1.6 The heme acquisition system Has
2.2.4 Structure solution
holoHasA-HasR
The rst structure to be determined was that of the HasA-HasR-heme complex. Although it was expected (and later conrmed by the structure) that the 3D structure of HasR was very similar to those of the siderophore receptors, the sequence similarity was too low for molecular replacement [76]. Therefore, selenomethionine labeled HasR was produced using an E. coli strain in which methionine synthesis was repressed. This labeled HasR
Figure 2.4: Fluorescence scan of the HasA-HasR-heme crystal used for phasing.
The energies suggested by the scan and indicated below the graph were used for data collection.
behaved exactly as the wild type in purication, complex formation and crystallization.
A uorescence scan (see Fig. 2.4) of a crystal conrmed the presence of selenomethio-nine and allowed determination of peak and inection wavelengths.
A complete MAD dataset was collected at the SLS beamline X06SA to a resolution of 3 Å. Table 2.1 shows the data statistics for all three wavelengths. The diraction statistics were good to around 3.1 Å resolution (see Tab. 2.1), whereas the anomalous signal was usable to 5 Å only (see Fig. 2.5).
Data processing
Data were processed with XDS [68] to a resolution of 3.1 Å in space group F222 with unit cell axes a = 157, b = 163 and c = 595. Due to a beamline problem, the data collection of the rst dataset (remote high) stopped after 135◦. After the beamline was in operation again, a second dataset was started to collect the missing 55◦. However, during data processing it became clear, that this second part scaled badly with the rst, indicating that the beam was not yet stable. Therefore, this second part was not used for phasing.
Redundancy 2.8 (2.1) 3.6 (2.8) 3.7 (2.9) 13.8 (9.4) 6.5 (4.9) Rmeas [%] 11.0 (32.8) 15.5 (40.7) 16.1 (46.0) 22.5 (102.1) 16.6 (98.1) Rmrgd-F [%] 13.3 (38.8) 14.9 (39.9) 15.8 (45.4) 14.7 (59.1) 16.7 (77.7)
Table 2.1: Data statistics for the three wavelength MAD dataset of the wild type HasA-HasR-heme complex and the native datasets for the wild type HasA-HasR-HasA-HasR-heme and HasA-HasR complexes
This explains the lower completeness and redundancy of the remote high data compared to the other two. The other statistics however are good, which can be explained by the high symmetry space group. The R-Factors and the signal-to-noise ratio are even best for the remote high dataset. This could be explained by the fact, that this dataset was collected rst. However, the crystal was big enough so it could be moved to a new spot for each of the other two datasets, which should have reduced this eect. The data were scaled with XSCALE with the remote high data set as reference.
Phasing
The programs SHELXC/D/E [115, 117119] were used for substructure determination using the GUI hkl2map [102].
The statistics from SHELXC suggested, that the anomalous signal was usable to 4.5-5 Å (see Fig. 2.5 (a)).
The easiest way to determine the optimal resolution cuto for the substructure deter-mination, is to simply run SHELXD with dierent input values and compare the results (see Fig. 2.5 (b)). In this case, it was run with inputs from 4 to 6 Å in 0.1 Å steps and the correlation coecient (CC) between observed and calculated structure factors of the best solution was noted for each input. The CC was highest for 5 Å.
A phasing attempt was undertaken with SHELXE and as seen in Fig. 2.5 (c) the correct hand can be determined. However, the density from SHELXE was very dicult to interpret and therefore initial phases were calculated with SHARP [19]:
The rst 16 of the heavy atom sites from SHELXE were used as input and SHARP was run with renement of sites and their occupancies, renement of f0 and f00 and solvent attening assuming 70 % solvens content. The resulting map was much more detailed
(a) SHELXC
(b) SHELXD
(c) SHELXE
Figure 2.5: Results from SHELXC/D/E
than that from SHELXE.
