Room-temperature serial synchrotron crystallography of the human phosphatase PTP1B

Abstract

Room-temperature X-ray crystallography provides unique insights into protein conformational heterogeneity, but obtaining sufficiently large protein crystals is a common hurdle. Serial synchrotron crystallography (SSX) helps to address this hurdle by allowing the use of many medium- to small-sized crystals. Here, a recently introduced serial sample-support chip system has been used to obtain the first SSX structure of a human phosphatase, specifically protein tyrosine phosphatase 1B (PTP1B) in the unliganded (apo) state. In previous apo room-temperature structures, the active site and allosteric sites adopted alternate conformations, including open and closed conformations of the active-site WPD loop and of a distal allosteric site. By contrast, in our SSX structure the active site is best fitted with a single conformation, but the distal allosteric site is best fitted with alternate conformations. This observation argues for additional nuance in interpreting the nature of allosteric coupling in this protein. Overall, our results illustrate the promise of serial methods for room-temperature crystallography, as well as future avant-garde crystallography experiments, for PTP1B and other proteins.

Room-temperature crystallography reveals altered binding of small-molecule fragments to PTP1B

“…One case involves essentially complete closing of the allosteric L16 site, but only partial closing (~50%) of the active-site WPD loop ( Figure 11 ) – in contrast to the previous paradigm in which the WPD loop and allosteric sites are precisely conformationally coupled ( Keedy et al, 2018 ). Similar (de)coupling was also seen recently with serial synchrotron crystallography of apo PTP1B ( Sharma et al, 2023 ). Thus, RT crystallography can add important nuance to our understanding of allosteric mechanisms in PTPs ( Choy et al, 2017 ; Cui et al, 2017 ; Hjortness et al, 2018 ) and likely other proteins.…”

High-resolution double vision of the archetypal protein tyrosine phosphatase

“…Of the ∼350 structures of PTP1B in the PDB, the majority exhibit a disordered and therefore unmodeled C-terminus. The ordering of the C-terminus is shown to be coupled, albeit only partially, to the conformation of the WPD loop and the allosteric L16 site (Keedy et al 2018; Sharma, Ebrahim, and Keedy 2023; Skaist Mehlman et al 2023). In a previously published closed-state structure of PTP1B, the C-terminus was modeled as ordered with the L16 site in the closed conformation ( Fig.…”

“…If properly modeled, they could likely help explain functional effects of mutations and ligands (Wankowicz et al 2022), allosteric mechanisms, and other phenomena for PTP1B and other systems. Moreover, other biophysical perturbations (Keedy 2019) such as variable temperature (Fraser et al 2011;Keedy et al 2014Keedy et al , 2018Fischer 2021;Stachowski et al 2022;Sharma, Ebrahim, and Keedy 2023;Skaist Mehlman et al 2023;Greisman, Dalton, et al 2023;Thorne 2023) or pressure (Urayama, Phillips, and Gruner 2002;Guerrero et al 2023) could reveal additional, previously "hidden" conformational heterogeneity or excited states that could provide further insights into biological mechanisms for PTPs as well as other proteins. These areas represent promising avenues for future study.…”

High-resolution double vision of the allosteric phosphatase PTP1B

“…Of the �350 structures of PTP1B in the PDB, the majority exhibit a disordered and therefore unmodeled C-terminus. The ordering of the C-terminus is shown to be loosely coupled to the conformation of the WPD loop and the allosteric L16 site (Keedy et al, 2018;Sharma et al, 2023;Skaist Mehlman et al, 2023). In a previously published closed-state structure of PTP1B, the C-terminus was modeled as ordered with the L16 site in the closed conformation (Fig.…”