Multisite phosphorylation is ubiquitous in cellular signaling and is thought to provide signaling proteins with additional regulatory mechanisms. Indeed, mathematical models have revealed a large number of mechanisms by which multisite phosphorylation can produce switchlike responses. The T cell antigen receptor (TCR) is a multisubunit receptor on the surface of T cells that is a prototypical multisite substrate as it contains 20 sites that are distributed on 10 conserved immunoreceptor tyrosine-based activation motifs (ITAMs). The TCR ζ-chain is a homodimer subunit that contains six ITAMs (12 sites) and exhibits a number of properties that are predicted to be sufficient for a switchlike response. We have used cellular reconstitution to systematically study multisite phosphorylation of the TCR ζ-chain. We find that multisite phosphorylation proceeds by a nonsequential random mechanism, and find no evidence that multiple ITAMs modulate a switchlike response but do find that they alter receptor potency and maximum phosphorylation. Modulation of receptor potency can be explained by a reduction in molecular entropy of the disordered ζ-chain upon phosphorylation. We further find that the tyrosine kinase ZAP-70 increases receptor potency but does not modulate the switchlike response. In contrast to other multisite proteins, where phosphorylations act in strong concert to modulate protein function, we suggest that the multiple ITAMs on the TCR function mainly to amplify subsequent signaling.

Many actin structures are nucleated and assembled by the barbed-end tracking polymerase formin family, including filopodia, focal adhesions, the cytokinetic ring and cell cortex. These structures respond to forces in distinct ways. Formins typically have profilin-actin binding sites embedded in highly flexible disordered FH1 domains, hypothesized to diffusively explore space to rapidly capture actin monomers for delivery to the barbed end. Recent experiments demonstrate that formin-mediated polymerization accelerates when under tension. The acceleration has been attributed to modifying the state of the FH2 domain of formin. Intriguingly, the same acceleration is reported when tension is applied to the FH1 domains, ostensibly pulling monomers away from the barbed end. Here we develop a mesoscale coarse-grain model of formin-mediated actin polymerization, including monomer capture and delivery by FH1, which sterically interacts with actin along its entire length. The binding of actin monomers to their specific sites on FH1 is entropically disfavored by the high disorder. We find that this penalty is attenuated when force is applied to the FH1 domain by revealing the binding site, increasing monomer capture efficiency. Overall polymerization rates can decrease or increase with increasing force, depending on the length of FH1 domain and location of binding site. Our results suggest that the widely varying FH1 lengths and binding site locations found in known formins could be used to differentially respond to force, depending on the actin structure being assembled. © 2016 Wiley Periodicals, Inc.

Signaling by surface receptors often relies on tethered reactions whereby an enzyme bound to the cytoplasmic tail of a receptor catalyzes reactions on substrates within reach. The overall length and stiffness of the receptor tail, the enzyme, and the substrate determine a biophysical parameter termed the molecular reach of the reaction. This parameter determines the probability that the receptor-tethered enzyme will contact the substrate in the volume proximal to the membrane when separated by different distances within the membrane plane. In this work, we develop particle-based stochastic reaction-diffusion models to study the interplay between molecular reach and diffusion. We find that increasing the molecular reach can increase reaction efficacy for slowly diffusing receptors, whereas for rapidly diffusing receptors, increasing molecular reach reduces reaction efficacy. In contrast, if reactions are forced to take place within the two-dimensional plasma membrane instead of the three-dimensional volume proximal to it or if molecules diffuse in three dimensions, increasing molecular reach increases reaction efficacy for all diffusivities. We show results in the context of immune checkpoint receptors (PD-1 dephosphorylating CD28), a standard opposing kinase-phosphatase reaction, and a minimal two-particle model. The work highlights the importance of the threedimensional nature of many two-dimensional membrane-confined interactions, illustrating a role for molecular reach in controlling biochemical reactions.

Immune receptors signal by recruiting (or tethering) enzymes to their cytoplasmic tails to catalyze reactions on substrates within reach. This is the case for the phosphatase SHP-1, which, upon tethering to inhibitory receptors, dephosphorylates diverse substrates to control T cell activation. Precisely how tethering regulates SHP-1 activity is incompletely understood. Here, we measure binding, catalysis, and molecular reach for tethered SHP-1 reactions. We determine the molecular reach of SHP-1 to be 13.0 nm, which is longer than the estimate from the allosterically active structure (5.3 nm), suggesting that SHP-1 can achieve a longer reach by exploring multiple active conformations. Using modeling, we show that when uniformly distributed, receptor-SHP-1 complexes can only reach 15% of substrates, but this increases to 90% when they are coclustered. When within reach, we show that membrane recruitment increases the activity of SHP-1 by a 1000-fold increase in local concentration. The work highlights how molecular reach regulates the activity of membrane-recruited SHP-1 with insights applicable to other membrane-tethered reactions.

Signalling by surface receptors often relies on tethered reactions whereby an enzyme bound to the cytoplasmic tail of a receptor catalyses reactions on substrates within reach. The overall length and stiffness of the receptor tail, the enzyme, and the substrate determine a biophysical parameter termed the molecular reach of the reaction. This parameter determines the probability that the receptor-tethered-enzyme will contact the substrate, in the volume proximal to the membrane, when separated by different distances within the membrane plane. In this work we develop particlebased stochastic reaction-diffusion models to study the interplay between molecular reach and diffusion. We find that increasing the molecular reach can increase reaction efficacy for slowly diffusing receptors, while for rapidly diffusing receptors increasing molecular reach reduces reaction efficacy. In contrast, if reactions are forced to take place within the 2D plasma membrane instead of the 3D volume proximal to it, or if molecules diffuse in 3D, increasing molecular reach increases reaction efficacy for all diffusivities. We show results in the context of immune checkpoint receptors (PD-1 dephosphorylating CD28), a standard opposing kinase-phosphatase reaction, and a minimal two-particle model. The work highlights the importance of the 3D nature of many 2D membraneconfined interactions, illustrating a role for molecular reach in controlling biochemical reactions.O.D. and S.A.I. contributed equally to this work.