De Novo Design of Peptides Targeted to the EF Hands of Calmodulin

Abstract

This report describes the use of the concept of inversion of hydropathy patterns to the de novo design of peptides targeted to a predetermined site on a protein. Eight-and 12-residue peptides were constructed with the EF hands or Ca 2؉ -coordinating sites of calmodulin as their anticipated points of interaction. These peptides, but not unrelated peptides nor those with the same amino acid composition but a scrambled sequence, interacted with the two carboxyl-terminal Ca 2؉ -binding sites of calmodulin as well as the EF hands of troponin C. The interactions resulted in a conformational change whereby the 8-mer peptide-calmodulin complex could activate phosphodiesterase in the absence of Ca 2؉ . In contrast, the 12-mer peptide-calmodulin complex did not activate phosphodiesterase but rather inhibited activation by Ca 2؉ . This inhibition could be overcome by high levels of Ca 2؉ . Thus, it would appear that the aforementioned concept can be used to make peptide agonists and antagonists that are targeted to predetermined sites on proteins such as calmodulin.Accumulating evidence suggests that a simple binary code of polar and nonpolar amino acids arranged in the appropriate order is sufficient to build helical bundle structures and artificial peptides with rudimentary function (for review see Ref. 1). Therefore, only the sequence location, not the identity, of the polar and nonpolar amino acids must be explicitly specified for the formation of a stable helical structure or biologically active peptide. Such binary coding has been successfully employed to produce biologically active analogs of corticotrophin and growth hormone-releasing hormone (2, 3), to design proteins that fold into compact ␣-helical bundles (4), and to develop computer programs that simulate or predict some aspects of protein folding (5). Considering that 20 different amino acids are encompassed by the binary code, one would expect a marked degree of sequence degeneracy for a given shape, since any one of a number of specific polar or nonpolar amino acids could occupy a given position in the sequence. Indeed, experimental evidence has confirmed that gross shape is degenerate with regard to sequence in that any number of different primary amino acid sequences with the same binary code can fold into compact ␣-helical structures (4).More recent studies have shown that, unlike simple helical structures, a five-letter amino acid alphabet is minimally required to build a well ordered, ␤-sheet containing protein architecture (6). In this particular instance, a small ␤-sheet protein, the SH3 domain, could be constructed with 95% of the residues being Ile, Lys, Glu, Ala, and Gly. Interestingly, the pattern of hydropathy of the wild type SH3 domain was largely maintained in the two sequence-simplified structures.1 This would seem once again to point to the importance of the pattern of hydropathy in building more complex structures as well as simple helical ones.If, as described above, the gross architecture of a peptide or protein is in part determined by ...

Specific modulation of calmodulin activity induces a dramatic production of superoxide by alveolar macrophages

“…14 We recently described a 12-mer peptide CALP2 with all characteristics necessary to define the role of CaM in the production of radicals by inflammatory cells. 15,16 The peptide binds EF hand motifs of the calcium-binding proteins CaM and troponin C. Moreover, we showed that CALP2 inhibited the action of CaM, 15,17 but increased [Ca 2 þ ] i in airway epithelial cells, the latter being accompanied by an increased NO production by these cells. 18 Moreover, the effects of CALP2 were likely due to the sustained opening of calcium channels present in epithelial cells.…”

“…Furthermore, we earlier showed that CALP2 did not bind to CaM in the hydrophobic core, which is typical for W7, 66 but rather interferes with the carboxyl-terminal calcium binding site of CaM. 15 Moreover, it was recently shown that, depending on the protein bound to CaM, CaM undergoes marked different conformational changes, leading to changed calcium-binding. 67 It is therefore assumed that W7 inhibits the action of CALP2 through a different inhibitory mechanism on CaM, leading to altered conformational changes.…”

“…In the present study, we showed that the calcium-regulatory protein CaM is essential in radical production by alveolar macrophages. To address this issue, the selective CaM inhibitor CALP2 15 was used, and we found that CALP2 is a very potent activator of macrophages. Interestingly, we have shown that CALP2 only slightly activates neutrophils, whereas eosinophils were not activated at all.…”

“…15 Interestingly, this resulted in an increase of [Ca 2 þ ] i . 18 Therefore, we hypothesized that the action of CALP2 on superoxide production by alveolar macrophages is due to the inhibition of CaM activation and therefore an increase of [Ca 2 þ ] i in these cells.…”

Attenuation of Very Late Antigen-5-Mediated Adhesion of Bone Marrow-Derived Mast Cells to Fibronectin by Peptides with Inverted Hydropathy to EF-Hands

“…Interestingly, enhanced affinity by increased reciprocity of the pattern of hydropathy and increased length of the peptide resulted in a change of functional activity. Whereas CALP1 shows biological effects similar to Ca 2ϩ , CALP2 acts as an antagonist for CaM (26). A possible role for Ca 2ϩ influx and CaM activation in Fc⑀RI-induced affinity modulation of VLA-5 for fibronectin combined with the assumption that, based on the pattern of hydropathy, the EF-hand-like domains of VLA-5 are potential binding sites for CALPs suggest several targets for these peptides to interfere with mast cell adhesion.…”