Abstract
Allostery is an inherent feature of proteins, but it remains challenging to reveal the
mechanisms by which allosteric signals propagate. A clearer understanding of this intrinsic circuitry
would afford new opportunities to modulate protein function. Here, we have identified allosteric
sites in protein tyrosine phosphatase 1B (PTP1B) by combining multiple-temperature X-ray
crystallography experiments and structure determination from hundreds of individual small-
molecule fragment soaks. New modeling approaches reveal ’hidden’ low-occupancy conformational
states for protein and ligands. Our results converge on allosteric sites that are conformationally
coupled to the active-site WPD loop and are hotspots for fragment binding. Targeting one of these
sites with covalently tethered molecules or mutations allosterically inhibits enzyme activity. Overall,
this work demonstrates how the ensemble nature of macromolecular structure, revealed here by
multitemperature crystallography, can elucidate allosteric mechanisms and open new doors for
long-range control of protein function.