Side chain conformational averaging in human dihydrofolate reductase
Lisa M Tuttle1, H Jane Dyson, Peter E Wright
1Department of Integrative Structural and Computational Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
Biochemistry
|February 7, 2014
Summary
Human dihydrofolate reductase (hDHFR) uses subtle side chain dynamics, not major backbone shifts, for its catalytic cycle. Unlike E. coli DHFR, hDHFR exhibits minimal conformational changes, relying on side chain flexibility for ligand flux.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Dihydrofolate reductase (DHFR) enzymes from Escherichia coli (ecDHFR) and Homo sapiens (hDHFR) share similar structures but differ in catalytic mechanisms.
- ecDHFR undergoes significant loop rearrangements, while hDHFR maintains a closed conformation during its reaction cycle.
Purpose of the Study:
- To investigate the structural and dynamic differences in side chain flexibility between hDHFR and ecDHFR.
- To understand the mechanisms underlying ligand flux and conformational changes in hDHFR.
Main Methods:
- Nuclear magnetic resonance (NMR) relaxation dispersion experiments to probe millisecond timescale dynamics.
- X-ray crystallography to compare hinge-open and hinge-closed states of hDHFR.
- Analysis of (3)JCγCO and (3)JCγN couplings to determine side chain rotamer populations.
Main Results:
- hDHFR side chains lack the significant millisecond timescale fluctuations observed in ecDHFR.
- Conformational changes in hDHFR involve the sliding of helix αF and are facilitated by side chain rotamer averaging.
- The Phe31 side chain rotamer is dependent on substrate/product binding, influencing active site opening for ligand entry.
Conclusions:
- hDHFR utilizes minimal backbone conformational changes, relying on subtle side chain dynamics for its enzymatic cycle.
- Ligand flux in hDHFR is mediated by specific side chain motions and conformational adjustments, such as helix αF movement.
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