Structural insights into the mechanism of peptide bivalency — ASN Events
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  3. Structural insights into the mechanism of peptide bivalency

Structural insights into the mechanism of peptide bivalency (#226)

Theo Crawford 1 , Venkatraman Ramanujam 1 , Ben Cristofori-Armstrong 1 , Jen R Deuis 2 , Xinying Jia 1 , Michael Maxwell 1 , Sina Jami 2 , Linlin Ma 3 , Irina Vetter 2 , Mehdi Mobli 1
  1. Centre for Advanced Imaging, The University of Queensland, St Lucia, QLD, Australia
  2. Institute for Molecular Biosciences, The University of Queensland, St Lucia, QLD, Australia
  3. Griffith Institute for Drug Discovery, Griffith University, Nathan, QLD, Australia

Bivalency is a highly advantageous property of biomolecules that results in high target specificity and enhanced binding kinetics (1). Recently there have been several reports of venom peptides, consisting of two independently folded disulfide-rich domains connected by a linker, that display a bivalent mode of action (2-6). These peptides demonstrate remarkable avidity, often described as irreversible binding, despite their component domains individually exhibiting relatively weak binding.

Efforts to engineer bivalency by covalently linking two independent peptides with flexible linkers has generally failed to achieve the enhanced and prolonged pharmacological effect observed in naturally evolved bivalent peptides. The role that the linker plays in favourably positioning the two peptide domains may have a greater impact on bivalency than previously considered.

We have investigated the impact of the linker structure and dynamics of double-knot toxin (DkTx), a venom peptide that activates the transient receptor potential vanilloid 1 (TRPV1) channel, using NMR spectroscopy. Structural studies by NMR spectroscopy can provide high-resolution structural details of pre-organisation of the peptide linker in solution and NMR spin-relaxation can provide direct measures of disorder.

The solution structure of DkTx reveals residual order in the peptide linker attributed to tyrosine stacking between the first domain and the linker. Disruption of this aromatic stacking increases linker disorder, observable in NMR spin-relaxation experiments. Increasing linker disorder has a direct effect on target engagement, resulting in a decrease in potency and loss of avidity. Interestingly, the loss of avidity can be overcome by applying the peptide at higher concentrations potentially overcoming the loss of domain pre-orientation bias.

Future engineering of novel bivalent peptides may benefit from reduced linker disorder, maximising the potential of simultaneous two-site interactions. Exploring linkers of naturally evolved bivalent peptides may be one avenue to generate linkers capable of achieving enhanced and prolonged pharmacological effects.

  1. Vauquelin G, Charlton SJ. Exploring avidity: understanding the potential gains in functional affinity and target residence time of bivalent and heterobivalent ligands. Br J Pharmacol. 2013;168(8):1771-85.
  2. Bohlen CJ, Priel A, Zhou S, King D, Siemens J, Julius D. A bivalent tarantula toxin activates the capsaicin receptor, TRPV1, by targeting the outer pore domain. Cell. 2010;141(5):834-45.
  3. Chassagnon IR, McCarthy CA, Chin YK, Pineda SS, Keramidas A, Mobli M, et al. Potent neuroprotection after stroke afforded by a double-knot spider-venom peptide that inhibits acid-sensing ion channel 1a. Proc Natl Acad Sci U S A. 2017;114(14):3750-5.
  4. Maxwell MJ, Thekkedam C, Lamboley C, Chin YK, Crawford T, Smith JJ, et al. A bivalent remipede toxin promotes calcium release via ryanodine receptor activation. Nat Commun. 2023;14(1):1036.
  5. van de Locht A, Lamba D, Bauer M, Huber R, Friedrich T, Kroger B, et al. Two heads are better than one: crystal structure of the insect derived double domain Kazal inhibitor rhodniin in complex with thrombin. EMBO J. 1995;14(21):5149-57.
  6. van de Locht A, Stubbs MT, Bode W, Friedrich T, Bollschweiler C, Hoffken W, et al. The ornithodorin-thrombin crystal structure, a key to the TAP enigma? EMBO J. 1996;15(22):6011-7.
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