Anesthetic Site on Protein Kinase Cd’s C1B Domain
2339
2. Miller, K. W. 2002. The nature of sites of general anaesthetic action.
Br. J. Anaesth. 89:17–31.
binding pocket is that one side is bounded by two water
molecules held in place by a hydrogen-bonding chain
between two residues. These waters move modestly to
accommodate the ligand, providing the pocket with some
elasticity. Although van der Waals interactions contribute
to binding, a hydrogen bond between the ligand and
Tyr-236 is essential, because mutation of Tyr-236 to Phe
abolishes ligand binding. The requirement to maintain
both hydrogen bonding and van der Waals interactions
imposes geometric constraints that account for why ethanol
does not bind. It is possible that the cyclopropane ring plays
a unique role as a p-acceptor for X–H hydrogen bonds. The
binding site, whose structure we have characterized, medi-
ates inhibition of phorbol-induced PKCd activity by anes-
thetics, as shown by site-directed mutagenesis studies of
the kinase itself.
3. Yamakura, T., E. Bertaccini, ., R. A. Harris. 2001. Anesthetics
and ion channels: molecular models and sites of action. Annu. Rev.
Pharmacol. Toxicol. 41:23–51.
4. Campagna, J. A., K. W. Miller, and S. A. Forman. 2003. Mechanisms of
actions of inhaled anesthetics. N. Engl. J. Med. 348:2110–2124.
5. Eckenhoff, R. G., and J. S. Johansson. 1997. Molecular interactions
between inhaled anesthetics and proteins. Pharmacol. Rev. 49:
343–367.
6. Bocquet, N., H. Nury, ., P. J. Corringer. 2009. X-ray structure of a pen-
tameric ligand-gated ion channel in an apparently open conformation.
Nature. 457:111–114.
7. Hilf, R. J., and R. Dutzler. 2008. X-ray structure of a prokaryotic pen-
tameric ligand-gated ion channel. Nature. 452:375–379.
8. Hilf, R. J., and R. Dutzler. 2009. Structure of a potentially open state of
a proton-activated pentameric ligand-gated ion channel. Nature. 457:
115–118.
9. Hilf, R. J., C. Bertozzi, ., R. Dutzler. 2010. Structural basis of open
channel block in a prokaryotic pentameric ligand-gated ion channel.
Nat. Struct. Mol. Biol. 17:1330–1336.
Coordinates
10. Nury, H., C. Van Renterghem, ., P. J. Corringer. 2011. X-ray struc-
tures of general anaesthetics bound to a pentameric ligand-gated ion
channel. Nature. 469:428–431.
The coordinates and structure factors have been deposited
in the Protein Data Bank (accession codes 3UEJ, 3UGL,
3UGI, 3UEY).
11. Baenziger, J. E., and P. J. Corringer. 2011. 3D structure and allosteric
modulation of the transmembrane domain of pentameric ligand-gated
ion channels. Neuropharmacology. 60:116–125.
12. Schoenborn, B. P., H. C. Watson, and J. C. Kendrew. 1965. Binding of
xenon to sperm whale myoglobin. Nature. 207:28–30.
SUPPORTING MATERIAL
13. Schoenborn, B. P. 1965. Binding of xenon to horse haemoglobin.
Nature. 208:760–762.
14. Sachsenheimer, W., E. F. Pai, ., R. H. Schirmer. 1977. Halothane
binds in the adenine-specific niche of crystalline adenylate kinase.
FEBS Lett. 79:310–312.
We thank Elizabeth Kelly for assistance with the anesthetic potency
measurements, Dr. Peter M. Blumberg for providing the PKCd C1B
construct and Dr. David Jones for providing us with the structure factor
file for his LUSH structure. Molecular graphics images were produced
using the University of California, San Francisco, Chimera package (59)
from the Resource for Biocomputing, Visualization, and Informatics at
the University of California, San Francisco (supported by NIH P41 RR-
01081) or using Pymol (60). S.S. thanks V. Stojanoff, M. Allaire, J. Jakon-
cic, and F. Yokaichiya of beam line X6A, and N. Sukumar of BM8, for their
support during data collection.
15. Franks, N. P., A. Jenkins, ., P. Brick. 1998. Structural basis for the
inhibition of firefly luciferase by a general anesthetic. Biophys. J.
75:2205–2211.
16. Bhattacharya, A. A., S. Curry, and N. P. Franks. 2000. Binding of the
general anesthetics propofol and halothane to human serum albumin.
High resolution crystal structures. J. Biol. Chem. 275:38731–38738.
17. Gursky, O., E. Fontano, ., D. L. Caspar. 1994. Stereospecific dihaloal-
kane binding in a pH-sensitive cavity in cubic insulin crystals. Proc.
Natl. Acad. Sci. USA. 91:12388–12392.
This research was supported by grants from the U.S. Public Health Service
to K.W.M. (GM 069726) and R.O.M. (AA018316), by a Foundation for
Anesthesia Education and Research award to W.S.S., by the Department
of Anesthesia, Critical Care & Pain Medicine, Massachusetts General
Hospital, and by funds provided by the State of California for medical
research on alcohol and substance abuse through University of California,
San Francisco, to R.O.M. This work is based upon research conducted at the
Northeastern Collaborative Access Team beam lines (BM8) of the
Advanced Photon Source, which is supported by award RR-15301 from
the National Center for Research Resources at the National Institutes of
Health. Research carried out (in whole or in part) at X6A beam line was
funded by the National Institute of General Medical Sciences, National
Institutes of Health, under agreement GM-0080. The National Synchrotron
Light Source, Brookhaven National Laboratory is supported by the U.S.
Department of Energy under contract no. DE-AC02-98CH10886.
18. Liu, R., P. J. Loll, and R. G. Eckenhoff. 2005. Structural basis for high-
affinity volatile anesthetic binding in a natural 4-helix bundle protein.
FASEB J. 19:567–576.
19. Slater, S. J., K. J. Cox, ., C. D. Stubbs. 1993. Inhibition of protein
kinase C by alcohols and anaesthetics. Nature. 364:82–84.
20. Hemmings, Jr., H. C., and A. I. Adamo. 1994. Effects of halothane and
propofol on purified brain protein kinase C activation. Anesthesiology.
81:147–155.
21. Rebecchi, M. J., and S. N. Pentyala. 2002. Anaesthetic actions on other
targets: protein kinase C and guanine nucleotide-binding proteins. Br.
J. Anaesth. 89:62–78.
22. Newton, A. C. 2001. Protein kinase C: structural and spatial regulation
by phosphorylation, cofactors, and macromolecular interactions.
Chem. Rev. 101:2353–2364.
23. Cho, W. 2001. Membrane targeting by C1 and C2 domains. J. Biol.
Chem. 276:32407–32410.
24. Kazanietz, M. G., J. J. Barchi, Jr., ., P. M. Blumberg. 1995. Low
affinity binding of phorbol esters to protein kinase C and its recombi-
nant cysteine-rich region in the absence of phospholipids. J. Biol.
Chem. 270:14679–14684.
REFERENCES
1. Hemmings, Jr., H. C., M. H. Akabas, ., N. L. Harrison. 2005.
Emerging molecular mechanisms of general anesthetic action. Trends
Pharmacol. Sci. 26:503–510.
25. Szallasi, Z., K. Bogi, ., P. M. Blumberg. 1996. Non-equivalent roles
for the first and second zinc fingers of protein kinase Cd. Effect of their
Biophysical Journal 103(11) 2331–2340