title: Neutron diffraction studies of Escherichia coli dihydrofolate reductase complexed with methotrexate.
abstract: Hydrogen atoms play a central role in many biochemical processes yet are difficult to visualize by x-ray crystallography. Spallation neutron sources provide a new arena for protein crystallography with TOF measurements enhancing data collection efficiency and allowing hydrogen atoms to be located in smaller crystals of larger biological macromolecules. Here we report a 2.2-A resolution neutron structure of Escherichia coli dihydrofolate reductase (DHFR) in complex with methotrexate (MTX). Neutron data were collected on a 0.3-mm(3) D(2)O-soaked crystal at the Los Alamos Neutron Scattering Center. This study provides an example of using spallation neutrons to study protein dynamics, to identify protonation states directly from nuclear density maps, and to analyze solvent structure. Our structure reveals that the occluded loop conformation [monomer (mon.) A] of the DHFR.MTX complex undergoes greater H/D exchange compared with the closed-loop conformer (mon. B), partly because the Met-20 and beta(F-G) loops readily exchange in mon. A. The eight-stranded beta sheet of both DHFR molecules resists H/D exchange more than the helices and loops. However, the C-terminal strand, betaH, in mon. A is almost fully exchanged. Several D(2)Os form hydrogen bonds with exchanged amides. At the active site, the N1 atom of MTX is protonated and thus charged when bound to DHFR. Several D(2)Os are observed at hydrophobic surfaces, including two pockets near the MTX-binding site. A previously unidentified D(2)O hydrogen bonds with the catalytic D27 in mon. B, stabilizing its negative charge.
authors.affiliation: Department of Biochemistry, Cellular and Molecular Biology, M407 Walters Life Sciences, University of Tennessee, Knoxville, TN 37996, USA.
authors.forename: Brad
authors.initials: B
authors.lastname: Bennett
authors.forename: Paul
authors.initials: P
authors.lastname: Langan
authors.forename: Leighton
authors.initials: L
authors.lastname: Coates
authors.forename: Marat
authors.initials: M
authors.lastname: Mustyakimov
authors.forename: Benno
authors.initials: B
authors.lastname: Schoenborn
authors.forename: Elizabeth E
authors.initials: EE
authors.lastname: Howell
authors.forename: Chris
authors.initials: C
authors.lastname: Dealwis
mesh_terms: D001665:Binding Sites; D003460:Crystallization; D041961:Deuterium Exchange Measurement; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D008727:Methotrexate / Q000737:chemistry* / Q000378:metabolism; D033363:Neutron Diffraction; D011522:Protons; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D029968:Escherichia coli Proteins; D011522:Protons; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate