bi9b00718_si_001.pdf (2.48 MB)
The Crystal Structure of Dph2 in Complex with Elongation Factor 2 Reveals the Structural Basis for the First Step of Diphthamide Biosynthesis
journal contribution
posted on 2019-10-14, 18:48 authored by Michael
K. Fenwick, Min Dong, Hening Lin, Steven E. EalickElongation
factor 2 (EF-2), a five-domain, GTP-dependent ribosomal
translocase of archaebacteria and eukaryotes, undergoes post-translational
modification to form diphthamide on a specific histidine residue in
domain IV prior to binding the ribosome. The first step of diphthamide
biosynthesis in archaebacteria is catalyzed by Dph2, a homodimeric
radical S-adenosylmethionine (SAM) enzyme having
a noncanonical architecture. Here, we describe a 3.5 Å resolution
crystal
structure of the Methanobrevibacter smithii (Ms) Dph2 homodimer bound to two molecules of MsEF-2, one of which is ordered and the other largely disordered. MsEF-2 is bound to both protomers of MsDph2, with domain IV bound to the active site of one protomer and
domain III bound to a surface α-helix of an adjacent protomer.
The histidine substrate of domain IV is inserted into the active site,
which reveals for the first time the architecture of the Dph2 active
site in complex with its target substrate. We also determined a high-resolution
crystal structure of isolated MsDph2 bound to 5′-methylthioadenosine
that shows a conserved arginine residue preoriented by conserved phenylalanine
and aspartate residues for binding the
carboxylate group of SAM. Mutagenesis experiments suggest that the
arginine plays an important role in the first step of diphthamide
biosynthesis.