Engineering of Three-Finger Fold Toxins Creates Ligands with Original Pharmacological Profiles for Muscarinic and Adrenergic Receptors

Protein engineering approaches are often a combination of rational design and directed evolution using display technologies. Here, we test “loop grafting,” a rational design method, on three-finger fold proteins. These small reticulated proteins have exceptional affinity and specificity for their diverse molecular targets, display protease-resistance, and are highly stable and poorly immunogenic. The wealth of structural knowledge makes them good candidates for protein engineering of new functionality. Our goal is to enhance the efficacy of these mini-proteins by modifying their pharmacological properties in order to extend their use in imaging, diagnostics and therapeutic applications. Using the interaction of three-finger fold toxins with muscarinic and adrenergic receptors as a model, chimeric toxins have been engineered by substituting loops on toxin MT7 by those from toxin MT1. The pharmacological impact of these grafts was examined using binding experiments on muscarinic receptors M1 and M4 and on the α1A-adrenoceptor. Some of the designed chimeric proteins have impressive gain of function on certain receptor subtypes achieving an original selectivity profile with high affinity for muscarinic receptor M1 and α1A-adrenoceptor. Structure-function analysis supported by crystallographic data for MT1 and two chimeras permits a molecular based interpretation of these gains and details the merits of this protein engineering technique. The results obtained shed light on how loop permutation can be used to design new three-finger proteins with original pharmacological profiles

Motions and structural variability within toxins: Implication for their use as scaffolds for protein engineering

Animal toxins are small proteins built on the basis of a few disulfide bonded frameworks. Because of their high variability in sequence and biologic function, these proteins are now used as templates for protein engineering. Here we report the extensive characterization of the structure and dynamics of two toxin folds, the “three-finger” fold and the short α/β scorpion fold found in snake and scorpion venoms, respectively. These two folds have a very different architecture; the short α/β scorpion fold is highly compact, whereas the “three-finger” fold is a β structure presenting large flexible loops. First, the crystal structure of the snake toxin α was solved at 1.8-Å resolution. Then, long molecular dynamics simulations (10 ns) in water boxes of the snake toxin α and the scorpion charybdotoxin were performed, starting either from the crystal or the solution structure. For both proteins, the crystal structure is stabilized by more hydrogen bonds than the solution structure, and the trajectory starting from the X-ray structure is more stable than the trajectory started from the NMR structure. The trajectories started from the X-ray structure are in agreement with the experimental NMR and X-ray data about the protein dynamics. Both proteins exhibit fast motions with an amplitude correlated to their secondary structure. In contrast, slower motions are essentially only observed in toxin α. The regions submitted to rare motions during the simulations are those that exhibit millisecond time-scale motions. Lastly, the structural variations within each fold family are described. The localization and the amplitude of these variations suggest that the regions presenting large-scale motions should be those tolerant to large insertions or deletions

Inhibition of [³H]-Prazosin binding to α_1A-adrenoceptor by MT7, MT1 and chimeric toxins.

<p>Binding experiments were performed by incubating α_1A-AR membrane fractions of receptor with [³H]-Prazosin (1.5 nM) and varying concentrations of toxin at room temperature and overnight. The results are expressed as the ratio of the specific [³H]-Prazosin binding measured with (B) or without toxin (B_o). All experiments were performed at least three times in duplicate.</p

Inhibition of [³H]-NMS binding to hM1 and hM4 receptors by wild-type MT7, MT1 and chimera toxins.

<p>Binding experiments were performed by incubating hM1 (A) or hM4 (B) membrane fractions of receptor with [³H]-NMS (0.5 nM) and varying concentrations of toxin at room temperature and overnight. The total specific binding in each experiment was 1500±300 cpm. The results are expressed as the ratio of the specific [³H]-NMS binding measured with (B) or without toxin (B_o). All experiments were performed at least three times in duplicate.</p

Sequence alignment of the toxins MT7, MT1, MT3 and ρ-Da1a compared to the various chimeras.

<p>The grafted residues of MT1 loop 1 are shown in green with conserved residues in a lighter shade generating the chimera of MT7-1/1. For MT7-1/2, MT7-1/2tip and MT7-1/2top the grafted residues of MT1 loop 2 are shown in cyan (top) and blue (tip) and the conserved residues in a lighter shade. For MT7-1/3, MT7-1/1+3 and MT7-1/2tip+3 the grafted residues of MT1 loop 3 are shown in yellow with the conserved residues in a lighter shade. The two extra mutations on the C-terminal section of MT7-1/1+3 that correspond to residues from MT1 are shown in magenta. Below the space-filling representation on the MT7 structure show the positioning of the grafted loops from MT1.</p

Affinity constants of wild-type MT7, MT1 and chimeric toxins for M1, M4 muscarinic receptors and α_1A-adrenoceptor.

<p>Affinity constants of wild-type MT7, MT1 and chimeric toxins for M1, M4 muscarinic receptors and α_1A-adrenoceptor.</p

Denmotoxin, a three-finger toxin from the colubrid snake Boiga dendrophila (mangrove catsnake) with bird-specific activity

10.1074/jbc.M605850200Journal of Biological Chemistry2813929030-29041JBCH