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.

Immune receptor signalling proceeds by the binding of enzymes to their cytoplasmic tails before they catalyse reactions on substrates within reach. Studies of binding and catalysis have led to a binding- induced allosteric activation model for enzymes, such as SHP-1, whose catalytic activity increases upon recruitment to inhibitory receptors, such as PD-1. However, the impact of molecular reach is poorly understood. Here, we use surface plasmon resonance to measure binding, catalysis, and molecular reach for tethered SHP-1 reactions. We find a molecular reach for SHP-1 (13.0 nm) that is smaller than a maximum stretch estimate (20.4 nm) but larger than an estimate from crystal structure of the active conformation (5.3 nm), suggesting that SHP-1 explores a spectrum of active conformations. The molecular reach confines SHP-1 to a small membrane-proximal volume near its substrates leading membrane recruitment to increase its activity by a 1000-fold increase in concentration with a smaller 2-fold activity increase by PD-1 binding-induced allostery. Lastly, we use mathematical modelling to quantify the degree of co-clustering required for PD-1-SHP-1 complexes to reach their substrates.