Beetle luciferases with naturally red- and blue-shifted emission

New crystal structures of red- and green blue–shifted beetle luciferases reveal that the color emission mechanism is dependent on the active site microenvironment affected by the conformation of loop regions.

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Reviewer #1 (Comments to the Authors (Required)): The manuscript describes the structures of luciferase enzymes from two species that have not previously been structurally characterized. The structures are used to inform mutagenesis studies to examine the role of several loops in influencing the wavelength of emitted light.
The structures include a red-shifted luciferase from Phrixotrix hirtus and a blue-shifted enzyme from A. vivianii. The structure of red-shifted RE(Ph) is at low resolution (3.0Å and 3.6Å), while the structure of the blue-shifted BG(Av) enzyme is at higher resolution (1.9Å). Both structures show high degrees of mobility of the C-terminal subdomain, a feature that has been observed previously in other luciferase structures, as well as other members of this broader enzyme family. Unfortunately, none of the structures contain bound ligands, a feature that also may relate to the high C-terminal subdomain disorder. The structural analysis is good; however the rather low resolution of the RE enzyme should be more fully discussed along with the potential limitations this provides for conclusions and analysis.
The use of organizational headings (Results, Discussion, etc...) would make it easier to read this manuscript.

Major concerns.
There is a great deal of discussion of the oligomeric state of the enzymes, including the octameric state of the RE enzyme observed in the P1 crystal. From the image, it appears this is simply a crystallographic packing of two tetramers. The authors should use the PISA informatics (through server or COOT) to suggest which of the subunit interactions are deemed to be biologically significant.
The disorder of the C-terminal subdomains has been observed before and should be noted more clearly that this is not unique to the current structures. Further, this raises the question of whether the "open" orientations observed are in fact real "states" or are simply positions where the crystal lattice stabilizes the position of the flexible sub-domain.
The paper ignores prior work that demonstrates the use of the second conformation for the oxidative reaction. This has been shown convincingly by both biochemical and structural work with the P. pyralis enzyme. On page 3, for example, the authors simply refer to "reversible opening and closing" without explaining that there are in fact two catalytic states. Further, the paper ignores some recent studies of red-shifting mutants of P. pyralis.
Pg 2 notes that "a single mutation, R11A, was sufficient to disrupt the octamer..." This is not shown by the data in figure S1c that shows that multiple mutations are required to shift the SEC peak to a monomer.
On middle paragraph of page 3, it is not clear what drove the authors to examine the 351-364 loop. Given the low resolution, how much confidence is there in the alternate positions observed. Further, how does this loop behave in alternate structures?
A statement (pg 4) like "We were able to discern with sufficient accuracy the structure of ..." suggests that the electron density here is likely weak. No density is shown in the manuscript and inclusion would allow the reader to judge for him or herself the quality of the data.
Minor concerns.
The authors should consider whether it is correct to use an adjective "N-terminal" to describe the domains. Either they are the N-and C-terminal domains or the N-and C-terminus. As the latter can refer simply to the two ends of the protein, I prefer including domains. The authors also use Ndomain and C-domain on pg 2; that is acceptable also as long as they are consistent. Table S2. Is the number of observations for the RE P1 merged structure (7.87 million) correct? For a low resolution structure that seems unlikely.
A sequence comparison, perhaps in the SI, for the structurally characterized proteins that inform the study should be considered. Additionally, this would be a good place to list the PDB codes for the structures being discussed from this paper and prior studies. The statement on page 3 that "RE is also the only beetle luciferase with an additional residue..." is not really conveyed by Figure  2e, as cited. An alignment would allow the reader to understand the context of the Arg insertion and would strengthen this statement. Further, a sequence alignment would give them an opportunity to label the catalytic lysine of the C-terminal domain, which would facilitate understanding of the discussion of residues K524, K526, and K529.
Supplemental Figure S1, the eluction times of standards should be shown. Chromatographic traces for each do not need to be included.
No PDB accession codes are provided for the deposited structures.
Reviewer #2 (Comments to the Authors (Required)): The article presents the first crystal structures of naturally red-emitting (REPh) and most blueshifted green-emitting (GBAv) luciferases and the significance for two conserved loops in bioluminescent color determination. The bioluminescent color change is an enigma in bioluminescent research. The finding of the interaction of R337 with loop(351-364) in GBAv for green emission is very important. I feel regret that the crystal structure complexed with any ligands are not determined. However authors investigate well the effect of the active site microenvironment with structural, mutational, computational, thermal analysis in bioluminescent color determination. The article is well written and explains the findings in an understandable and appropriate way, and should be interest to the community.

