Minimally disruptive optical control of protein tyrosine phosphatase 1B

Abstract

Supplementary Appendix 1. Analysis of the crystal structure of PTP1BPS. We used X-ray crystallography to examine the structural integrity of PTP1B within the PTP1BPS chimera (Supplementary Table 3). Crystals of PTP1BPS exhibited three surprising features ( Fig. 1d and Supplementary Fig. 2): (i) Their unit cell and space group were indistinguishable from those of previously collected crystals of PTP1B and PTP1B-ligand complexes. (ii) They permitted resolution of PTP1B, but not LOV2. (iii) They showed PTP1B with a wild-type conformation (i.e., the root-mean-square deviation of aligned atoms between the catalytic domains of PTP1BPS and wild-type PTP1B was 0.30 Å). These features, considered alone, might indicate that LOV2 is absent from our crystals; four additional crystallographic attributes, however, contradict this interpretation: (i) Our crystals were yellow, a color derived from LOV2 (a consequence of its FMN cofactor 1,2 ; Supplementary Figures 2a and 2b). (ii) When exposed to 455-nm light, the crystals turned clear, an indication that LOV2 remains capable of forming a cysteine adduct with FMN ( Supplementary Figures 2d and 2e). We note: Solutions of PTP1BPS(C450M), which cannot form the cysteine adduct, do not photoswitch ( Supplementary Figure 2f). (iii) The unit cell had a gap near the ⍺7 helix, the attachment point of LOV2 ( Supplementary Figure 2c). (iv)Previously examined apo structures of PTP1B in which the ⍺7 helix is disordered possess the same unit cell and space group as our crystals. 3 These features, taken together, suggest that disorder in the ⍺7 helix of PTP1B causes variability in the orientation of LOV2 within the crystal lattice. Broadly, PTP1B crystallizes with the same space group, unit cell, and conformation in the presence and absence of LOV2 and, thus, appears to be structurally unperturbed (to the extent detectable with X-ray crystallography) by this light-sensitive fusion partner.

Light-regulated allosteric switch enables temporal and subcellular control of enzyme activity

“…Application of LightR approach to different classes of enzymes suggests its broad applicability for the regulation of a wide variety of protein functions in living cells. Thus, unlike other light-regulated allosteric switches ( Wu et al, 2009 ; Hongdusit et al, 2020 ), this method combines multiple important advantages of optogenetic regulation with its broad applicability to a wide range of enzymes.…”

“…Multiple broadly applicable approaches have been developed to enable tight regulation of protein localization and protein interactions ( Guntas et al, 2015 ; Wang et al, 2016 ; Strickland et al, 2012 ; Kawano et al, 2015 ); however, the direct control of enzymatic activity remains a challenge. Only a few existing strategies achieve direct regulation of enzymatic activity, but they either lack the ability to activate enzymes locally within a cell, or they are difficult to apply to multiple enzyme classes ( Wang et al, 2019 ; Dagliyan et al, 2016 ; Zhou et al, 2017 ; Diaz et al, 2017 ; Wu et al, 2009 ; Hongdusit et al, 2020 ), thus limiting their use for addressing mechanistic questions in cell biology. This highlights the need for an optogenetic approach which is broadly applicable, easy to implement and provides all the advantages of optogenetics in one tool including tight temporal and local subcellular regulation.…”

“…Only four optogenetic methods for allosteric regulation of activity have been reported so far and they suffer from critical limitations ( Dagliyan et al, 2016 ; Wu et al, 2009 ; Hongdusit et al, 2020 ; Winkler et al, 2015 ; Reynolds et al, 2011 ). Two of these strategies achieve subcellular control of certain targeted proteins but they cannot be applied to many other enzymes due to their design ( Wu et al, 2009 ; Hongdusit et al, 2020 ). One approach is broadly applicable but it does not achieve local control and triggers inactivation rather than activation of the protein ( Dagliyan et al, 2016 ).…”

“…Only a few existing strategies achieve direct regulation of enzymatic activity, but they either lack the ability to activate enzymes locally within a cell, or they are difficult to apply to multiple enzyme classes [5][6][7][8][9][10] , thus limiting their use for addressing mechanistic questions in cell biology. This highlights the need for an optogenetic approach which is broadly applicable, easy to implement and provides all the advantages of optogenetics in one tool including tight temporal and local subcellular regulation.…”

“…Also, allosteric switch domains are genetically encoded into the targeted protein simplifying the application of the tool. Only four optogenetic methods for allosteric regulation of activity have been reported so far and they suffer from critical limitations 6,9,10,12,13 . Two of these strategies achieve subcellular control of certain targeted proteins but they cannot be applied to many other enzymes due to their design 9,10 .…”