Effect of detergent binding on cytochrome P450 2B4 structure as analyzed by X-ray crystallography and deuterium-exchange mass spectrometry.
Multiple crystal structures of CYP2B4 have demonstrated the binding of the detergent 5-cyclohexyl-1-pentyl-β-D-maltoside (CYMAL-5) in a peripheral pocket located adjacent to the active site. To explore the consequences of detergent binding, X-ray crystal structures of the peripheral pocket mutant CYP2B4 F202W were solved in the presence of hexaethylene glycol monooctyl ether (C8E6) and CYMAL-5. The structure in the presence of CYMAL-5 illustrated a closed conformation indistinguishable from the previously solved wild-type. In contrast, the F202W structure in the presence of C8E6 revealed a detergent molecule that coordinated the heme-iron and extended to the protein surface through the substrate access channel 2f. Despite the overall structural similarity of these detergent complexes, remarkable differences were observed in the A, A', and H helices, the F-G cassette, the C-D and β4 loop region. Hydrogen-deuterium exchange mass spectrometry (DXMS) was employed to probe these differences and to test the effect of detergents in solution. The presence of either detergent increased the H/D exchange rate across the plastic regions, and the results obtained by DXMS in solution were consistent in general with the relevant structural snapshots. The study provides insight into effect of detergent binding and the interpretation of associated conformational dynamics of CYP2B4.
School of Pharmacy, University of Connecticut, Storrs, CT 06269, United States. Electronic address: manish.shah@uconn.edu.
Manish B
MB
Shah
School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea.
Hyun-Hee
HH
Jang
School of Pharmacy, University of Connecticut, Storrs, CT 06269, United States.
P Ross
PR
Wilderman
The Department of Medicine, University of California, San Diego, La Jolla, CA 92093, United States.
David
D
Lee
The Department of Medicine, University of California, San Diego, La Jolla, CA 92093, United States.
Sheng
S
Li
The Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, United States.
Qinghai
Q
Zhang
The Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, United States.
C David
CD
Stout
School of Pharmacy, University of Connecticut, Storrs, CT 06269, United States.
James R
JR
Halpert
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D000072467:Cytochrome P450 Family 2 / Q000737:chemistry / Q000235:genetics; D003902:Detergents / Q000737:chemistry* / Q000494:pharmacology; D041961:Deuterium Exchange Measurement; D005960:Glucosides / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D003902:Detergents; D005960:Glucosides; C400643:cyclohexyl-pentyl-maltoside; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit)
Cytochrome P450; Hydrogen-deuterium-exchange mass spectrometry; X-ray crystallography