Specific comments:
Sequence alignment of two loops with REPh, GBAv, GLc and GPp will assist in understanding your results. Page 3, line 10 : The authors should define "an angle between two domains". S12 Sup Table1, REPh, No of reflections : The value 33877 is very low. The author will make a mistake in writing of the values of no. of reflections and unique reflections. S12 Sup Table1, Refinement : Resolution and Rwork/Rfree need units of Å and %, respectively. S18 Sup Fig1(a) : I can't understand the dashed lines and arrows.
Reviewer #3 (Comments to the Authors (Required)): In this paper, for the first time, The crystal structures of two beetle luciferases with red-and blueshifted light relative to the green-yellow light have been reported. They have shown that the structure of a blue-shifted green-emitting luciferase from the firefly Amydetes vivianii is monomeric with a structural fold similar to the previously reported firefly luciferases. The crystal structure of only known naturally red-emitting luciferase from the glow-worm Phrixotrix hirtus as tetramers and octamers.
1. The only main issue which should be taken to consideration is the real oligomer/monomer state. I am very keen to know if the protein has been formed in dimer or tetramer form upon purification or its real structural base. 2. A SDS-PAGE and PAGE image of purified protein could be crucial. 3. Specific activity of the enzyme compared to other reported firefly luciferases like P.pyralis or L. turkestanicus should be reported if is available. 4. There are more reports on the role of flexible loop of 352-364 in its critical role in water accessibility to the luciferin binding site. Is is in line with your investigation? There are some minor language problem which should be corrected .
1st Authors' Response to Reviewers: July 26, 2018 Review from referee 1. The manuscript describes the structures of luciferase enzymes from two species that have not previously been structurally characterized. The structures are used to inform mutagenesis studies to examine the role of several loops in influencing the wavelength of emitted light. The structures include a red-shifted luciferase from Phrixotrix hirtus and a blue-shifted enzyme from A. vivianii. The structure of red-shifted RE(Ph) is at low resolution (3.0Å and 3.6Å), while the structure of the blue-shifted BG(Av) enzyme is at higher resolution (1.9Å). Both structures show high degrees of mobility of the C-terminal subdomain, a feature that has been observed previously in other luciferase structures, as well as other members of this broader enzyme family. Unfortunately, none of the structures contain bound ligands, a feature that also may relate to the high C-terminal subdomain disorder. The structural analysis is good; however the rather low resolution of the RE enzyme should be more fully discussed along with the potential limitations this provides for conclusions and analysis.
We included in different parts of the manuscript the fact that REPh was acquired at low resolution. Although the resolution is low, a high-quality electron density maps were acquired for the P1 and P3121 crystal forms of the REPh. Supplementary Fig. 1 shows different electron density maps and we added the following text to emphasize the low resolution of REPh structures in the manuscript.
Page 2: "The crystal structure of WT REPh was determined at low resolution by molecular replacement from two different crystal forms in the space groups P1 and P3121 at resolution of 3.05 Å and 3.60 Å, respectively (Supplementary Table 1)." Page 2: "With the low resolution of the REPh structure, site directed mutagenesis clearly confirms the interface interactions, where the single mutation, R11A, was sufficient to disrupt the octamer of REPh and resulted exclusively in monomers in solution ( Supplementary Fig.  2c)." Page 3: "The loop 348-361 in REPh, albeit at low resolution, was modeled in the electron-density map ( Supplementary Fig. 1c)." Page 4: "Despite the low resolution of the REPh structure, the high-quality electron density map allowed to model this loop region in the P3121 crystal form ( Supplementary Fig. 1d), where……" The use of organizational headings (Results, Discussion, etc...) would make it easier to read this manuscript.
We have included the headings as suggested.
Major concerns. 1) There is a great deal of discussion of the oligomeric state of the enzymes, including the octameric state of the RE enzyme observed in the P1 crystal. From the image, it appears this is simply a crystallographic packing of two tetramers. The authors should use the PISA informatics (through server or COOT) to suggest which of the subunit interactions are deemed to be biologically significant.
The oligomeric state of the wild-type (WT) REPh has been addressed extensively and confirmed through site directed mutagenesis and gel filtration analysis. These give a strong evidence that the WT REPh is an oligomer in solution. As pointed here by the reviewer, the REPh in the P1-crystal arrangement is a dimer of tetramers, with the tetramers being the relevant biological conformation or at least the most stable in the in vitro conditions used here. The octamer with dimer of tetramers arrangement could also be concentration dependent, where the concentrations used in the crystallization trial might be similar to the biological concentration in the railroad worm. The PISA analysis did not yield an assembly that could be stable in solution. Nevertheless, one of the main factors taken into consideration by PISA calculations are water accessible surfaces, which are determined by the interactions of water molecules present in the structure. The absence of waters in the REPh structure due to the low resolution is introducing a bias in the PISA calculations.
