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

• Published in: Journal of the American Chemical Society Vol. 137; no. 28; pp. 9128 - 9135
• Main Authors: Cammarata, Michael B, Thyer, Ross, Rosenberg, Jake, Ellington, Andrew, Brodbelt, Jennifer S
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 22-07-2015
• Subjects: Binding Sites; Escherichia coli - chemistry; Escherichia coli - enzymology; Escherichia coli - metabolism; Folic Acid Antagonists - chemistry; Folic Acid Antagonists - pharmacology; Ligands; Mass Spectrometry - methods; Methotrexate - chemistry; Methotrexate - pharmacology; Models, Molecular; NADP - chemistry; NADP - metabolism; Photochemical Processes; Tetrahydrofolate Dehydrogenase - chemistry; Tetrahydrofolate Dehydrogenase - metabolism; Ultraviolet Rays
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/jacs.5b04628

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.