Beetle luciferases with naturally red- and blue-shifted emission

Supplementary Note 1

In the structure of RE_Ph, we were able to build the C-terminal domain of only one of the four molecules that were observed in the asymmetric unit of the P3₁21 crystal form. The C-terminal domain showed higher average B factors (69.4 Å², with several regions with values above 100) relative to the N-terminal domain within the same crystal form (60.3 Ų). The electron density maps of the C-terminal domains in some of the molecules were incomplete, and in other cases they showed very little to no definite electron density. The redundancy imposed by the non-crystallographic symmetry aided in the building and refinement of a reliable model, particularly of the N-terminal domain, and improved the agreement between the structural model and the determined electron density (Supplementary Fig. 1a and 1b).

Supplementary Note 2

The crystal structure of RE_Ph is an octamer in the P1 crystal form and a tetramer in the P3₁21 crystal form (Fig. 1a and 1b; Supplementary Fig. 2a and 2b). The octamer of RE_Ph is composed of dimers that are joined at the tetramer interface (Fig. 1c and 1d; Supplementary Fig. 2b). The tetramer is formed by interactions between M152, Y153 and F162 from the dimers (Fig. 1d). The degree of oligomerization of RE_Ph in solution was confirmed by using size-exclusion chromatography (SEC; Supplementary Fig. 2c and 2d). The estimated molecular weight (MW) of the wild-type (WT) RE_Ph from the SEC analysis was ≈ 240 kDa, which corresponds to the tetramer found in the P3₁21 crystal. The octamer with a predicted MW of 480 kDa was not observed in solution, which is probably a result of weak inter-tetramer interactions that dissociate in solution, but maybe stabilized at higher protein concentrations represented in the crystallization drops.

Mutations at the tetramer interface, which include double mutant Y153A and F162A, produced dimers with MW of 122 kDa. The monomer was not detected for any of the mutations at the tetramer interface. However, the monomer units within the dimers are held strong polar interactions at two contact points on the dimer interface, where R11 from one of the monomers forms H-bonding interactions with Y26, Y30, and N179 of the other monomer (Fig. 1c). The single mutant R11A in the dimer interface was sufficient to disrupt all interface interactions and exclusively generated monomers of RE_Ph with a MW of 61 kDa as determined by SEC analysis (Supplementary Fig. 2c). Interestingly, disrupting the dimer interface was sufficient to disassemble the tetramer interface. These results indicate that the overall interactions across the dimer interface are relatively stronger than those across the tetramer interface, and that the dimer is more stable than the tetramer. This can also explain why the octamer was not observed in solution as it might be less stable than the tetramer with more hydrophobic interface interactions.

Table S2 TD-DFT/MM emission for electronic transition between S1 and S0 for the model GBAv-closed-insert-R356 (resulting from the insertion of Arg 356). TD-DFT/MM done with 6-311G(2d,p) basis set and B3LYP functional on structure optimized at the same level of theory.

Supplementary Note 3

The origin of the difference in color of emitted light was investigated by introducing mutations in RE_Ph and GB_Av at the following positions:

• (1) Loop^348‒361 in RE_Ph, which corresponds to loop^351‒364 in GB_Av

• (2) Loop^521‒528 in RE_Ph, which corresponds to loop^523‒530 in GB_Av

• (3) Single mutants in the C-terminal domain, which includes S462F, E486K, and E486V in RE_Ph, and F465S, E488K, and E488V in GB_Av, which were found previously to alter the color emitted by luciferase from Luciola mingrelica (Modestova et al, 2014; Modestova & Ugarova, 2016).

Multiple mutants of the C-terminal domain introduced here did not alter the emission of both RE_Ph and GB_Av luciferases. However, loop^348‒361 was found to play an important role in determining the color of emitted light of RE_Ph and GB_Av. This loop is tightly packed against the N-terminal domain of GB_Av and other green-emitting luciferases relative to RE_Ph (Fig. 2c; Supplementary Fig. 6). Its high mobility in RE_Ph is a consequence of the restructuring of the hydrogen bonding as a result of the natural substitutions and insertions (Fig. 2c).

