Scaffold proteins: hubs for controlling the flow of cellular information

Abstract

The spatial and temporal organization of molecules within a cell is critical for coordinating the many distinct activities carried out by the cell. In an increasing number of biological signaling processes, scaffold proteins have been found to play a central role in physically assembling the relevant molecular components. Although most scaffolds use a simple tethering mechanism to increase the efficiency of interaction between individual partner molecules, these proteins can also exert complex allosteric control over their partners and are themselves the target of regulation. Scaffold proteins offer a simple, flexible strategy for regulating selectivity in pathways, shaping output behaviors, and achieving new responses from preexisting signaling components. As a result, scaffold proteins have been exploited by evolution, pathogens, and cellular engineers to reshape cellular behavior.

Fig. 1. Scaffold proteins organize cellular information flow

A) Spatial organization is necessary to achieve high fidelity intracellular information transfer. Proteins can be assembled into specific complexes by compartmentalization (organelle targeting), membrane localization, and by scaffold proteins. B) Intracellular signaling pathways often use scaffold proteins. Canonical examples include Ste5, essential to the yeast mating MAPK pathway, and KSR, which directs signaling in the mammalian Ras-Raf-MEK-MAPK pathway. C) Scaffold proteins also play an important role in organizing cell-cell communication junctions, such as neuronal synapses. The PDZ scaffold, PSD-95, controls NMDA and AMPA glutamate receptor targeting to the synapse. D) Assembly line processes such as protein folding use scaffold proteins. The HOP protein promotes transfer of unfolded proteins between Hsp70 and Hsp90 chaperones.

Fig. 2. Scaffold proteins can mediate pathway regulation and feedback to shape complex signaling responses

A) Scaffold proteins are analogous to circuit boards - modular platforms that wire together components and direct the flow of information - and can program complex signaling behaviors. B) Scaffold proteins function to wire pathway input and output through alternative possible routes. C) Scaffold proteins can mediate branching of pathways to multiple outputs. D) Scaffold proteins are themselves the targets of regulation. In T-cell signaling, activation of the T-cell receptor causes phosphorylation of the LAT and Slp76 scaffolds by Zap70, and phosphorylation-dependent recruitment of substrates leads to PLCγ activation and PIP2 hydrolysis. E) Scaffold proteins can be the target of feedback phosphorylation that tunes pathway responses. Feedback phosphorylation of the KSR scaffold by activated ERK blocks Raf (MAPKKK) binding and attenuates MEK activation, thereby decreasing pathway output. Plot adapted from (24).

Fig. 3. Benefits and Costs of Scaffold Tethering Mechanisms

A) By co-localizing enzyme and substrate, scaffold proteins can lower the entropic cost of signaling interactions – the loss of independent translational and rotational degrees of freedom is paid through binding interactions with the scaffold. The size of the advantage gained depends on the flexibility of the scaffold structure. B) By restricting the conformational freedom of interacting proteins, scaffolds can orient these molecules to enhance the rate of signal transfer. The rigid Cullin scaffold proteins tether E2 ubiquitin conjugating enzymes and their substrates. If the Cullin backbone is made flexible by mutation, the rate of substrate ubiquitination is greatly decreased. C) Tethering has potential drawbacks: at high concentrations scaffolds may titrate enzyme and substrate away from one another. D) Increased affinities can restrict substrate release and diffusion throughout the cell, potentially limiting signal amplification and spatial redistribution (e.g. nuclear localization).

Fig. 4. Allosteric regulation by scaffold proteins

A) Scaffolds can allosterically modulate the conformation of enzymes and substrates to gate information flow. B) In MAPK ERK signaling, KSR can directly bind to the MAPKKK Raf and influence its activity toward the MAPKK MEK. The pseudokinase domain of KSR dimerizes with Raf, altering the conformation of the C-helix on Raf so that its kinase domain becomes catalytically active (thereby allowing Raf to phosphorylate MEK). C) The VWA domain of Ste5 promotes phosphorylation of the MAPK Fus3 by the MAPKK Ste7. The scaffold may unlock the activation loop of the MAPK Fus3 to make it a better substrate for MAPKK Ste7.

Fig. 5. Scaffold proteins are modular and can be used as platforms for redirecting information in evolution and engineering

A) Pathogens can use scaffold-like proteins to rewire host signaling responses. The YopM scaffold from Yersinia pestis forces the interaction of the host Rsk1 and Prk2 kinases. The inappropriate activation is necessary for virulence. Viral scaffold proteins, such as HIV Vif, can target antiviral host proteins, such as the cytidine deaminase APOBEC3G, for degradation by targeting them to Cullin-E2 ubiquitin ligases. B) Engineered scaffolds can direct new cell signaling behaviors. A chimera of the Ste5 and Pbs2 yeast MAPK scaffold proteins can redirect mating pathway input to osmolarity pathway output. C) Synthetic feedback loops can be engineered by controlling recruitment of positive and negative effectors to the Ste5 MAPK scaffold protein. Such loops can be used to precisely shape the dynamics and dose-response of the yeast mating MAPK pathway to produce a wide range of signaling behaviors. D) Natural metabolic pathways are often organized into multi-enzyme complexes that function like an assembly line to enhance the rate and yield of metabolite production. Engineered scaffold proteins can link together novel combinations of metabolic enzymes to more efficiently synthesize desired chemical products. Adapted from (51).

Box 1. Structure, Interactions and Mechanisms of Ste5: MAPK Pathway Scaffold Protein