Structural Characterization of Dihydrofolate Reductase Complexes by Top-Down Ultraviolet Photodissociation Mass Spectrometry

Abstract

The stepwise reduction of dihydrofolate to tetrahydrofolate entails significant conformational changes of dihydrofolate reductase (DHFR). Binary and ternary complexes of DHFR containing cofactor NADPH, inhibitor methotrexate (MTX), or both NADPH and MTX were characterized by 193 nm ultraviolet photodissociation (UVPD) mass spectrometry. UVPD yielded over 80% sequence coverage of DHFR and resulted in production of fragment ions that revealed the interactions between DHFR and each ligand. UVPD of the binary DHFR·NADPH and DHFR·MTX complexes led to an unprecedented number of fragment ions containing either an N- or C-terminal protein fragment still bound to the ligand via retention of noncovalent interactions. In addition, holo-fragments retaining both ligands were observed upon UVPD of the ternary DHFR·NADPH·MTX complex. The combination of extensive holo and apo fragment ions allowed the locations of the NADPH and MTX ligands to be mapped, with NADPH associated with the adenosine binding domain of DHFR and MTX interacting with the loop domain. These findings are consistent with previous crystallographic evidence. Comparison of the backbone cleavage propensities for apo DHFR and its holo counterparts revealed significant variations in UVPD fragmentation in the regions expected to experience conformational changes upon binding NADPH, MTX, or both ligands. In particular, the subdomain rotation and loop movements, which are believed to occur upon formation of the transition state of the ternary complex, are reflected in the UVPD mass spectra. The UVPD spectra indicate enhanced backbone cleavages in regions that become more flexible or show suppressed backbone cleavages for those regions either shielded by the ligand or involved in new intramolecular interactions. This study corroborates the versatility of 193 nm UVPD mass spectrometry as a sensitive technique to track enzymatic cycles that involve conformational rearrangements.

Ion Activation Methods for Peptides and Proteins

“…263 The resulting UVPD mass spectra of the native complexes gave numerous diagnostic product ions that provided 80% sequence coverage of DHFR, in addition to many fragment ions that retained the NADPH or MTX ligands. 263 The collection of holo (ligand-containing) and apo (ligand-free) product ions offered a means to determine the binding site of each ligand. Comparisons of the fragmentation maps for the various complexes (DHFR alone versus DHFR/NADPH complex versus DHFR/methotrexate complex versus ternary DHFR/NADPH/methotrexate complex) showed significant variations in fragmentation efficiency that were attributed to structural changes.…”

“…In essence, backbone cleavages were enhanced in the more flexible regions of the protein, and backbone cleavages were reduced in regions that were “shielded” by the ligand in a way suggestive of formation of new stabilizing intramolecular interactions. 263 …”

Structural Characterization of Native Proteins and Protein Complexes by Electron Ionization Dissociation-Mass Spectrometry

“…A single 193 nm photon deposits 6.4 eV of energy, which is sufficient to cause dissociation by directly accessing excited electronic states, leading to a diversity of fragmentation pathways (e.g., a/b/c- and x/y/z-product ions) 12 . Extensive sequence information with retention of labile modifications and non-covalent ligand-bound fragment ions has been observed 22, 24, 26 . Sequence coverage for native and denatured proteins by UVPD was found to be independent of the charge state of the precursor ions 12 .…”

High-Throughput Analysis of Intact Human Proteins Using UVPD and HCD on an Orbitrap Mass Spectrometer

“…32, 33 UVPD exhibits impressive performance for both denatured proteins and native-like proteins, the latter typically generated in low charge states which have proven more challenging to characterize by collision- and electron-based methods. 32–36 To date, UVPD has only had limited usage for high-throughput top-down analysis of complex protein mixtures, such as cell lysates. 10 Previously, Cannon et al .…”