SUPPORTING INFORMATION
Structure and functional analysis of the siderophore periplasmic binding protein from the
fuscachelin gene cluster of Thermobifida fusca
Kunhua Li,1 and Steven D. Bruner1*
1
Department of Chemistry, University of Florida, Gainesville, Florida 32611, United States
*
Correspondence to: Steven D. Bruner, Department of Chemistry, University of Florida, PO box
117200, Gainesville, Florida 32611, United States. E-mail: bruner@ufl.edu
S1. Cloning of full length FscJ, expression and localization
Full-length fscJ (fscJ_F) was amplified by using PCR from T. fusca genomic DNA with the
primers fscJ_N1 (GCG GGATCC ATG GGG TTG GGA AAG) and same fscJ_Cstop. FscJ_F
was expressed under similar conditions as the truncated proteins. 1 L Cell lysate was centrifuged
at 10,000 g for 2 h and the pelleted inclusion body discarded. Cellular membrane was harvested
with an additional 1 h centrifugation at 25,000 g. The membrane was washed with 800 mM
NaCl, 20 mM HEPES, pH 8.0, and resuspended in 120 mM NaCl 20 mM HEPES, pH 8.0, with
0.05% dodecylmaltoside. FscJ_F was purified through a Ni-NTA based IMAC method. Histag
Western Blot was used to confirm the expression; purified proteins from the membrane fraction
were analyzed with SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF)
membrane (Bio-Red). Poly-histidine monoclonal antibody (Sigma) was used as the primary
antibody and goat anti-rabbit IgG-HRP was used as the secondary antibody. Western Blot signal
1
was developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Forma) and
detected on a KODAK RP X-OMAT film (Figure S1D).
S2. FscJ SAD-MR based experimental phase improvement
FscJ shares a low sequence identity compared with all other published homolog structures (Table
S1) and direct molecular replacement do not yield any potential solution. FscJ has only two
methionines in the total sequence of ~300 amino acids which both located on the N terminus of
the protein, making standard SAD a challenge. Harvested SeMet-FscJ crystals were screened for
anomalous signal and the best data set was indexed to 3.2 Å resolution in space group I222. The
anomalous measurability of the data set was 0.1 at 8.3 Å resolution and 0.02 at 6.2 Å resolution
from phenix.xtriage analysis. Se sites identification was initiated with phenix.hyss at 6.6 Å
resolution and phases were calculated with an initial figure of merit of 0.38, which can be
improved to 0.64 with density modification at 5.6 Å. A poly-alanine model was built manually
into the 5.6 Å electron density map and combined model and heavy atom sites yield an
interpretable 4.5 Å map from phenix.autosol. Final 3.2 Å map was achieved through combining
the HA sites from 5.6 Å and model from 4.5 Å, and corresponding full-length poly-Ala model
was built in Coot based on predicted protein 3D structure from Rosetta server (RMSD = 3.6 Å
compare with the final structure). With a relatively low overall resolution and inaccurate phase
information, the poly-alanine model can be refined to Rfree/work =0.44/0.49% and the assignment
of the corresponding sidechain is rather difficult. A detailed SAD-MR strategy adapted from
EMBL-Hamburg Auto-Rickshaw was used to improve the model. The Rfree value dropped below
40% after six rounds of MR-SAD (Table S3), and the resultant model was applied into molecular
replacement for native data sets in higher resolution.
2
Figure S1 (A) size exclusive column purification of FscJ (with hexa-Histag). FscJ elution time is
similar to proteins with molecular weight ~ 60 kDa; (B) SDS-PAGE of purified FscJ
with/without hexa-Histag (left/right line) with an observed molecular weight between 43 ~ 56
kDa; (C) MALDI-TOF-MS analysis of FscJ (with hexa-Histag) provide a single mass of 35.6
kDa and; (D) SDS-PAGE and Western Blotting analysis of full-length FscJ.
Figure S2 The anomalous difference map (3.0 σ contour level) calculated from (A) FscJ Se-Met
data set (blue); and (B) FscJ Hg soaking data set (magenta). Hg atoms bind with His168 on the
protein outer surface.
3
Figure S3 (A) Hexa-Histag stabilizes FscJ dimer interface; (B) conformation changes near
central substrate binding site in F1 (light blue) and F2 (green) structures.
4
Figure S4 ITC binding analysis of purified ferric-fuscachelin A complex against hexa-Histag
free FscJ. (A) wild type FscJ; (B) E279A/N280A mutant; and (C) E302A/D310A mutant.
Table S1 Protein structure comparison using the Dali Server. FscJ monomer (P41-A) was used as
the search model and top results are shown according to the Z-score and alignment root-meansquare deviation. (RMSD, Å)
pdb
Z-score RMSD
lali
n. res
%id
Siderophore (Type)
3tny-A
23.0
3.4
251
280
28
Schizokinen (Hydroxamate)
4fkm-A
21.3
3.4
247
256
19
Ferrioxamine-B (Hydroxamate)
4b8y-A
20.9
3.7
250
277
22
(Hydroxamate)
3lhs-A
20.7
3.9
258
291
21
Staphyloferrin A (Hydroxamate)
3mwf-A
20.7
3.8
250
292
26
Staphyloferrin B (Hydroxamate)
3be5-A
20.7
3.8
255
294
24
(Unknown)
3eiw-A
20.6
4.0
260
292
20
Staphyloferrin A (Hydroxamate)
5
Table S2 Primers for FscJ site-directed mutagenesis
Mutant #
Site(s)
Primers
1
E279AN280A CAGCAGCCGGCGGGGgcggccTTCGCTGCCTTCTAC
2
E302A
GTCATCTTCTACgcgACCGACGCCCAG
2
D310A
CAGGAGAACCCCgccCCGTTCACCGAG
Table S3 Rwork/free in FscJ MR-SAD phasing and building cycles
Cycle
Built resn. # (BACANNEER)
Rwork/free
0 (poly-Ala)
-
0.4399 / 0.4951
1
474
0.3859 / 0.4873
2
522
0.3682 / 0.4834
3
537
0.3317 / 0.4593
4
546
0.3491 / 0.4653
5
549
0.3338 / 0.4303
6
556
0.3079 / 0.3859
6