Fig. 1: Minimally disruptive photocontrol of PTP1B.
From: Minimally disruptive optical control of protein tyrosine phosphatase 1B
a Left: An alignment of a competitively inhibited structure of PTP1B (orange, pdb entry 2f71) and an apo structure of PTP1B (yellow, pdb entry 3a5j) highlights an allosteric control system. Closure of the WPD loop (black) over an inhibitor orders the α7 helix; opening of the loop (light blue) hinders this ordering. Right: An alignment of the LOV2 domain from A. sativa (blue) and an N-terminal segment of the same domain of A. thaliana (white) that is identical between the two proteins (pdb entries 2v0w and 4hhd, respectively). Two terminal α-helices (gray and white) are stable in the dark state, but not the light state. b Design of a photoswitchable chimera. Light-induced unwinding of the A’α helix of LOV2 destabilizes the α7 helix of PTP1B, causing an allosteric conformational change that inhibits catalysis. We attached the C-terminal α7 helix of PTP1B to the N-terminal A’α helix of LOV2 at crossover points in a primary sequence alignment (1–8). These points are highlighted in blue (PTP1B) and red (LOV2) in (a). c Assays on 4-methylumbelliferyl phosphate (4MUP) show the results of chimera optimization. Construct 7 has the largest dynamic range (DR) of the crossover variants; 7.1 has a higher activity than 7, and 7.1(T406A), termed PTP1BPS, has a larger DR than 7.1. The dashed gray and blue lines denote values for 7.1 and 7.1(T406A), respectively. The plotted data depict the mean, SE, and associated estimates of DR for n = 6 independent experiments. d Aligned catalytic domains of PTP1B in three structures: photoswitchable (6ntp), apo (3a5j), and competitively inhibited (2f71, α6 and α7 only). e An analysis of the activity of PTP1BPS on p-nitrophenyl-phosphate (pNPP) indicates that light affects kcat, but not Km (kcat-dark/kcat-light = 2.50 ± 0.04). Error bars denote SE for n = 3 independent reactions. f The DR of PTP1BPS is similar for substrates of different sizes. The plotted data depict the mean, SE, and associated estimates of DR for n ≥ 3 independent reactions. g Structures of pNPP, 4MUP, and a peptide (PEP) derived from epidermal growth factor receptor (EGFR). Source data are provided as a Source Data file.
