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Four--Helix Bundle with Designed Anesthetic Binding Pockets. Part I: Structural and Dynamical Analyses

Dejian Ma ^*, Nicole R. Brandon ^*, Tanxing Cui ^*, Vasyl Bondarenko ^*, Christian Canlas ^*, Jonas S. Johansson  ^¶, Pei Tang ^*   and Yan Xu ^* 

^* Department of Anesthesiology, Department of Pharmacology, and Department of Computational Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, 15260; Department of Anesthesiology and Critical Care, and ^¶ Johnson Research Foundation, Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Correspondence: Address reprints to Professor Yan Xu or Professor Pei Tang, 2048 Biomedical Science Tower 3, 3501 Fifth Avenue, Pittsburgh, PA 15260. Tel.: 412-648-9922; Fax: 412-648-8998; E-mail: xuy{at}anes.upmc.edu or tangp@anes.upmc.edu.

The four--helix bundle mimics the transmembrane domain of the Cys-loop receptor family believed to be the protein target for general anesthetics. Using high resolution NMR, we solved the structure (Protein Data Bank ID: 2I7U) of a prototypical dimeric four--helix bundle, (A₂-L1M/L38M)_2, with designed specific binding pockets for volatile anesthetics. Two monomers of the helix-turn-helix motif form an antiparallel dimer as originally designed, but the high-resolution structure exhibits an asymmetric quaternary arrangement of the four helices. The two helices from the N-terminus to the linker (helices 1 and 1') are associated with each other in the dimer by the side-chain ring stacking of F12 and W15 along the long hydrophobic core and by a nearly perfect stretch of hydrophobic interactions between the complementary pairs of L4, L11, L18, and L25, all of which are located at the heptad e position along the helix-helix dimer interface. In comparison, the axes of the two helices from the linker to the C-terminus (helices 2 and 2') are wider apart from each other, creating a lateral access pathway around K47 from the aqueous phase to the center of the designed hydrophobic core. The site of the L38M mutation, which was previously shown to increase the halothane binding affinity by 3.5-fold, is not part of the hydrophobic core presumably involved in the anesthetic binding but shows an elevated transverse relaxation (R₂) rate. Qualitative analysis of the protein dynamics by reduced spectral density mapping revealed exchange contributions to the relaxation at many residues in the helices. This observation was confirmed by the quantitative analysis using the Modelfree approach and by the NMR relaxation dispersion measurements. The NMR structures and Autodock analysis suggest that the pocket with the most favorable amphipathic property for anesthetic binding is located between the W15 side chains at the center of the dimeric hydrophobic core, with the possibility of two additional minor binding sites between the F12 and F52 ring stacks of each monomer. The high-resolution structure of the designed anesthetic-binding protein offers unprecedented atomistic details about possible sites for anesthetic-protein interactions that are essential to the understanding of molecular mechanisms of general anesthesia.

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				T. Cui, V. Bondarenko, D. Ma, C. Canlas, N. R. Brandon, J. S. Johansson, Y. Xu, and P. Tang Four-{alpha}-Helix Bundle with Designed Anesthetic Binding Pockets. Part II: Halothane Effects on Structure and Dynamics Biophys. J., June 1, 2008; 94(11): 4464 - 4472. [Abstract] [Full Text] [PDF]