Substrate binding to histone deacetylases as shown by the crystal structure of the HDAC8-substrate complex.
Histone deacetylases (HDACs)-an enzyme family that deacetylates histones and non-histone proteins-are implicated in human diseases such as cancer, and the first-generation of HDAC inhibitors are now in clinical trials. Here, we report the 2.0 A resolution crystal structure of a catalytically inactive HDAC8 active-site mutant, Tyr306Phe, bound to an acetylated peptidic substrate. The structure clarifies the role of active-site residues in the deacetylation reaction and substrate recognition. Notably, the structure shows the unexpected role of a conserved residue at the active-site rim, Asp 101, in positioning the substrate by directly interacting with the peptidic backbone and imposing a constrained cis-conformation. A similar interaction is observed in a new hydroxamate inhibitor-HDAC8 structure that we also solved. The crucial role of Asp 101 in substrate and inhibitor recognition was confirmed by activity and binding assays of wild-type HDAC8 and Asp101Ala, Tyr306Phe and Asp101Ala/Tyr306Phe mutants.
Vannini
Alessandro
A
Istituto di Ricerche di Biologia Molecolare P. Angeletti, Via Pontina Km 30.600, 00040 Pomezia, Rome, Italy.
Volpari
Cinzia
C
Gallinari
Paola
P
Jones
Philip
P
Mattu
Marco
M
Carfí
Andrea
A
De Francesco
Raffaele
R
Steinkühler
Christian
C
Di Marco
Stefania
S
D001224:Aspartic Acid / Q000378:metabolism; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry; D006655:Histone Deacetylases / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D012097:Repressor Proteins; D001224:Aspartic Acid; C409470:HDAC8 protein, human; D006655:Histone Deacetylases