After detailed structural and sequence analysis of the luciferases, only few natural substitutions were found within the active site. Introduction of I351L, one of the natural substitutions found, did not change the emission of WT GB_Av (Fig. 3b). However, introduction of R337L red-shifted the emission of WT GB_Av from 538 to 580 nm (Fig. 3c), and it drastically decreased its intensity. Furthermore, the intensity of R337L was slightly enhanced after the introduction of a second mutation, I351L, which did not alter the emission of WT GB_Av on its own. Similar to the single mutant (R337L), the double mutant (R337L/I351L) shifted the emission from 538 to 580 nm. Therefore, R337 is clearly important the red-emission, and the substitution I351L enhances the intensity without affecting the emission energy.

Various mutations were introduced at position L334 in RE_Ph to investigate its role in color emission. All mutants at L334 blue-shifted the emission of RE_Ph from 623 nm for the WT enzyme to 610‒606 nm for L334R, L334K, L334E, L334Q, and L334F (Fig. 2e and 3a). The single mutant with the strongest blue shift (from 623 to 600 nm) is L334Q. The contribution of a second amino acid L348 does not appear to be significant in the fine-tuning of the color, because introduction of L348I mutation in RE_Ph did not alter the WT emission energy (Fig. 3a and 3c).

The role of loop^523‒530 in the C-terminal domain was investigated by characterizing the role of K524, T527, and K529 in the stability and color emission of GB_Av and its corresponding positions in RE_Ph, K522, T525, and K527, respectively (Supplementary Fig. 4). Loop^523‒530 is important for the opening and closing of the active site, and could play a role in the color of the emitted light and the enzyme stability. None of the mutants introduced in loop^521‒528 of RE_Ph, which includes K522R, K522E, K522Q, T525A, K527R, K527E, K527Q, and K527A, have altered the WT emission (Fig. 3C). Although K524A red-shifted the emission of GB_Av, T527A and K529A did not alter its emission (Fig. 3C; Supplementary Fig. 7b). Furthermore, residue K524 plays an important role in the stabilization of the closed conformation. In the closed conformation of G_LC in complex with oxyluciferin and AMP (PDB code: 2D1R) (Nakatsu et al, 2006), K526 in the C-terminal domain interacts with T292 in the N-terminal domain. The tight H-bonding interactions between K526 and T292 contribute to the microenvironment and tight packing around the active site of G_LC as is the case for GB_Av (Supplementary Fig. 4e). These interactions are absent in RE_Ph (Supplementary Fig. 4d) because T292 of GB_Av and G_LC is substituted with P287 in RE_Ph.

Thermal unfolding analysis by differential scanning calorimetry (DSC) measurements revealed different thermodynamic stabilities with calculated melting points (T_m) of 35°C for WT RE_Ph and 43 °C for WT GB_Av. Although similar thermodynamic stability for the GB_Av enzyme was determined in the absence or presence of substrates (T_m ≈ 43°C; Supplementary Fig. 7e and 7h), the addition of substrates changed the shape of thermograms and increased the stability of the WT RE_Ph from 35°C to 41°C with luciferin (Supplementary Fig. 7c and 7g). Two transitions were observed for the melt of RE_Ph in the presence of ATP with T_m values of 36°C and 41°C.

The introduction of mutant K524A in GB_Av slightly decreased its thermal stability relative to the WT enzyme with similar overall shapes of the thermograms (Supplementary Fig. 7e and 7f). However, the introduction of mutant K522A changed the overall shape of the thermogram of WT RE_Ph (Supplementary Fig. 7c and 7d). In addition, the thermal melt of K522A RE_Ph was not detectable in the absence of substrates and the signal was detectable only after the addition of substrate, luciferin or ATP.

