Results
AlphaFold2 models help solve crystal structure of the inner
membrane reductase FoxB (CASP: T1058) by molecular replacement – by
IJ and
HT.
From email to the CASP Prediction Center: The model you sent me
(from the leading group) worked for molecular replacement and we finally
solved the structure by MR-SAD. I am still astonished that the human
expert model worked, while none of the server models we tried did (as
they were rather similar). Henning Tidow
Brief description of the target
Most microorganisms rely on the bioavailability of iron for their
survival. Due to the low solubility of ferric iron, they often use
secreted siderophores for the chelation and uptake of iron. In
Gram-negative bacteria, siderophores are usually taken up by
TonB-dependent transporters (TBDTs) located in the bacterial outer
membrane. The route of ferric-siderophores across the inner membrane
(IM) is less straightforward and differs across many bacterial species
and siderophore chemistries. Ferric-siderophore complexes are either
recognized by the dedicated periplasmic-binding proteins for delivery to
IM transporters for uptake into the cytoplasm or the iron is released
from the ferric-siderophore complexes by a reduction mechanism. The
Gram-negative bacterium Pseudomonas aeruginosa (an opportunistic
human pathogen) is able to take up Fe-siderophore complexes called
ferrioxamines via a dedicated TBDT FoxA in an act of siderophore piracy16. For several years
we also worked towards the structure determination of FoxB, another
protein of unknown function located in the same operon as FoxA. With the
help of the AlphaFold2 model generated in the course of the CASP14
competition, we were able to determine the structure of FoxB. It
possesses a novel fold with the transmembrane domain harboring two heme
molecules indicating a role as inner membrane reductase involved in
Fe-siderophore uptake and processing17.
Workflow of how an AlphaFold2 model helped to solve the
structure
Native FoxB crystals obtained in decyl-maltopyranoside (DM) diffracted
to approximately 5 Å resolution on average. Most of the crystals
belonged to the P212121space group. All crystals were obtained in 30% PEG 600, 0.1 M BICINE pH
9, 0.1 M ZnSO4. Use of a lipid-like peptide (LLP7) as
additive allowed us to collect several datasets extending to 3.4-3.5 Å18.
All molecular replacement attempts using distant homologs and homology
models thereof failed. We acquired Se-Met anomalous data to 3.5 Å
resolution, with anomalous signal to 4.5 Å as well as anomalous data at
the Fe edge with anomalous signal to 4 Å, as we knew from spectroscopic
characterization that FoxB most likely contained at least one heme
group. Combining all anomalous data provided some experimental phases
and allowed partial model building. A single FoxB was present in the
asymmetric unit. However, phasing power was only sufficient to build
approximately 60-70% of the backbone structure (Fig. 1). Although two
heme groups could be successfully placed, further tracing of the protein
backbone and confident sequence assignment was prevented by the low
number of Met residues (5/382) and low resolution of the datasets.
Lysozyme fusion at the N-terminus also resulted in crystals diffracting
to approximately 4.5 Å.