Figure 1.
MetJ activation and SAM binding mode.
(A) Schematic representation of MetJ activation. Binding of SAM increases the DNA affinity of MetJ manifold. (B) Chemical structure of SAM (top) and SAH (bottom). The molecules mainly differ in the positive charge on the sulfur atom. (C) Fit of crystal structures of MetJ in apo (1CMC, green) and holo (1CMB, blue) form. The only major difference between the structures is the conformation of the loops underlayed in gray. These loops are in direct contact with other MetJ molecules in the crystal lattice, and thus their conformation is unlikely to represent the true in-vivo configuration. The bound co-repressor SAM is shown in red.
Figure 2.
Force distribution at the protein - DNA interface.
Force in (A) MetJ-dna and (B) MetJ is distributed to specifically targeted key residues on the protein-DNA interface. Only Arg40 and the loop formed by residues 50–53 show significant response to SAM binding. Large parts of helix A and the are not part of the allosteric regulatory mechanism. Colors for the protein surfaces range from blue for
to red for high
; the DNA is displayed as sticks. For better overview, DNA was plotted into MetJ as well. (C) Correlations of changes in residue wise forces
for MetJ and MetJ-dna. Both systems show a highly similar force distribution pattern, with a correlation of R = 0.74. The line displays the fit to a linear model.
Figure 3.
(A) Changes in atomic forces, , mapped onto a cartoon representation of the protein structure. Colors range from blue for elements outside the allosteric network with
to red for force transducing elements with high
. Helix identifiers are printed onto the structure. (B) Network-like representation of pronounced changes in inter-atomic forces observed upon SAM binding. Edges connect non-bonded atom pairs with
. Forces between helix A and B are mainly propagated via side chain interactions. Propagation of the allosteric signal is highly anisotropic and directed, targeting individual residues at the protein-DNA interface while leaving large parts of the protein unaffected. (C) Changes in normalized pair-wise forces
plotted along the MetJ sequence. The secondary structure is marked as gray bars. The vertical line indicates the start of the second monomer. (D) SAM interacts with a specific set of MetJ residues. Plotted are the forces exerted by SAM on MetJ. Numbers of strongly affected residues are plotted, residues in dimer 2 are marked with a stroke. Error bars show the standard error over the whole simulation time. Arg42 and Glu39 are among the most affected residues. Residues 64′–67′ are located far away from the binding site, close to the N-terminal end of helix B.
Figure 4.
Subtle conformational changes induced by SAM binding.
(A) Force distribution for backbone hydrogen bonds of helix B indicates helix bending. Hydrogen bonds are plotted as sticks, with red for increasing and blue for decreasing O-H Coulomb interaction. Spheres show the atoms of Ala64, Cys58 and Asn53. The angle between these atoms increases
upon SAM binding. (B) Force transmission via a buried salt bridge and quenching of side chain fluctuations for MetJ-dna (left) and MetJ (right). Sticks display average coordinates over 300 ns in the apo (red) and holo (colors by atom type) configuration. Bending of helix B, supported by direct interaction with SAM, puts strain on the salt bridge formed by Glu59 and Arg43, visible as a small conformational rearrangement and high changes in pairwise forces. Fluctuations of Glu39 and Arg42 are quenched due to strong interaction with SAM, see also Fig. 4. (C) Relocation and stiffening of the Arg40 side chain for MetJ-dna (left) and MetJ (right). We measured tighter packing of the Thr37, Arg40 and Asn53 side chains and increased Arg40-DNA salt bridge formation.
Figure 5.
(A, B) MetJ-dna (A) and MetJ (B) show quenching of fluctuations upon SAM binding. Plotted are differences in backbone root mean square fluctuations between apo and holo structures for both monomers (red and blue curves). Positive values indicate stiffening upon SAM binding. Loop 1 and helix C are underlaid in gray. Differences in
can be explained by the fact that in the crystal structure, DNA is only in direct contact with loop 1 residues of one monomer. (C) Regions with decreased root mean square fluctuations (RMSF) color coded on the MetJ-dna structure. Colors range from blue for no change to red for strongly decreased fluctuations. Strong stiffening is observed for helix C (C′) and loop 1. Side-chain fluctuations of Glu39 and Arg42 are quenched due to direct interaction with SAM (zoom), whereas the stiffening of Arg40 is an indirect effect, compare to Fig. 3D. Stiffening spreads to large parts of helix A. (D) The most dominant mode of fluctuation derived from MD simulations of MetJ-dna mapped on a cartoon representation. The first three eigenvectors were used to generate the trajectory. The two overlaid structures show the extreme positions when projecting along these eigenvectors. Amplitudes of fluctuations were exaggerated for better visibility. Loop 1 and helix C are marked red. (E) The network propagating fluctuations between helix C and loop 1. PCA on residue averaged pair-wise forces
for apo MetJ-dna reveals a network of coupled interactions (see Methods). Edges represent residue pairs for which the first eigenvector is
. Edges within the first monomer are colored blue, those within the second red.
Table 1.
Changes in entropy upon co-repressor binding.
Figure 6.
Differences between SAM and SAH.
(A) Changes in residue-wise forces for MetJ-dna when replacing SAM by SAH. As expected, the strongest differences are observed for residues in close proximity to the charged sulfur atom. (B) Increased quenching of dynamics upon SAM binding. Plotted are differences in backbone RMSF for MetJ-dna in complex with SAM and SAH along the protein sequence for both monomers (red and blue). Positive values indicate increased stiffening for MetJ-SAM. The secondary structure is marked in gray. (C) Difference in residue wise forces
for MetJ-dna when substituting SAM by SAH for both monomers (red and blue). The secondary structure is marked in gray. Tyr11 and Ile28 (marked with arrows) show a high
in the second monomer for which the DNA is fully resolved in the crystal structure.