Today I will briefly discuss one of the landmark papers in protein dynamics. This paper is called Reprogramming Control of an Allosteric Signaling Switch Through Modular Recombination, by John Dueber and Wendell Lim from UCSF, published in Science in 2003.
Allosteric signaling switch has been a focus in synthetic biology, as proteins that belong to the signaling pathways are often regulated by allosteric gating mechanisms. Some of these mechanisms are governed by simple binding domains, in which the switch is under autoinhibited state when proper ligand is absent. In this paper, Dueber and his group proposed that combinations of domains could give rise to a diversified number of gating behaviours in response to nonphysiological inputs. This story is highly significant because their results indicate that simple catalytic and interaction domains within a single polypeptide could give rise to different proteins of complex properties. It also advances our knowledge to how proteins of complex functions could be derived.
To approach the hypothesis, Dueber had developed two key major experimental procedures: (i) switch proteins; and (ii) actin polymerization assays: pyrene-labeled and carboxylated polystyrene bead. There were three types of switch proteins developed: single heterologous ligand; chimeric switch; and heterlogous switch. To develop switch proteins, the ligand motifs and the linker region were PCR-amplified from existing plasmids. These proteins were then expressed with an affinity tag (either 6xHis tag or GST tag), followed by purification and tag removal by Ni beads and TEV protease or glutathione agarose resin. Next, actin polymerization assays were performed. In the case of pyrene-labeled actin assays, pyrene-labeled actin was incubated with Arp2/3, switch proteins and ligand proteins to initiate polymerization. Then the rate of actin polymerization could be measured by fluorescene measurement. Next, bead actin polymerization assay was performed. Carboxylated polystyrene beads were coated with GST fusions to input ligands and PDZ ligand. After incubation, the beads were washed and incubated with Xenopus oocyte extractsand rhodamine-labeled actin, followed by microscopic analysis on rhodamine.
Dueber developed a signaling switch gated by single ligand. This switch was consisted of N-WASP output domain, GBD and B-motif. At the autoinhibited state, GBD and B motif interacted with output and Arp2/3 to suppress output to interact with actin filament. To initiate actin polymerization, Cdc42 and PIP2 bind to GBD and B motif respectively to relieve autoinhibition. This model was consistent with the observation that increasing PDZ ligands could activate the switch. For the second designs, they developed AND-gate switches by linking two domains with an output. There were two classes of designs developed: chimeric and heterlogous switches. Chimeric switch design was regulated by PDZ ligand and Cdc42. The modular domains of this particular design were PDZ and GBD domains, in which Cdc42 could disrupt interaction of GBD and output. Heterologous switch was regulated by PDZ and SH3 ligands. The modular domains were composed of PDZ and SH3 domains. Both classes of switches were subdivided into behavioural classes. Out of 34 switches, 2 showed antagonistic gating, 2 showed OR gate behaviour, and 5 showed AND gate behaviour. From analyzing the behaviours, the basic design principle was identified, in which the affinity of autoinhibition was correlated with the basal repression and input sensitivity. This principle was supported by 2 observations: (1) reducing the PDZ ligand-affinity for switch C11, the switch then resembled AND gate; and (2) increasing PDZ ligand affinity turned H15 switch from behaving like OR gate to AND gate. Their results also indicated the importance of linker region to switch behaviour, as the increase of linker region from 5 to 20 residues could increase the sensitivity of inputs. Furthermore, in the case of antagonistic switches, PDZ ligand was found to act as an activator while SH3 increased the basal level of repression. PDZ was responsible for the autoinhibitory interaction with output, and SH3 interaction was responsible for destabilizing the PDZ interaction. In the case of positive integrating switches, both domains worked together to downregulate the output activity. Interestingly, disruption of both was required to activate the output function.
Dueber and his group have effectively addressed the main question. The results from simple binding domains and corresponding ligands had supported the notion that complex signaling circuits could be derived from simple domains, and more importantly, domain recombination could potentially derive proteins with novel regulated functions. For future direction, the geometry of the output domain must be investigated. In order for the output domain to be regulated by other domains and to carry its native function, certain geometry must be preserved. To understand the preservation, X-ray crystallography and mass spectroscopy should be conducted on samples: output domain bound to GBD and output domain bound to actin filament.