Abstract
Protein-tyrosine phosphatase 1B (PTP1B) is the canonical enzyme for investigating how distinct structural elements influence enzyme catalytic activity. Although it is recognized that dynamics are essential for PTP1B function, the data collected thus far have not resolved whether distinct elements are dynamically coordinated or, alternatively, whether they fulfill their respective functions independently. To answer this question, we performed a comprehensive 13C-methyl relaxation study of Ile, Leu, and Val (ILV) residues of PTP1B, which, because of its substantially increased sensitivity, provides a comprehensive understanding of the influence of protein motions on different time scales for enzyme function. We discovered that PTP1B exhibits dynamics at three distinct time scales. First, it undergoes a distinctive slow motion that allows for the dynamic binding and release of its two most N-terminal helices from the catalytic core. Second, we showed that PTP1B 13C-methyl group side chain fast time-scale dynamics and 15N backbone fast time-scale dynamics are fully consistent, demonstrating that fast fluctuations are essential for the allosteric control of PTP1B activity. Third, and most importantly, using 13C ILV constant-time Carr-Purcell-Meiboom-Gill relaxation measurements experiments, we demonstrated that all four catalytically important loops-the WPD, Q, E, and substrate-binding loops-work in dynamic unity throughout the catalytic cycle of PTP1B. Thus, these data show that PTP1B activity is not controlled by a single functional element, but instead all key elements are dynamically coordinated. Together, these data provide the first fully comprehensive picture on how the validated drug target PTP1B functions.
Highlights
Protein-tyrosine phosphatase 1B (PTP1B, PTPN1) was the first non–receptor-bound protein-tyrosine phosphatase (PTP) isolated [1]
We recently reported the 15N-based sequence specific backbone assignment of the folded catalytic domain of PTP1B (;35 kDa; residues 1–301; hereafter referred to as PTP1B) and leveraged these data to define how 15N fast time-scale dynamics contributes to PTP1B activity, especially allostery [20]
The relatively large size of PTP1B, together with the inability to concentrate the protein to !250 mM, made 15N constanttime Carr–Purcell–Meiboom–Gill data analysis, which reports on intermediate events, statistically inaccurate and unable to provide insights into the role of intermediate time-scale dynamics for PTP1B function
Summary
Protein-tyrosine phosphatase 1B (PTP1B, PTPN1) was the first non–receptor-bound protein-tyrosine phosphatase (PTP) isolated [1]. These reports highlighted the importance of structural rigidity in the extended WPD loop, for proline residue Pro185 [7] This proline is essential for PTP1B activity because it controls an indispensable CH/ p switch that associates either with Trp179 within the WPD loop (closed state) or Phe269 from helix a6 (open state; Fig. S1). PTP1B helix a3 was identified as the mechanical/dynamics support that drives the transition between the open and closed states of the WPD loop and serves as the connector between the WPD loop and the allosteric pocket and helix a7 [7] Together, these structural and dynamics data have revealed many key aspects of PTP1B activity and regulation. Our data confirm our previous discovery (using only 15N-based protein backbone NMR measurements) that fast motions are critical
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