Tropomyosin has discrete actin-binding sites with sevenfold and fourteenfold periodicities.

Analysis of the periodic distribution of amino acids in tropomyosin has revealed the presence of seven or 14 quasi-equivalent actin-binding sites. We tested the hypothesis of periodic actin-binding sites by making deletions of chicken striated alpha-tropomyosin cDNA using oligonucleotide-directed mutagenesis. The deletions corresponded to one-half (amino acid residues 47 to 67), two-thirds (residues 47 to 74) and one actin-binding site (residues 47 to 88), on the basis of there being seven sites. The mutant cDNAs were expressed as fusion and non-fusion proteins in Escherichia coli and analyzed for actin binding and regulatory function. Fusion tropomyosin binds to actin with an affinity similar to that of muscle tropomyosin. Of the mutant fusion tropomyosins, only that with a full site deleted retained actin affinity and the ability to inhibit the actomyosin S1 ATPase, though it was less effective than wild-type. We conclude that an integral number of half-turns of the tropomyosin coiled-coil, and the consequential sevenfold periodicity, as well as the correct orientation of the ends with respect to each other, are important for actin binding. On the other hand, non-fusion tropomyosin binds well to actin only in the presence of troponin, and the binding is calcium-sensitive. Assay of non-fusion mutant tropomyosins showed that mutants with deletion of one-half and one actin binding site both had high affinity for actin, equal to or slightly less than wild-type. The ability of these two mutants to regulate the actomyosin or acto-S1 ATPase with troponin in the absence of calcium was indistinguishable from that of the wild-type. The normal regulatory function of the mutant with a 1/14 deletion (removal of a quarter turn or half a site) indicates that a 14-fold periodicity is adequate for regulation, consistent with the presence of two sets of seven alpha and seven beta quasi-equivalent actin-binding sites. An alternative explanation is that the alpha-sites are of primary importance and that proper alignment of the alpha-sites in every second tropomyosin, as when half a site is deleted, is sufficient for normal regulatory function. Deletion of a non-integral period (2/3 of a site) severely compromised actin-binding and regulatory function, presumably due to the inability of the mutant to align properly on the actin filament.

Interleukin-1 (IL-1) receptors on murine splenic B cells and the 70Z/3 pre-B-cell line appear to be identical and bind human IL-1 alpha and IL-1 beta differentially.

In this study murine splenocytes were found to possess specific, high-affinity IL-1 receptors (IL-1R) capable of binding radiolabeled human IL-1 alpha with a Kd of 2-6 x 10(-10) M. Experiments performed with purified splenic B cells demonstrated that B cells express low levels of IL-1R (100-200 receptors per cell). Separation of splenic B cells into high- (resting) and low-density (in vivo-activated) fractions showed that low-density B cells expressed 2-fold more IL-1R compared with high-density B cells suggesting that IL-1R are upregulated on B-cell activation. In vitro stimulation of B cells with mitogens resulted in a 5- to 10-fold increase in IL-1R expression compared to IL-1R levels on unstimulated B cells. Receptors on the murine pre-B-cell line 70Z/3, which possesses type II IL-1R, and murine splenic B cells appear to be identical. Cross-linking studies demonstrated that the 125I-labeled IL-1/IL-1R complex on B cells and 70Z/3 IL-1R was found to block binding of IL-1 to IL-1R on 70Z/3 and splenic B cells, but not to type I IL-1R on murine EL4 thymoma cells. Competitive inhibition experiments showed differential binding of human IL-1 beta to splenic B cells and 70Z/3 cells as a function of temperature. The IL-1R on 70Z/3 and splenic B cells bound human IL-1 alpha, murine IL-1 alpha, and murine IL-1 beta with high affinity at both 4 degrees and 37 degrees C. In contrast, the affinity of IL-1R on these same cell types for human IL-1 beta was significantly reduced at 37 degrees C compared to 4 degrees C. The reduced binding affinity for human IL-1 beta was due to an increased off-rate at 37 degrees C compared with 4 degrees C. Characterization of IL-1R on murine splenic B cells will help clarify the role of IL-1 in the regulation of the immune response.

Structure-function analysis of human IL-1 alpha: identification of residues required for binding to the human type I IL-1 receptor.

Using oligonucleotide-directed mutagenesis, the binding site on human interleukin-1 alpha (IL-1 alpha) for the human type I IL-1 receptor (IL-1R) has been analyzed. Substitution of seven amino acids (Arg12, Ile14, Asp60, Asp61, Ile64, Lys96 and Trp109) resulted in a significant loss of binding to the receptor. Based on crystallographic information, the side chains of these residues are clustered in one region of IL-1 alpha and exposed on the surface of the protein. Five of the residues in the IL-1 alpha binding site align with the binding residues previously determined in human IL-1 beta, demonstrating that the type I IL-1R recognizes homologous regions in both ligands. Unexpectedly, only three of the aligned residues are identical between IL-1 alpha and IL-1 beta. These observations suggest that the composition of contact residues in the binding site is unique for each ligand-receptor complex in the IL-1 system.