We performed four different simulations of LOV2-Rac1b system: combinations of light and dark states of the LOV2 domain with active and inactive states of the Rac1b domain. When comparing the dark-inactive to the dark-active state (only activating the Rac1b domain), there are several changes induced in the Rac1b do- main. These changes do not extend to the LOV2 domain. In the Rac1b domain, the p-loop stays in place, while both switch 1 and switch 2 move outwards, simul- taneously making a hydrogen bond between them. This hydrogen bond between switch 1 and switch 2, in both active and inactive states, is shown in figure 6.2.8a as an interaction between the Asp56Rac1 and Phe36Rac1 residues. The 19-amino
acid insertion region extends further, looping around the LOV2 domain so that the LOV2 domain sits tightly on top of the 19-amino acids insertion. The β2 and β3 sheets move outwards as well, breaking their connection to the α5 helix. Inter- estingly, the complexation partners of the Mg2+ ion change significantly. In the
dark-inactive state, magnesium is complexed to 2 phosphate groups, Thr17Rac1 and
2 water molecules from the solvent. In the dark-active state, however, magnesium complexes two phosphate groups, Thr35Rac1 and Asp56Rac1 with no water molecules
bound.
There are no significant changes induced in the LOV2 domain of the LOV2-Rac1b fusion protein, upon activating only the Rac1b domain. The Jα helix as well as the FMN surrounding show no major movement or events happening during the
Figure 6.2.6 Comparison of characteristic atom-pair distances between residues (A) Arg102p67 and
Asn26Rac1 in p67phox-Rac1, (B) Arg102p67 and Asn26Rac1 in p67phox-Rac1b, (C) Arg102p67 and
Ser22Rac1 in p67phox-Rac1, (D) Arg102p67 and Ser22Rac1 in p67phox-Rac1b, (E) Asp67p67 and
Gly30Rac1in p67phox-Rac1 and (F) Asp67p67and Gly30Rac1in p67phox-Rac1b. The inactive states are
6.2 Results 81
Figure 6.2.7 Comparison of characteristic atom-pair distances between (A) Gly81Rac1and Thr24Rac1,
(B) Lys82Rac1 and Tyr23Rac1, (C) Asn104p67 and Gln181Rac1 and (D) Ala27Rac1 and Gln181Rac1
Another important feature of the light-induced molecular mechanism of LOV2 is the rotational reorientation of the glutamine residue. This difference in states is visualized in figures 6.2.8c and 6.2.8d. It has been shown numerous times [16, 57– 61] that this flip of the Gln513LOV 2 residue is a critical step in LOV2 signaling.
Additionally, activating the Rac1b domain in the light-activated LOV2 domain, causes switch 1 to move outwards and switch 2 to move inwards, thus making a hydrogen bond between them. The p-loop, the 19-amino acid insertion and both β2 and β3 regions show no significant change when comparing light-inactive versus light-active simulations.
In the same fashion, activating the LOV2 domain connected to an activated Rac1b domain, causes a number of characteristic changes on the contact interface between the LOV2 and Rac1b domains. Most notably, when comparing dark-active to light- active states, the breakage of the Jα helix causes a significant contraction of the 19-amino acid insertion region. There is also noticeable twisting of the β2 and β3 regions, which causes a movement of switches 1 and 2 towards the solvent, making them more exposed.
The center of mass distances of the Rac1b and LOV2 subunits of the protein con- struct are shown in figure 6.2.9. As can be seen in the graphs, the distance between the two units is dictated by the Rac1b activation state. This is contrary to the ex- pected LOV2-control of the distance between the units. In the cases of dark-inactive and light-inactive states, the distances stay constant over the simulation times, while in the dark-active and light-active states, the distance between the LOV2 and Rac1b subunits decreases over time.
Figure 6.2.10 shows electrostatic potentials of the LOV2 and Rac1b subunits in different simulations. The most noticeable regions are the 19-amino acids insertion, from the Rac1b unit, which is positively charged, while the saddle area of the LOV2 domain, that sits on top of the 19-amino acid insertion, is negatively charged. These oppositely charged regions further strengthen the interaction between the subunits, through electrostatic contacts.
6.2 Results 83
Figure 6.2.8 Structural changes of LOV2-Rac1b fusion protein regions. (A) Hydrogen bond distance between switch 1 and switch 2 regions of the Rac1b domain. The inactive state is shown in black, while the active state is shown in red. (B) Structural changes of the Jα helix in the dark (blue) and light (red) states. Rest of the LOV2-Rac1b protein shown in green in the background. (C) Hydrogen bonding network around the FMN in the dark state, including the Gln513LOV 2 structural orientation together
with Asn482LOV 2and Asn492LOV 2. Structural changes of the same residues in the surrounding of the
Figure 6.2.9 Comparison of the center of mass distances between Rac1b and LOV2 domains in (A) dark-inactive (black) and dark-active states (red) and (B) light-inactive (black) and light-active (red) states.
Figure 6.2.10 APBS surfaces of the LOV2 and the Rac1b units of the 100ns structures from (A) dark-inactive, (B) dark-active, (C) light-inactive and (D) light-active simulations.
6.3 Discussion 85
6.3 Discussion
To analyze the early signaling mechanism of Rac1 and its spliced variant, Rac1b, we performed a number of simulations of these two proteins and their protein complexes and fusion constructs with other domains, in their inactive and active states. We will start the discussion with the analysis of the molecular mechanisms of isolated Rac1 and Rac1b domains, which we will further expand on with the analysis of their constructs.