Pyruvate dehydrogenase k
inase (PDHK) regulates the act
iv
ity of the pyruvate dehydrogenasemult
ienzyme complex. PDHK
inh
ib
it
ion prov
ides a route for therapeut
ic
intervent
ion
in d
iabetes andcard
iovascular d
isorders. We report crystal structures of human PDHK
isozyme 2 complexed w
ithphys
iolog
ical and synthet
ic l
igands. Several of the PDHK2 structures d
isclosed have C-term
inal
crossarms that span a large trough reg
ion between the N-term
inal regulatory (R) doma
ins of the PDHK2 d
imers.The structures conta
in
ing bound ATP and ADP demonstrate var
iat
ion
in the conformat
ion of the act
ives
ite l
id, res
idues 316-321, wh
ich enclose the nucleot
ide
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if" BORDER=0 > phosphates at the act
ive s
ite
in theC-term
inal catalyt
ic doma
in. We have
ident
if
ied three novel l
igand b
ind
ing s
ites located
in the R doma
inof PDHK2. D
ichloroacetate (DCA) b
inds at the pyruvate b
ind
ing s
ite
in the center of the R doma
in,wh
ich together w
ith ADP,
induces s
ign
if
icant changes at the act
ive s
ite. Nov3r and AZ12
inh
ib
itors b
indat the l
ipoam
ide b
ind
ing s
ite that
is located at one end of the R doma
in. Pfz3 (an alloster
ic
inh
ib
itor)b
inds
in an extended s
ite at the other end of the R doma
in. We conclude that the N-term
inal doma
in ofPDHK has a key regulatory funct
ion and propose that the d
ifferent
inh
ib
itor classes act by d
iscretemechan
isms. The structures we descr
ibe prov
ide
ins
ights that can be used for structure-based des
ign ofPDHK
inh
ib
itors.