The emission of WT GB_Av was pH-dependent, with a blue shift from 550 nm at pH 7.0 to 538 nm at pH 8.0 (Supplementary Fig. 7b). The introduction of K524A in GB_Av red-shifted the WT emission by ≈ 18 nm, from 550 to 573 nm at pH 7.0 and from 538 to 555 nm at pH 8 (Supplementary Fig. 7b). Increasing the pH above 8.0 did not alter the emission of GB_Av, but a decrease in pH below 7.0 inactivated the enzyme. The emission of RE_Ph was pH-independent for both WT and the K522A mutant with λ_max = 623 nm (Supplementary Fig. 7a).

Table S3 TD-DFT/MM emission for electronic transition between S1 and S0 for the model GBAv-closed-I351L (resulting from the I351L mutation in GBAv-closed before MD). The TD-DFT/MM calculations were performed with the 6-311G(2d,p) basis set with the B3LYP functional on a structure optimized at the same level of theory.

Table S4 TD-DFT/MM–calculated emission for electronic transition between S1 and S0 for the model GBAv-closed-R337L (resulting from the R337L mutation in GBAv-closed before MD). The TD-DFT/MM calculations were performed with the 6-311G(2d,p) basis set and the B3LYP functional on a structure optimized at the same level of theory.

Table S5 TD-DFT/MM emission for electronic transition between S1 and S0 for the model GBAv-closed-R337L/I351L (resulting from double R337L and I351L mutations in GBAv-closed before MD). The TD-DFT/MM calculations were performed with the 6-311G(2d,p) basis set and the B3LYP functional on a structure optimized at the same level of theory.

Supplementary Note 4

Computational study of WT GB_Av.

To understand the natural blue shift of the light observed for GB_Av relative to common green-yellow emitting luciferases, computational analyses were performed with one of the monomers extracted from the crystal structure of GB_Av. The model was generated by following the steps outlined in the method section.

As noted in the main text, GB_Av has its C-terminal domain in an open conformation. Two models were analyzed: one model with an open C-terminal domain (“GB_Av-open”) and one model was constructed with its C-terminal domain closed (“GB_Av-closed”; Supplementary Fig. 9). The closure of the C-terminal domain was simulated by using an umbrella sampling classical molecular dynamics (MD), as described in the method section. Random snapshots were extracted from 10 ns classical MD simulations on both models, and the value of the fluorescence emission maximum was calculated by time-dependent density functional theory/molecular mechanics (TD-DFT/MM).

The emission calculated from snapshots in GB_Av-open shows a red shift relative to the experimental value (Supplementary Table 2). The most pronounced difference is 15 nm (0.06 eV). These results — accounting for the level of theory employed (TD-DFT/MM) and the intrinsic error of the method — show good agreement with the experimental emission recorded for GB_Av. However, the calculated values are also close to the experimental emissions of other green-emitting firefly luciferases, which include the luciferases of Luciola cruciata at 560 nm (PDB code: 2D1R) and Photinus pyralis at 558 nm (PDB code: 4G36). Therefore, the crystal structure of GB_Av in the open conformation did not show the expected blue-shifted emission of GB_Av.

The umbrella sampling classical MD simulations used to close the C-terminal domain and to obtain GB_Av-closed has a flat free-energy profile, which reveals the absence of an energy barrier. The TD-DFT/MM-calculated emission of the resulting GB_Av-closed model is slightly blue-shifted relative to the experimental value and further from the green-emitting firefly luciferase wavelengths. The largest difference is 13 nm (0.06 eV) and therefore the closed model better reproduced the experimental blue shift observed with GB_Av luciferase.

During the classical MD simulation performed on GB_Av-closed, the oxyluciferin adopts a different position in the cavity than the one observed in simulations with models of green-emitting firefly luciferases (Supplementary Fig. 10). The reorganization of the electrostatic potential exerted by the residues that surround the active site in GB_Av leads to a blue shift in emission. As a result of its new position, oxyluciferin is far from S250 in GB_Av, the only residue of the active site that differs from the green-emitting luciferases (Supplementary Fig. 8). No hydrogen bond was observed between S250 and the oxyluciferin in GB_Av-closed. Consequently, the sole presence of S250 alone cannot explain the observed blue shift in GB_Av luciferase.

