 |
PDBsum entry 2pse
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Oxidoreductase
|
PDB id
|
|
|
|
2pse
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
References listed in PDB file
|
|
|
Key reference
|
|
|
Title
|
|
Crystal structures of the luciferase and green fluorescent protein from renilla reniformis.
|
|
|
Authors
|
|
A.M.Loening,
T.D.Fenn,
S.S.Gambhir.
|
|
|
Ref.
|
|
J Mol Biol, 2007,
374,
1017-1028.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
Due to its ability to emit light, the luciferase from Renilla reniformis (RLuc)
is widely employed in molecular biology as a reporter gene in cell culture
experiments and small animal imaging. To accomplish this bioluminescence, the
37-kDa enzyme catalyzes the degradation of its substrate coelenterazine in the
presence of molecular oxygen, resulting in the product coelenteramide, carbon
dioxide, and the desired photon of light. We successfully crystallized a
stabilized variant of this important protein (RLuc8) and herein present the
first structures for any coelenterazine-using luciferase. These structures are
based on high-resolution data measured to 1.4 A and demonstrate a classic
alpha/beta-hydrolase fold. We also present data of a coelenteramide-bound
luciferase and reason that this structure represents a secondary conformational
form following shift of the product out of the primary active site. During the
course of this work, the structure of the luciferase's accessory green
fluorescent protein (RrGFP) was also determined and shown to be highly similar
to that of Aequorea victoria GFP.
|
|
|
|
|
|
|
|
Figure 2.
Fig. 2. Structure of RrGFP. The condition used is labeled as
RrGFP:PEG/MPD in Table 1. Residues 7–226 (of 233 total) were
identified in the data. (a) Cartoon representation of a single
unit cell of the RrGFP crystal. The four protomers in each unit
cell are labeled I–IV. For each protomer, the N-terminus is
shown in blue and the C-terminus is shown in red. (b)
Superposition of RrGFP and AvGFP. The molecule at the center of
the β-barrel is the fluorophore. The primary sequences of the
two GFPs are 28% identical and 50% similar. PDB code 1EMA was
used for the AvGFP structure.^58 (c) Close-up cartoon
representation of the RrGFP fluorophore. The gray mesh
represents a σ[A]-weighted F[o]−F[c] difference map before
the inclusion of the fluorophore in the model phases, contoured
at 2.0 σ.
|
|
Figure 3.
Fig. 3. (a) The topology of RLuc8's α/β-hydrolase fold
domain. α-Helices are shown in blue, and β-sheets are shown in
red. Numbering/lettering of the sheets/helices is done with
respect to the standard for α/β-hydrolases,^33 and the
locations of the presumptive catalytic residues are marked. The
cap domain is an excursion from the fold pattern composed of
residues 146–230 in the luciferase. (b) The domains of RLuc8.
Shown are the locations of the cap domain (in gray) and
α/β-hydrolase fold domain (blue to red) in the context of the
crystal structure. (c) Close-up stereo cartoon representation of
the active site of the RLuc8:diammonium structure. The
presumptive active site residues are color coded with respect to
the average degree of enzymatic perturbation mutagenesis at the
site yields, based on published data.^22^,^23 Mutations at
green-, yellow-, and orange-colored residues were associated
with <1%, 1–10%, and 10–100%, respectively, of full
enzymatic activity.
|
|
|
|
|
|
The above figures are
reprinted
from an Open Access publication published by Elsevier:
J Mol Biol
(2007,
374,
1017-1028)
copyright 2007.
|
|
|
|
|
|
|
