WWamides

Definition

Wwamides are biologically active heptapeptide amides that were originally isolated from ganglia of the African giant snail, Achatina fulica.

Discovery

H.Minakata et al., in 1993 isolated WWamide-1, -2 and -3 novel neuromodulatory peptides from ganglia of the African giant snail, Achatina fulica 1. Fu Y et al., in 2006 reported backbone perturbations in a β-sheet protein employing A-to-E and A-to-O mutations. Both mutations, deleting the same H-bond in the hydrophobic core, lead to a pronounced decrease in protein stability. The folding free energies of the ester and olefin mutants, together with the transfer free energies measured on relevant model compounds, afford an estimation of 0.3 kcal/mol for the O-O electrostatic repulsion term in the context of a β-sheet H-bond network. The determined value of ΔGO-Orep should enable more accurate H-bond strength measurements utilizing amide-to-ester mutations. From this data, the H-bond between F23 and R14 in the Pin WW domain is determined to be worth 1.3 kcal/mol 2. Structural thermodynamic parameters are calculated for aqueous solutions of secondary amides of carboxylic acids. Specific and nonspecific contributions to the total energy of intermolecular interactions are determined and the boundaries of concentration regions for a various structural organization of solutions are found 3.

Structural Characteristics

Backbone hydrogen bonds (H-bonds) are prominent features of protein structures; however, their role in protein folding remains controversial because they cannot be selectively perturbed by traditional methods of protein mutagenesis. Authors have assessed the contribution of backbone H-bonds to the folding kinetics and thermodynamics of the PIN WW domain, a small -sheet protein, by individually replacing its backbone amides with esters. Amide-to-ester mutations site-specifically perturb backbone H-bonds in two ways: a H-bond donor is eliminated by replacing an amide NH with an ester oxygen, and a H-bond acceptor is weakened by replacing an amide carbonyl with an ester carbonyl. They perturbed the 11 backbone H-bonds of the PIN WW domain by synthesizing 19 amide-to-ester mutants. Thermodynamic studies on these variants show that the protein is most destabilized when H-bonds that are enveloped by a hydrophobic cluster are perturbed. Kinetic studies indicate that native-like secondary structure forms in one of the protein's loops in the folding transition state, but the backbone is less ordered elsewhere in the sequence.  Aminoacid sequence of WWamide-1 is H-Trp-Lys-Glu-Met-Ser-Val-Trp-NH2. WWamide-2 sequence is H-Trp-Arg-Glu-Met-Ser-Val-Trp-NH2 andthat of WWamide-3 is H-Trp-Lys-Gln-Met-Ser-Val-Trp-NH2 1.

Mode of Action

The synthetic WWamide-1 displayed an inhibitory activity on a central neuron of the snail and exhibited peripherally modulatory effects on muscular contractions in various tissues of the snail and other molluscs1. It has been proposed that an amide-to-E-olefin (A-to-O) mutation is the ideal peptide bond perturbation.  An A-to-O mutation in a protein eliminates one H-bond donor (NH) and one H-bond acceptor (CO) without introducing electrostatic repulsions. However, this strategy has rarely been realized due to the difficulties associated with stereospecific synthesis of alkene-containing isosteres and incorporating them into proteins. Recently, our group has reported a convenient protocol for the preparation of the Phe-Phe E-olefin dipeptide isostere and its incorporation into proteins 4.   Perturbation of the Phe22-Phe23 amide bond in the Pin WW domain employing both A-to-O and A-to-E mutations (energetic comparison of the ester mutant and E-olefin mutant enables to quantify the repulsive O-O interaction introduced by A-to-E mutations and to establish the H-bond energy. Fumihiro M et al.,in 2006 identified in the animal a tachykinin-like peptide (ThTK), two FRFamides and a WWamide. ThTK induced contractions in the esophagus, prostate gland and capsule gland, while FRFamide and WWamide had an inhibitory action on tissues. Immunostaining using specific antibodies demonstrated the distribution of these peptides in the central nervous system.  

Functions

Inhibitory activity, the synthetic WWamide-1 displayed an inhibitory activity on a central neuron of the snail 1.

Iso-structural moiety, since backbone hydrogen bonds are formed between main chain amides, they can be perturbed by replacing the amide bond of interest in a protein with an iso-structural moiety which has reduced or lacks H-bonding capacity 2.

An amide-to-ester (A-to-E) mutation eliminates the H-bond donor (N-H) and weakens the H-bond acceptor (C=O). A-to-E mutations are conservative in that the trans conformation of the linkage is maintained, as well as the, ψ dihedral angle preference of the flanking substructure 2.

Neural regulation, Fumihiro M et al suggested that FRFamide and WWamide peptides mediate the neural regulation of motility in reproductive organs.

References

Minakata H, Ikeda T, Muneoka Y, Kobayashi M, Nomoto K (1993). WWamide-1, -2 and -3: novel neuromodulatory peptides isolated from ganglia of the African giant snail, Achatina fulica. FEBS Lett., 323(1-2):104-108.

2.Fu Y, Gao J, Bieschke J, Dendle MA, Kelly JW (2006). Amide-to-E-olefin versus amide-to-ester backbone H-bond perturbations: Evaluating the O-O repulsion for extracting H-bond energies.  J Am Chem Soc., 128(50):15948–15949.

Zaichikov AM (2007). Structural thermodynamic parameters and intermolecular interactions in aqueous solutions of secondary amides. Journal of Structural Chemistry, 48(1):94-103.

Fu Y, Bieschke J, Kelly JW (2005).  E-olefin dipeptide isostere incorporation into a polypeptide backbone enables hydrogen bond perturbation: probing the requirements for Alzheimer's amyloidogenesis. J. Am. Chem. Soc., 127(44):15366-15367.