Reprogramming Control of an Allosteric Signaling Switch Through Modular Recombination

Abstract

Many eukaryotic signaling proteins are composed of simple modular binding domains, yet they can display sophisticated behaviors such as allosteric gating and multi-input signal integration, properties essential for complex cellular circuits. To understand how such behavior can emerge from combinations of simple domains, we engineered variants of the actin regulatory protein N-WASP (neuronal Wiskott-Aldrich syndrome protein) in which the "output" domain of N-WASP was recombined with heterologous autoinhibitory "input" domains. Synthetic switch proteins were created with diverse gating behaviors in response to nonphysiological inputs. Thus, this type of modular framework can facilitate the evolution or engineering of cellular signaling circuits.

Design of a directed molecular network

“…This constraint is evident particularly in the context of the highly evolved isotropic biological networks where substantial mutations, protein deletions, or network tampering can often result in the disruption of vital cellular functions. Therefore, as an alternative possibility, we reasoned that rationally designed synthetic self-organized molecular systems might provide useful model networks for the study and better understanding of complex system behavior (18)(19)(20)(21)(22)(23). In this account we describe the rationale, de novo design, graph estimation, and analyses of a relatively simple synthetic molecular system that seem to bear many of the basic properties one commonly ascribes to complex self-organized networks.…”

Switching desaturase enzyme specificity by alternate subcellular targeting

“…3); the interchangeable tissue-specific roles of the transcription factors WER and GL1 in plant epidermal hair development (4); the modification of laccase͞peroxidase activity in the extracellular matrix by specific dirigent proteins (reviewed in ref. 5); the altered gating properties of the actin regulatory protein N-WASP associated with different ''input'' domains (6); or the alternative responses to steroid hormones, which depend on the cell-type distribution of various steroid receptors (reviewed in ref. 7).…”

Synthetic modular systems – reverse engineering of signal transduction

“…Creating such gating function in chimeric proteins would be useful both to provide exquisite control over their activation, and also to explore the properties and evolution of physiological circuits. The first step towards this goal was recently made by Lim and colleagues [45], who created chimeric proteins focused on a region (VCA) from the N-Wasp polypeptide which binds the Arp2/3 complex and thereby promotes branching actin polymerization. By flanking the VCA region with interaction domains (i.e., SH3, PDZ) and their peptide ligands, they created a working, dual input, synthetic protein switch, refer to Fig.…”

“…By flanking the VCA region with interaction domains (i.e., SH3, PDZ) and their peptide ligands, they created a working, dual input, synthetic protein switch, refer to Fig. 2 [45]. The regulation of this switch is based on autoinhibitory intramolecular interactions that are alleviated by competing binding events, in the form of exogenous peptides.…”