1.6 The heme acquisition system Has
2.3.2 The structure of the apo-HasA-HasR complex
The overall structure of the HasA-HasR complex (apo complex) is the same as is seen for the HasA-HasR-heme complex (holo complex). The most striking dierence is the side chain orientation of HasA-H83, that in the holo complex occupies a position, that would lead to a clash with the porphyrin ring if the heme were in its binding site on HasA. In the apo complex, it is in the "normal" position, i.e. has the same orientation as in the crystal structure of holoHasA alone.
(a) Electrostatic surface of HasR
(HasA is shown for orientation). (b) Electrostatic surface of HasA (HasR is shown for orientation).
(c) Electrostatic surface of
the contact areas on HasR. (d) Electrostatic surface of
contact area 1 on HasA. (e) Electrostatic surface of contact area 2 on HasA.
Figure 2.11: Electrostatic surfaces of the HasA-HasR-heme complex as calculated by Pymol (positive potential blue, negative red).
(a) HasA-HasR-heme complex (b) HasA-HasR complex
(c) Heme pocket of the
HasA-HasR-heme complex (d) Heme pocket of the HasA-HasR
complex
Figure 2.12: Comparison of the holo and apo complexes. The overall structures are almost indistinguishable except for the heme molecule. In the close up view of the heme binding site the dierent orientations of HasA-H83 can be seen.
3.1 Isoleucine 671
3.1.1 Motivation
To get an idea about what might be the force driving the heme transfer from HasA to HasR, we analyzed a superposition of the HasA-HasR-heme complex with the holoHasA structure to check whether there are clashes between HasR in the complex conformation and HasA in its solution conformation. Except for loop HasA-A1, there is no clash with HasR and no dierences in the conformation of HasA except for the side chain orientation of HasA-H83, as mentioned in 2.3.1.
There is only one residue of HasR that would make a clash with the heme if it were bound to HasA: isoleucine 671 sterically clashes with the C ring of the porphyrin. We hypothesized that during binding of holoHasA to HasR this residue might be "kicking"
the heme out of its binding site in HasA and down towards HasR.
Therefore, this residue was mutated to glycine, alanine, valine and leucine and the heme uptake properties of the resulting mutants analyzed. The I671G mutant grows on heme as the sole iron source, but is not able to extract heme from HasA, although it does bind the hemophore, suggesting that the proteins bind but the heme is not transferred. The I671A, I671V and I671L mutants behave more like the wild type, i.e. can take up heme from HasA.
3.1.2 Methods
The I671G and I671A mutants were chosen for crystallization. The mutant complexes were formed, puried and crystallized in the same way as the wild type complexes. The sharing of work between Philippe Delepelaire and the author was the same as described above (see 2.2.1).
for the wild type complex. The crystals had the same space group and very similar cell parameters, so there was no need for molecular replacement. The data were directly rened against a model containing the 4 protein chains from from the wild type HasA-HasR-heme complex but without the heme. Immediately after rigid body renement, positive dierence density indicating the position of the heme showed up in the binding site of HasA in case of the HasA-HasR(I671G)-heme complex and in both heme binding sites in case of the HasA-HasR(I671G)-bisheme complex. Same as in the wild type, the position of the iron could be clearly seen. However, the orientation of the porphyrin was less clear. The heme molecules were therefore placed in the same position as the one in the holoHasA structure (PDB code 1DK0). During renement, the side chain of HasA-H83 ipped back to the position that is also seen in holoHasA. For the hemes in the binding site on HasR, the same position was chosen as for the wild type complex. Table 3.1 shows the data statistics and Table 3.2 the renement statistics for the HasA-HasR(I671G)-heme complex and the HasA-HasR(I671G)-bisheme complex.
I671A
The HasA-HasR(I671A)-heme complex also crystallized in the same space group and a dataset was collected, but could only be processed to around 3.2 Å. Positive dierence density was visible in the heme binding site on HasA. However, the electron density map was very poor and the R-Factors did not improve to values lower than 30 and 33 % for Rwork and Rfree, respectively. Therefore, we do not have condence in this structure.
3.1.3 Results
I671G
As can be seen in Figure 3.1 (a) the secondary structure elements of both proteins are the same as in the wild type complexes, and also the interfaces are very similar.
The heme is bound to HasA and has not yet been transferred to HasR. It is coordinated only by HasA-Y75 which is stabilized by a hydrogen bond from HasA-H83. This coordi-nation is the same as in holoHasA, but as in the wild type complexes the loop HasA-A1 is
Dataset I671G I671G2
Wavelength [Å] 1.016 1.000
Resolution [Å] 40-2.80 (2.87-2.80) 50-2.60 (2.75-2.60)
I /σ 13.11 (0.70) 9.77 (1.68)
Reections 1130832 (28161) 761743 (87683) Completeness [%] 98.0 (79.4) 99.6 (98.0) Redundancy 12.2 (5.12) 6.4 (4.7) Rmeas[%] 20.2 (238.7) 12.5 (64.8) Rmrgd-F [%] 19.9 (261.4) 12.1 (75.9)
Table 3.1: Data statistics for the HasR(I671G)-heme complex and the HasA-HasR(I671G)-bisheme complex.
disordered. Figure 3.1 (b) shows the position of the heme on HasA with the sixth coor-dination site lacking so that the heme is coordinated only by one protein residue. Figure 3.1 (c) shows the same binding site but with the holoHasA structure superimposed for clarity. It can be seen that the position of the coordinating residues is the same in both structures except for loop HasA-A1. The occupancy of the heme was rened to 90 %.
I671G "bis-heme-complex"
From the structure of the wild type complex we concluded that it is not possible to have two hemes bound to the complex, because the one bound to HasA is too close to I671.
This restriction should then not hold for the I671G mutant. Therefore, we crystallized a complex of holoHasA and holoHasR(I671G). The crystals were of a darker red color than those of the complexes containing only one heme molecule and positive dierence density was seen in both heme binding sites. The occupancy of the heme was rened and all four heme molecules in the asymmetric unit are occupied to > 90 %.
(a) Overview
(b) Heme binding site (c) Heme binding site compared to that in holoHasA (PDB code 1DK0, gray)
Figure 3.1: Structure of the HasA-HasR(I671G)-heme complex
(a) Overview
(b) Heme binding site (c) Heme binding site compared to that of the HasA-HasR(I671G)-heme complex (gray)
Figure 3.2: Structure of the HasA-HasR(I671G)-bisheme complex
Renement I671G I671G2
resolution [Å] 39.2-2.8 (2.83-2.80) 49.4-2.6 (1.63-2.59) no. of reections 92482 (2123) 118245 (3403)
completeness [%] 98.1 (71) 99.6 (92)
Rwork [%] 22.6 (46.6) 22.99 (35.0)
Rfree [%] 26.2 (48.3) 26.03 (36.5)
model composition
protein residues 1831 1848
heme atoms 86 172
water molecules 13 43
B-factors
HasR 101.8 68.3
HasA 119.3 89.4
Heme on HasR 100.5
Heme on HasA 121.3 89.9
deviation from ideal values
bond lengths [Å] 0.01 0.07
bond angles [◦] 1.08 0.71
Ramachandran plot
favored regions [%] 89.7 94.0
allowed regions [%] 99.1 99.5
Table 3.2: Renement statistics for the HasR(I671G)-heme complex and the HasA-HasR(I671G)-bisheme complex.