2) The disorder of the C-terminal subdomains has been observed before and should be noted more clearly that this is not unique to the current structures. 3) The paper ignores prior work that demonstrates the use of the second conformation for the oxidative reaction. This has been shown convincingly by both biochemical and structural work with the P. pyralis enzyme. On page 3, for example, the authors simply refer to "reversible opening and closing" without explaining that there are in fact two catalytic states. Further, the paper ignores some recent studies of red-shifting mutants of P. pyralis. Thanks for making us aware of the two catalytic conformations. We indicated the two catalytic conformations in the manuscript: Page 3: "Together with the structural data, these results confirm the pronounced mobility of the C-terminal domain of beetle luciferases, which is capable of reversible opening and closing of the active site through two catalytic conformations during the bioluminescence reaction. The two catalytic conformations are stimulated by rotation on the C-terminal domain of firefly luciferases (Sundlov, et al, 2012)." 4) Pg 2 notes that "a single mutation, R11A, was sufficient to disrupt the octamer..." This is not shown by the data in figure S1c that shows that multiple mutations are required to shift the SEC peak to a monomer. The size-exclusion chromatography (SEC) data for R11A of REPh has been added as Supplementary  Fig. 2c, the new panel is shown below. The estimated size of the single mutant R11A of REPh is similar to the monomeric GBAv luciferase. The single mutant R11A and the triple mutant R11A, Y153A, and F162A of REPh yielded similar retention time as the monomeric GBAv luciferase ( Supplementary Fig. 2c). The data shown here support our statement that the introduction of the single mutant R11A was sufficient to disrupt the oligomeric packing of REPh and produced monomers.
The following sentence have been included to clarify the interest in investigating the importance of this loop in the color tuning mechanism: Page 3: "The presence of the only known insertion in beetle luciferases (R353 in REPh), its proximity to both the active site and several key substitutions around the benzothiazole moiety that were previously found to have an impact in the color tuning (Viviani et al, 2007), drove us to further investigate its relevance in the color-tuning mechanism." Below is Supplementary Fig. 6 that has been generated to show the conformational differences of loop [351][352][353][354][355][356][357][358][359][360][361][362][363][364] in REPh in comparison to the same loop in GBAv and other green emitting luciferases.
6) A statement (pg 4) like "We were able to discern with sufficient accuracy the structure of ..." Page 5 of 7 suggests that the electron density here is likely weak. No density is shown in the manuscript and inclusion would allow the reader to judge for him or herself the quality of the data. Supplementary fig. 1 has been added to show the general quality of the data and electron density maps, particularly for the loops mentioned in the manuscript.
Minor concerns. 1) The authors should consider whether it is correct to use an adjective "N-terminal" to describe the domains. Either they are the N-and C-terminal domains or the N-and C-terminus. As the latter can refer simply to the two ends of the protein, I prefer including domains. The authors also use Ndomain and C-domain on pg 2; that is acceptable also as long as they are consistent. The text has been corrected to reflect the N-terminal domain or C-terminal domain throughout the manuscript.
2) Table S2. Is the number of observations for the RE P1 merged structure (7.87 million) correct? For a low resolution structure that seems unlikely. This is a typo, and the correct number of total reflections is 517,157.
3) A sequence comparison, perhaps in the SI, for the structurally characterized proteins that inform the study should be considered. Additionally, this would be a good place to list the PDB codes for the structures being discussed from this paper and prior studies. The statement on page 3 that "RE is also the only beetle luciferase with an additional residue..." is not really conveyed by Figure 2e, as cited. An alignment would allow the reader to understand the context of the Arg insertion and would strengthen this statement. Further, a sequence alignment would give them an opportunity to label the catalytic lysine of the C-terminal domain, which would facilitate understanding of the discussion of residues K524, K526, and K529. The sequence alignment figure has been added as Supplementary Figure 5. Figure S1, the elution times of standards should be shown. Chromatographic traces for each do not need to be included. The included SEC chromatograms are only for mutants that changed the oligomerization state of the REPh in addition to the WT GBAv that has been used as a reference for the monomeric structures. The included traces are important to show the different oligomerization states of REPh mutant enzymes that disrupted the packing of the oligomerization state of the WT REPh. 5) No PDB accession codes are provided for the deposited structures. The structures are submitted to the PDB and the following accession codes have been assigned: 6AAA, 6ABH and 6AC3 (Supplementary Table 1).

