Insertion of various functional groups including cyclopropyls and propargyls into monoamine oxidase substrates convert these compounds into mechanism-based inactivators [38
]. Substitutions at the epsilon position of Lys-4 of histone H3 peptides including propargylamine, aziridine, and cyclopropylamine have been synthesized and the propargylamine containing peptide behaves as a classical mechanism-based inactivator [40•
]. Based on a combination of mass spectrometry and optical and NMR spectroscopy, an inactivation mechanism in which the propargylamine is initially oxidized to the conjugated imine which undergoes Michael addition with the flavin cofactor has been proposed [24
] (). The resultant N5
flavin adduct generated leads to a stably bound enzyme-inhibitor complex. Taking advantage of this behavior, a recent 2.7 Å crystal structure was obtained of LSD1 after treatment with a N-methylpropargylamine analog induced inactivation [41•
]. In order to see electron density for the peptide moiety, it was necessary to treat the inactivated complex with sodium borohydride prior to crystallization, which presumably led to reduction of the linker double bonds between the flavin and peptide inactivator (). The importance of borohydride treatment was rationalized as increasing the resistance of the adduct to hydrolysis as well as eliminating the potential heterogeneity of the double bond stereoisomers associated with the parent adduct.
Mechanism-based inactivation of LSD1
The structure of the LSD1-inactivator complex, obtained in the presence of CoREST, was noteworthy in several respects. First, only residues 1-7 of the histone tail peptide gave sufficiently strong electron density to be modeled (). Weak density for H3 residues 8-21 may have been detected extending toward the SWIRM domain but it was insufficient to accurately model. The importance of residues beyond 7 for demethylase action is established [33•
] so it is likely that the LSD1-inactivator structure is incomplete in detailing biologically important recognition. Nevertheless, the H3 residues visualized formed a very unusual series of three gamma turns in the secondary structure [41•
] (). Gamma turn structure involves backbone hydrogen bond interactions between the i and i+2 residues in a protein chain providing a pseudo 7-membered ring [42
] (). Based on related structural studies, most enzymes involved in post-translational modifications recognize extended conformations of peptide substrates proximal to the targeted residue. To our knowledge, this interaction between LSD1 and the tail of H3 is unique among characterized enzyme-peptide substrate complexes.
LSD1 substrate specificity revealed by an LSD1-inactivator crystal structure
Given this unusual conformation observed in the LSD1 complex, it is appropriate to question its biological relevance, especially since it was obtained with a suicide inactivator. However, the crystal structure reveals a series of hydrogen bond and van der Waals interactions between the side chains of H3 residues and those of LSD1, effectively predicting the behavior of enzyme mutants and alternative substrates [41•
]. For example, the LSD1 structure shows that the free N-terminal amino group of H3 makes a key electrostatic interaction with Asp-555 of LSD1. Replacement of the amino group by a methyl group, charge masking by an acetyl group, or mutagenesis of Asp-555 all result in a major loss in substrate processing. Analogous experiments confirm the key interactions between Arg-2 of histone H3 and LSD1 residues Asp-556 and Trp-552. Overall, the LSD1 inactivated structure reveals an elegant basis for the highly specific demethylation for Lys-4 of histone H3. The ability of amino terminal acetylation to reduce processing efficiency by LSD1 may have biological significance for interpretation of the histone code since histone N-terminal acetylation is likely to occur reversibly in vivo