After one round of density modication using PIRATE [32], several programs were tried for automated model building. Of all programs tested only RESOLVE [125] gave usable results. It built approximately 20 % of the cα trace of the barrel and large parts of HasA. Also, and maybe even more importantly, it gave a map that was considerably more clear allowing manual model building in COOT [42] to complete the model of HasR.
Also, HasA could be placed in this map using PHASER [97].
Renement was done by REFMAC [99], CNS [21] and PHENIX [1, 2].
The nal R-Factors reached with these Se-Met-data were Rwork = 27% and Rfree = 30 % at 3.1 Å resolution.
Later, a native dataset was collected at the ESRF beamline ID23-1 to a resolution of 2.7 Å and used for nal renement by PHENIX. The renement statistics are shown in Table 2.2.
model composition
protein residues 1832 1832
heme atoms 86
water molecules 57 19
B-factors
HasR 95.7 80.7
HasA 112.1 95.4
heme 90.7
deviation from ideal values
bond lengths [Å] 0.01 0.01
bond angles [◦] 0.61 1.13
Ramachandran plot
favored regions [%] 92.4 89.6
allowed regions [%] 99.2 99.5
Table 2.2: Renement statistics for the wild type HasA-HasR complexes
apoHasA-HasR
Proteins for the apoHasA-HasR complex were obtained as described in section 2.2.2. The complex was crystallized in the same condition (50 mM Tris pH 8.0, 2 M NaCl) as the holo complex and gave similar looking crystals (except for the color, see Fig. 2.3 (b)). One crystal diracted to 3 Å resolution (see Table 2.1) and could be processed in the same space group (F222 with a = 158, b = 165, c = 597). After processing with XDS, the test set for the Rfree reections was transferred using the program CAD from the CCP4 suite.
The data were rened with PHENIX against a model from the holo complex containing only the protein chains and the model was adjusted manually in COOT. The renement statistics of the resulting model are shown in Table 2.2.
(a) density from sharp after
sol-vent attening (b) density from sharp after solvent attening (detail)
(c) density after resolve (d) density after resolve (detail)
(e) nal rened map (f) nal rened map (detail) Figure 2.6: Maps from dierent stages of the structure determination process.
The success of the phase improvement by RESOLVE (c and d) in comparison to the outcome of phasing by SHARP (a and b) is very pronounced. Also, the phase improvement achieved during renement is nicely seen by comparing (d) to (f).
The nal model contains one molecule HasR (residues 113-865), one molecule HasA (residues 1-28 and 39-174) and one heme molecule. Figure 2.7 (a) shows an overview of the structure. HasR ts to the overall structure known from the siderophore receptors:
a 22-strandedβ-barrel closed by an N-terminal plug domain. The model starts at residue N113, the N-terminal extension as well as the TonB box could not be modelled due to missing electron density.
The overall secondary structure arrangement of HasA matches that in the solution structure of apoHasA and the crystal structure of holoHasA. The loop HasA-A1, which is seen in two dierent orientations in the apo and holo structures (see 1.10), is disordered in the complex, i.e. has no density.
A superposition of the holoHasA structure on HasA in this complex reveals only one dierence: the side chain of H83 is ipped in the complex and the H-bond to HasA-Y75 is broken. The position of HasA-H83 as it is seen in the complex would produce a clash with the heme if it were in its binding site on HasA.
The heme is bound to HasR and coordinated by the two histidines H189 and H603 as shown in Figure 2.7 (b). In addition, there is one H-bond between the porphyrin and residue T359 of HasR. Apart from that, there are no bonds stabilizing the heme in its binding pocket. Consequently, the density of the porphyrin ring is rather diuse: the position of the iron itself could be determined well, but the exact position of the ring and the orientation of the propionate groups was dicult to determine. All 8 orientations were tried in parallel during renement and the one that gave the best results was kept for further renement. It also is the one that seemed most convincing physiologically, for its propionates point towards the solvent. Based on an analysis of heme-binding proteins in the Protein Data Bank [10] the iron-N2 distances were restrained to 2.2 ± 0.1 Å.
