The authors have declared that no competing interests exist.
In bacteria that live in hosts whose terminal sugar is a sialic acid, Glucosamine-6-phosphate deaminase (NagB) catalyzes the last step in converting sialic acid into Fructose-6-phosphate. These bacteria then use the Fructose-6-phosphate as an energy source. The enzyme NagB exists as a hexamer in Gram-negative bacteria and is allosterically regulated. In Gram-positive bacteria, it exists as a monomer and lacks allosteric regulation. Our identification of a dimeric Gram-negative bacterial NagB motivated us to characterize the structural basis of two closely related oligomeric forms. We report here the crystal structures of NagB from two Gram-negative pathogens,
Sialic acids are nine-carbon acidic sugars universally present on all mammalian cell surfaces as terminal sugars of glycoproteins and glycolipids [
Protein structures are often oligomers, and the oligomeric state of the protein has implications for its function. Usually, proteins with similar sequences that catalyze the same reaction have similar quaternary structures. In the sialic acid catabolism pathway, we recently reported on the variations in the quaternary structure of
The secondary structural elements are marked based on the structure of Pm-NagB. The residues marked in black bars are residues in the dimer interface. The residues marked in blue bars are the residues in the interface of subunits in the trimer interface.
Proteins were expressed from the recombinant plasmids, synthesized using gateway cloning technology [
Hanging-drop vapor-diffusion experiments were performed using a Mosquito robot (TTP Labtech). Crystals of
Tasciminate pH 5.0, 0.1 M Sodium Citrate tribasic pH 5.6, 16% PEG 3350 with 0.1 M Strontium Chloride. 6H2O as additive.
Diffraction data were collected to 2.3 Å resolution from a single crystal of
Values in parentheses are for the highest resolution shell.
Data Set | HiNagB | PmNagB |
---|---|---|
Data processing | ||
Space group | P21 | P21 |
|
103.41 144.30 131.14 | 84.80 79.57 85.30 |
|
90.00 92.07 90.00 | 90.00 109.13 90.00 |
Wavelength (Å) | 0.98 | 0.98 |
Resolution (Å) | 48.67–3.0 (3.1) | 41.91–2.30 (2.4) |
|
0.170(0.530) | 0.036 (0.203) |
|
0.11 (0.34) | 0.02 (0.14) |
Completeness (%) | 99.5 (99.4) | 99.2 (99.35) |
Mean I/sigma(I) | 6.45 (2.42) | 17.96 (4.41) |
CC1/2 | 0.98 (0.79) | 0.99 (0.98) |
Total No. of reflections | 143943 (14006) | 90060 (8289) |
No. of unique reflections | 76608 | 47456 |
Multiplicity | 1.9 (1.8) | 1.9 (1.8) |
|
46.1 | 42.7 |
Refinement statistics | ||
Resolution (Å) | 38.78–3.0 (3.1–3.0) | 37.1–2.3 (2.4–2.3) |
No. of reflections | 76519 | 47397 |
No. of reflections, test set | 3814 | 2275 |
|
0.19/0.24 | 0.20/0.23 |
No. of non-H atoms | ||
Protein | 3132 | 1072 |
macromolecules | 25020 | 8517 |
R.m.s. deviations | ||
Bond lengths (Å) | 0.008 | 0.004 |
Bond angles (°) | 1.30 | 1.01 |
Average |
||
Overall | 45.56 | 51.30 |
Ramachandran plot | ||
Favored (%) | ||
Outliers (%) | 97 | 96 |
PDB-ID | 0 | 0 |
7LQN | 7LQM |
The phases of the
Protein was purified as described for crystallography. 3 μl of
We used Molecular Dynamics (MD) simulations to computationally test our hypothesis and elucidate the roles of residues in the oligomerization of NagB. The
The monomer folds of both
The presence of alpha-8 helix (in
The overall topology of the monomer resembles a modified NADH-binding domain similar to
The RMS deviations are in Å units. The numbers in the parenthesis are the number of C-alpha atoms that were superposed. Superposition was carried out using the ‘super’ command in Pymol. The two monomeric proteins have a C-terminal helix missing. Hex- in the names corresponds to proteins that are hexamers, and mono- for those that are monomeric.
H.flu (hex) | Bb (hex) | E.coli (hex) | Pm(dimer) | Sm (mono) | |
---|---|---|---|---|---|
H.flu (hex) | |||||
Bb (hex) | 0.50(210) | ||||
E.Coli (hex) | 0.48(228) | 0.57(207) | |||
Pm (dimer) | 0.41(218) | 0.58(230) | 0.44(232) | ||
Sm (mono) | 0.87(176) | 0.69(177) | 1.0(190) | 0.86(181) | |
Bs (mono) | 0.99(220) | 1.0(218) | 1.0(218) | 0.94(218) | 0.97(185) |
H.flu–
A: Cartoon representation of the
To ensure that the purified proteins with quaternary structures were active deaminases, we conducted steady-state kinetic studies to check the activity of the enzymes. We used an ammonia Assay Kit–Modified Berthelot, Colorimetric detection from Abcam (ab102509), to study the release of ammonia. The results showed that the enzymes were active. A comparison of the KM values of the deaminases revealed very similar values (
Quaternary Structure | KM (mM) | |
---|---|---|
Hexamer | 0.55 ± 0.03 [ |
|
Dimer | 0.96 ± 0.2 | |
Hexamer | 0.83 ± 0.3 | |
Monomer | 0.13 ± 0.02 [ |
|
Monomer | 0.21 ± 0.03 [ |
We further investigated whether the hexamer seen in the
The crystallographic hexamer model was fit into the map. The model fits the map well and confirms the hexameric nature of HiNagB.
To understand the molecular basis for the observed difference in the quaternary structure of
First, we analyzed the trimeric interface and observed that the interface stabilizing energy for
To investigate the crucial role of these two residues on trimer formation, we performed in-silico mutations of these two residues, E164Q-Q210L, in the trimeric interface of the
Simultaneously, we compared the dimer and trimer interface of
A: Total stabilizing energy of trimer interfaces of
One would generally think a molecule made of the same sequence would crystallize in a space group where the molecular symmetry coincides with the crystallographic symmetry. In the case of
Furthermore, the relationship is not just a pure rotation from A to B but also a translation of 0.38Å along the rotation axis. In the case of A to C, the translation vector is 0.14Å, and in the case of B to C, it is 0.54Å. These are not just rotations but also include a translation along the axis. The combination of rotation and translation results in a slight asymmetry in the interactions between the A, B, and C subunits. This asymmetry is not conserved among the four different trimers in the asymmetric unit. One could say that while the protein’s oligomeric state is hexameric, there is some shear between the subunits. It is currently difficult to say if this shear impacts the activity or the function of the enzyme. The calculations of the angles and axis were performed using the draw_rotation_axis script in Pymol [
In the case of
We thank SOLEIL Synchrotron (Proxima-1 beamline) for providing the beamtime for data collection. We would like to thank the national cryoEM Facility at inStem. We would like to thank Dr. Sucharita Bose for her help with the initial data processing and guidance during the writing of the manuscript.