To further understand the effect of residue S250 on the emission of GB_Av, the corresponding in silico mutation F251S was included in the model G_Lc derived from the luciferase of Luciola cruciata (PDB code: 2D1R). The calculated emission of the F251S mutant of the G_Lc model shows a surprisingly small red shift, probably as a result of formation of a H-bond network between S251, a water molecule, and oxyluciferin. The calculated value of the emission is 566 nm (2.19 eV). The result indicates that the substitution of phenylalanine in G_Lc to serine in GB_Av is not responsible for the experimentally observed blue shift.

Computational study of mutations in GB_Av and comparison with RE_Ph.

To identify the reasons behind the red-shifted emission of RE_Ph relative to GB_Av, selected amino acid residues in GB_Av were mutated to those found in RE_Ph. Three residues were found to be important for the difference between these two luciferases. Two mutations are on residues located in the active site, R337 and I351 in GB_Av that correspond to L334 and L348 in RE_Ph, respectively. To model the effect of these substitutions, we took snapshot 1 of the GB_Av-closed model and manually replaced the residues to perform a 10 ns classical MD (three different simulations, with R337L, I351L, and double mutant R337L/I351L). Several snapshots were extracted from the MD calculation and the respective fluorescence electronic transitions were calculated.The single mutation, I351L, has nearly no effect on the emission of GB_Av-closed, similar to the experimental results (Fig. 3 and Supplementary Table 3). As predicted and in accordance with the similar properties of these residues, the mutant I351L did not alter the emitted color. For the other single mutant, R337L, a red shift in emission was observed after a simulation time of 6.8 ns, with a red shift between 25 and 45 nm relative to GB_Av-closed (Supplementary Table 4). The geometry of the active site was conserved, and no major changes in the oxyluciferin binding pocket were observed. However, during the MD simulation, R218 moved and formed a hydrogen bond with oxyluciferin (Supplementary Fig. 10). An analysis of the emission of the calculated value for single mutant R337L and the experimental values of the emission of single mutant R337L and double mutant R337L/I351L showed good agreement (snapshot 4 gives a value of 577 nm that is less than 5 nm different from the experimental value of 580 nm; Supplementary Table 4). As expected, the substitution of a charged residue close to oxyluciferin with an aliphatic residue has a strong effect on the emission.

The double mutant, R337L/I351L, shifted the emission to the red after a MD simulation time of 7 ns (Supplementary Table 5). A large red-shift (>50 nm) was observed for this double mutant relative to GB_Av-closed. This shift is not related to the geometry of the active site, which was conserved during the MD simulation without major changes in the position of oxyluciferin. However, displacement of R218 to form hydrogen bond with oxyluciferin was observed again. The calculated emissions are in a good agreement with the experimental results of double mutant R337L/I351L (See Table 5 in which experimental value = 580 nm, and calculated value = 595 nm). In the double mutation, mutation R337L appears to be more important relative to I351L to explain the red shift.

Finally, the natural insertion R353 in loop^348‒363 of RE_Ph was studied in GB_Av-closed_. The presence of R353 appears as the most viable reason for the conformational changes observed for loop^348‒363, including its high B factor. To model this motion, arginine was inserted at position 356 in snapshot 1 of GB_Av-closed. A longer MD simulation of 30 ns was performed to extract several snapshots. A small red shift of ≈ 15 nm after 20 ns was observed (Supplementary Table 6). Even after a simulation time of 30 ns, the insertion of R356 did not change the conformation of the loop, in contrast with its position in the crystal structure of RE_Ph. This result may be due to multiple amino acid substitutions that are responsible for the motion of loop^351‒364, which includes the substitution of E354 in GB_Av with N351 in RE_Ph (Fig. 2e and 2f).