The total interface area between the two proteins is 1743 Å2 and can be interpreted as being composed of two spatially separate contact areas. The rst is formed by loops L6-9 and the second by loops L2, 3, 10 and 11. Contact area one contributes 1108 Å2, contact area two 646 Å2. Six hydrogen bonds are formed by loop L2, two by L3, one by L6, one by L7, ve by L8, six by L9 and two by L10. In addition, there is one salt bridge formed by L9. A complete list of bonds between HasA and HasR can be found in Table 2.3.
Figure 2.8 shows the complex with the loops colored according to their assignment to
(a) Overview (b) Heme binding site
Figure 2.7: Structure of the HasA-HasR-heme complex (HasR barrel blue, plug orange, HasA pink, heme green). The coordinating histidines (H189 and H603) of HasR as well as HasA-Y75 and HasA-H83 are shown.
HasA distance [Å] HasR loop
Table 2.3: List of H-bonds and salt bridge in the HasA-HasR interface calculated by PISA [78].
the contact areas: loops of contact area one are colored lightpink, those of contact area two lightblue. L7, which contains the histidine 603, is colored cyan and loops not making contact to HasA (i.e. loops 1, 4 and 5) are colored black. Contact area one is larger than contact area two and was found by binding studies to be more important for the binding of HasA to HasR, although both need to be intact to allow heme uptake by cells from holoHasA [7].
(a) Overview (b) Close up view of the contact areas with H-bonds indicated by gray dots and the salt bridge by red dots
Figure 2.8: Interface between HasA and HasR.
Contact area one lightpink, contact area two lightblue, L7 cyan, non contacting loops black.
Figure 2.9: Regions on HasA and on HasR that have been identied by mutational studies [7, 86] to be important for binding of HasA to HasR
Mutational studies have been performed both on HasA and on HasR to determine the areas important for binding of the two proteins. On HasA, twoβ-strands were identied, that are both necessary for binding to HasR (see section 1.6.3 and [86]). On HasR, ve amino acid deletions have been made in all predicted extracellular loops [7] and it has been found that the loops most important for HasA-binding are L6, L8 and L9, which form the contact area one (see 2.3.1). The structure of the complex shows, that the regions identied in these studies interact directly with each other: loop L8 contacts strand HasA-S3 and loop L9 contacts strand HasA-S5. All ve regions identied by these mutagenesis studies are highlighted in yellow in Figure 2.9.
The two girdles of aromatic residues that are typical for all membrane proteins are present in HasR as well, as can be seen in Figure 2.10 (a). The aromatic residues (shown in yellow) are thought to determine the exact position of the protein in the membrane, i.e. the aromates lie at the interface between lipid and headgroup. A striking feature of the HasR receptor surface (see Fig. 2.10 (b)) is a band of basic residues just outside the outer aromatic girdle. These basic residues could be well suited to interact with the phosphates of the LPS layer.
The extracellular loops of HasR have a strong positive charge, as shown in Figure 2.11 (a). The interface contains a total of 16 positively charged residues (9 lysines and 7 argininges) and only 5 negatively charged ones. Four Lysines are located at the top of L6, 8 and 9. One Lysine residue is located at the top of L3. This strong positive potential
(a) Cartoon representation with the two girdles of aromatic residues colored yellow.
(b) Electrostatic surface (positive po-tential blue, negative red). Note the band of positive potential just outside the membrane region.
Figure 2.10: Surface properties of the HasA-HasR-heme complex.
The black lines mark the position of the lipid layers of the outer membrane, the LPS layer is outside the black lines and reaches at least as far as the band of positive potential seen in (b).
2.11 (d) and (e).
The heme binding pocket of HasR (see Fig. 2.11 (c)) is rather apolar as might be expected for the mainly apolar heme molecule, but with a few negatively charged residues to one side of the binding pocket. This might help in accommodating the propionate groups.