Formation of an enol-arginine ion pair in a region of low dielectric constant may account for an incremental increase in affinity of 2-3 orders of magnitude in the 4-4-20 molecule relative to other members of an idiotypic family of monoclonal antifluorescyl antibodies. The phenyl carboxyl group of fluorescein appears to be hydrogen bonded to the phenolic hydroxyl group of tyrosine 37 of the light chain. A molecule of 2-methyl-2,4-pentanediol (MPD), trapped in the interface of the variable domains just below the fluorescein binding site, may be partly responsible for the decrease in affinity for the hapten in MPD.

We used mapping with synthetic overlapping peptides in combination with molecular modeling to analyze the IgG antibodies that humans naturally produce against human T-cell receptor .8 chains and to localize the recognized peptide autoantigens in the three-dimensional structure of the molecule. Healthy individuals produce low levels of antibodies against T-cell receptor peptides, and these can be increased in autoimmune diseases. We characterized the reactivities in detail because IgG molecules reactive with self peptides occur in preparations of intravenous immunoglobulin and can be isolated by immunoaffinity chromatography. Natural IgG antibodies were directed against three major peptides. One corresponds to the first complementarity-determining region of the variable region. A second corresponds to the third framework of the variable region. The third is located in the constant region and is predicted to be a loop that extends out of the 18-barrel structure. This peptide is one that would give a characteristic structural distinction between the fl-chain constant region and the constant regions of immunoglobulin light chains to which 13 chains are homologous. The capacity to bind these peptides is found in small fractions of normal polyclonal IgG, which contains both Kc chains and A chains. The activity is antibody-like in being confined to the Fab fragment and in its capacity to discriminate among homologous synthetic peptides corresponding to distinct f-chain variable-region genes.We propose that a recognition and regulatory process naturally occurs that parallels the immune network for the regulation of the production of antibodies.Humans produce antibodies to a variety of self antigens without noticeable ill effects (1-4). The production of antibodies themselves is regulated in part by autoimmune responses to defined portions of the antibodies, most notably combining site-related markers or idiotypes (5, 6) and constant-region markers detected by IgM antibodies termed rheumatoid factors (7). Because of the crucial importance of immunoglobulin-like T-cell receptors (TCRs) in the initiation of specific immunity, we chose to focus on natural human antibodies to synthetic peptides based on human TCR /3-chain gene sequence. We assessed by enzyme-linked immunosorbent assay (ELISA) the capacity ofsera to react with synthetic TCR peptide autoantigens. These sera were obtained from ostensibly healthy individuals 20-90 years of age and from patients with rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE). Hexadecapeptides representing the TCR YT35 ,3 chain, which contains V,8, D1.1, J01.2, and Cp1 segments (8), were overlapped by 5 residues to model the antigenic structures (9, 10) of the TCR P fragment homologous to immunoglobulin A light chain (11).We found that reaction of human IgG with particular synthetic TCR autoantigens occurs in low levels in healthy individuals and can rise in autoimmune diseases such as RA. We present immunochemical data characterizing the nature and locations of the T...

Antigen-specific IgG Abs 1 in autoimmune and alloimmune disease are described to catalyze chemical reactions (1-3). Examples of catalytic Abs raised by routine experimental immunization with ordinary antigens have also been published (4 -7). However, no consensus has developed whether naturally occurring catalytic Abs represent rare accidents arising from adaptive sequence diversification processes or genuine enzymes with important functional roles. The major reason is that the turnover (k cat ) of antigen-specific IgG Abs is low. Some catalytic Abs express catalytic efficiencies (k cat /K m ) comparable to conventional enzymes, but this is due to high affinity recognition of the antigen ground state (reviewed in Ref. 8).Certain enzymes cleave peptide bonds by a mechanism involving the formation of a transient covalent intermediate of the substrate and a nucleophilic residue present in the active site. The nucleophiles are generated by intramolecular activation mechanisms, e.g. the activation of Ser/Thr side chain hydroxyl groups by hydrogen bonding to His residues, and can be detected by covalent binding to electrophilic phosphonate diesters (9, 10). Using these compounds as covalently reactive analogs of antigens (CRAs), we observed that IgG Abs express nucleophilic reactivities comparable to trypsin (11). Despite their nucleophilic competence, IgG Abs display low efficiency proteolysis, presumably due to deficiencies in steps occurring after formation of the acyl-Ab intermediate, viz., water attack on the intermediate and product release. Occupancy of the B cell receptor (BCR, surface Ig complexed to ␣ and ␤ subunits along with other signal transducing proteins) by the antigen drives B cell clonal selection. Proteolysis by the BCR is compatible with clonal selection, therefore, only to the extent that the release of antigen fragments is slower than the rate of antigeninduced transmembrane signaling necessary for induction of cell division. Immunization with haptens mimicking the charge characteristics of the transition state (12) has been suggested as a way to surmount the barrier to adaptive improvement of catalytic rate constants. Catalysis by designer IgG Abs obtained by these means, however, also proceeds only slowly.In mice and humans, the initial Ab repertoire consists of ϳ100 heritable VL and VH genes. Adaptive maturational processes expand the repertoire by several orders of magnitude. The initial BCR complex on the pre-B cell surface contains V-(D)-J rearranged Ig chains as a complex with surrogate L chains (reviewed in Ref. 13). Precise assignment of the B cell differentiation stage at which cell division becomes antigen-dependent is somewhat ambiguous, but it is generally believed that non-covalent antigen binding to the pre-BCR is not required for initial cell growth. / chains replace the surrogate L chain at the later stages of antigen-driven B cell differentiation, which is accompanied by diversification via somatic hypermutation processes and continued gene rearrangements (14,15). V-(D)-J gene ...

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