Conformational changes in Akt1 activation probed by amide hydrogen/deuterium exchange and nano-electrospray ionization mass spectrometry.
Amide hydrogen exchange coupled to nano-electrospray ionization mass spectrometry (nano-ESI-MS) has been used to identify and characterize localized conformational changes of Akt upon activation. Active or inactive Akt was incubated in D(2)O buffer, digested with pepsin, and analyzed by nano-ESI-MS to determine the deuterium incorporation. The hydrogen/deuterium (H/D) exchange profiles revealed that Akt undergoes considerable conformational changes in the core structures of all three individual domains after activation. In the PH domain, four beta-strand (beta1, beta2 beta5 and beta6) regions containing membrane-binding residues displayed higher solvent accessibility in the inactive state, suggesting that the PH domain is readily available for the binding to the plasma membrane for activation. In contrast, these beta-strands became less exposed or more folded in the active form, which is favored for the dissociation of Akt from the membrane. The beginning alpha-helix J region and the C-terminal locus (T450-470P) of the regulatory domain showed less folded structures that probably enable substrate entry. Our data also revealed detailed conformational changes of Akt in the kinase domain due to activation, some of which may be attributed to the interaction of the basic residues with phosphorylation sites. Our H/D exchange results indicating the conformational status of Akt at different activation states provided new insight for the regulation of this critical protein involved in cell survival.
Guo
Mingquan
M
Laboratory of Molecular Signaling, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD 20892-9410, USA.
Huang
Bill X
BX
Kim
Hee-Yong
HY
D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D003903:Deuterium / Q000737:chemistry*; D004789:Enzyme Activation; D006859:Hydrogen / Q000737:chemistry*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051057:Proto-Oncogene Proteins c-akt / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D000577:Amides; D006859:Hydrogen; D003903:Deuterium; D051057:Proto-Oncogene Proteins c-akt