MicroED Structure of a Protoglobin Reactive Carbene Intermediate
Microcrystal electron diffraction (MicroED) is an emerging technique that has shown great potential for describing new chemical and biological molecular structures. Several important structures of small molecules, natural products, and peptides have been determined using ab initio methods. However, only a couple of novel protein structures have thus far been derived by MicroED. Taking advantage of recent technological advances, including higher acceleration voltage and using a low-noise detector in counting mode, we have determined the first structure of an Aeropyrum pernix protoglobin (ApePgb) variant by MicroED using an AlphaFold2 model for phasing. The structure revealed that mutations introduced during directed evolution enhance carbene transfer activity by reorienting an α helix of ApePgb into a dynamic loop, making the catalytic active site more readily accessible. After exposing the tiny crystals to the substrate, we also trapped the reactive iron-carbenoid intermediate involved in this engineered ApePgb's new-to-nature activity, a challenging carbene transfer from a diazirine via a putative metallo-carbene. The bound structure discloses how an enlarged active site pocket stabilizes the carbene bound to the heme iron and, presumably, the transition state for the formation of this key intermediate. This work demonstrates that improved MicroED technology and the advancement in protein structure prediction now enable investigation of structures that was previously beyond reach.
© 2023 The Authors. Published by American Chemical Society. Attribution 4.0 International (CC BY 4.0). E.D. thanks the Wenner-Gren Foundations for their support through the Wenner-Gren Postdoctoral Fellowship. This study was supported by the National Institutes of Health P41GM136508. The Gonen laboratory is supported by funds from the Howard Hughes Medical Institute. N.J.P. thanks Merck and the Helen Hay Whitney Foundation for their support through the Merck-Helen Hay Whitney Foundation Postdoctoral Fellowship. This publication is based on work supported by the United States Army Research Office under Contract W911NF-19-0026 for the Institute for Collaborative Biotechnologies and the G. Harold and Leila Y. Mathers Charitable Foundation. The authors declare no competing financial interest.
Published - ja2c12004.pdf
Supplemental Material - ja2c12004_si_001.pdf