Review from referee 2.
The article presents the first crystal structures of naturally red-emitting (REPh) and most blueshifted green-emitting (GBAv) luciferases and the significance for two conserved loops in bioluminescent color determination. The bioluminescent color change is an enigma in bioluminescent research. The finding of the interaction of R337 with loop(351-364) in GBAv for green emission is very important. I feel regret that the crystal structure complexed with any ligands are not determined. However authors investigate well the effect of the active site microenvironment with structural, mutational, computational, thermal analysis in bioluminescent color determination. The article is well written and explains the findings in an understandable and appropriate way, and should be interest to the community.
Specific comments: Page 6 of 7 1) Sequence alignment of two loops with REPh, GBAv, GLc and GPp will assist in understanding your results. The sequence alignment figure has been added as Supplementary Figure 5.
2) Page 2, line 30 : " Supplementary Fig. 1d" is not in Supplementary Figures. Thanks for pointing this out. It should be Supplementary Fig. 2c. It has been changed in the manuscript.
3) Page 2, line 42 : "RSMD" may be RMSD. Thanks for pointing this out. It has been corrected to RMSD. Review from referee 3. In this paper, for the first time, the crystal structures of two beetle luciferases with red-and blue-shifted light relative to the green-yellow light have been reported. They have shown that the structure of a blue-shifted green-emitting luciferase from the firefly Amydetes vivianii is monomeric with a structural fold similar to the previously reported firefly luciferases. The crystal structure of only known naturally red-emitting luciferase from the glow-worm Phrixotrix hirtus as tetramers and octamers.
1. The only main issue which should be taken to consideration is the real oligomer/monomer state. I am very keen to know if the protein has been formed in dimer or tetramer form upon purification or its real structural base. The WT REPh elute as an oligomer from size exclusion chromatography (SEC) with molecular weight equivalent to the tetramer in solution. The introduction of mutation in the tetramer interface produced only dimers based on SEC analysis ( Supplementary Fig. 2c). However, the single mutant, R11A, in the dimer interface was sufficient to disrupt the interactions in both interfaces to produce exclusively monomers (the SEC profile of R11A mutant has been added to panel 1c).
Based on the SEC analysis of the REPh mutants, the interactions in the dimer interface are stronger than those in the tetramer interface, as a result of the polar nature of the interactions in the dimer interface in comparison to weaker hydrophobic interactions in the tetramer interface.
2. A SDS-PAGE and PAGE image of purified protein could be crucial. The SDS-PAGE of all proteins shown in Supplementary Fig. 2c has been added as Supplementary  Fig. 2d. All proteins purified in this manuscript have purity >90% based on Coomassie-staining.
3. Specific activity of the enzyme compared to other reported firefly luciferases like P.pyralis or L. turkestanicus should be reported if is available. The specific activities of these enzymes have been previously reported and since the kinetic analysis on the luciferase reaction will not improve the understanding of the color emission mechanism, we did not perform kinetic measurements in this manuscript as the main focus was on the understanding of the fine color-tuning mechanism.
4. There are more reports on the role of flexible loop of 352-364 in its critical role in water accessibility to the luciferin binding site. Is it in line with your investigation? This is a very important finding in our manuscript as we strongly believe that the microenvironment of the active site is directly related to the color emission of beetle luciferases. As a result, the amount of water in the active site can be one of the factors that affect the active site microenvironment in addition to the charge of the amino acids occupying the active site. Unfortunately, due to the low resolution of the REPh structure no water molecules could be modeled to support/refute previous findings.
There are some minor language problem which should be corrected. Thank you for submitting your revised manuscript entitled "Beetle luciferases with naturally redand blue-shifted emission". As you will see, the reviewers are pleased with the performed revisions, and we would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
Please proofread your manuscript one more time to address the comment made by reviewer #1. Please also mention all figures and S figures and their individual panels in the manuscript text (currently missing eg: Fig4, SFig6, 8, 9 and 10 and STable 3, 5, and 6 as well as some individual panels). Furthermore, please provide all S Tables and S figures as individual figure files. The current suppl docx file can remain, please simply upload the figures in an appropriate format in addition.
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Thank you for this interesting contribution, we look forward to publishing your paper in Life Science Alliance. The revised manuscript has addressed my prior concerns. There are still a handful of typographical errors that the authors should address.