Influence of the Loop between Residues 223-235 in Beetle Luciferase Bioluminescence Spectra: A Solvent Gate for the Active Site of pH-Sensitive Luciferases, The

ABSTRACT

Beetle luciferases emit a wide range of bioluminescence colors, ranging from green to red. Firefly luciferases can shift the spectrum to red in response to pH and temperature changes, whereas click beetle and railroadworm luciferases do not. Despite many studies on firefly luciferases, the origin of pH-sensitivity is far from being understood. Through comparative site-directed mutagenesis and modeling studies, using the pH-sensitive luciferases (Macrolampis and Cratomorphus distinctus fireflies) and the pH-insensitive luciferases (Pyrearinus termitilluminans, Phrixotrix viviani and Phrixotrix hirtus) cloned by our group, here we show that substitutions dramatically affecting bioluminescence colors in both groups of luciferases are clustered in the loop between residues 223-235 (Photinus pyralis sequence). The substitutions at positions 227, 228 and 229 (P. pyralis sequence) cause dramatic redshift and temporal shift in both groups of luciferases, indicating their involvement in labile interactions. Modeling studies showed that the residues Y227 and N229 are buried in the protein core, fixing the loop to other structural elements participating at the bottom of the luciferin binding site. Changes in pH and temperature (in firefly luciferases), as well as point mutations in this loop, may disrupt the interactions of these structural elements exposing the active site and modulating bioluminescence colors.

INTRODUCTION

Beetle luciferases emit a wide range of bioluminescence colors, ranging from green to red (1). Click beetle and railroadworm luciferases emit a single bioluminescence color (2), however firefly luciferases can modulate the proportion of green and red bioluminescence through a pH-sensitive mechanism (3).

Recent theoretical and experimental studies suggest that the mechanism of bioluminescence color determination by luciferase involves the polarization of oxyluciferin phenol and keto/enol groups, under the influence of active site residues, and the rigidity of the active site (4-6). The primary sequences of several beetle luciferases (7-18) are known. The three-dimensional structure has been solved for the North American firefly luciferase, in the absence of substrates (19), and more recently for the Japanese Luciola cruciata firefly luciferase in the presence of either the luciferyl-adenylate analog 5'-O-[N-(dehydroluciferyl)-sulphamoil] adenosine (DLSA), and of oxyluciferin and adenosine monophosphate (AMP) (20). Some luciferin binding-site residues were identified by modeling studies (21,22), site-directed mutagenesis (23-26) and by direct inspection of the three-dimensional structure in the presence of analogs (20). However, several other residues distributed seemingly randomly over the primary structure of firefly luciferases are known to affect bioluminescence colors, many of them resulting in red mutants (27-31).

One approach to investigate the structure/function relationships in proteins is through comparative site-directed mutagenesis of homolog proteins. To investigate the relationship between luciferase structure, bioluminescence colors and pH-sensitivity, we previously cloned the pH-insensitive click beetle and railroadworm luciferases (15-17). In contrast to firefly luciferases, much fewer residues were found to affect the bioluminescence colors of the pH-insensitive luciferases (32-34): none of them displaying the large redshifts observed in firefly luciferases (35). A set of residues differing between pH-sensitive and pH-insensitive luciferases was identified (16), and site-directed mutagenesis of several residues was performed. However, none of these substitutions conferred pH-sensitivity to a pH-insensitive luciferase. Only the substitution of T226 in pH-insensitive luciferases and the analogous N229 in pH-sensitive luciferases, caused dramatic redshift in both groups of luciferases (33-35). We also cloned two new firefly luciferases (36,37) and found that the substitution E354N is responsible for the presence of a shoulder in the red region of the spectrum of Macrolampis luciferase (37). From these studies, a network of interacting residues which could be involved with pH-sensitivity determination, including the residue N229, was identified (37).

Considering that N229 (T229 in pH-insensitive luciferases) and the substitutions R226A and V227A that were suggested to affect bioluminescence color in click beetle luciferases (38), are located in the loop between residues 223-235, we decided to investigate the influence of this loop in bioluminescence color determination, using the pH-sensitive (Cratomorphus, Macrolampis) (36,38) and pH-insensitive (Pyrearinus, Phrixotrix spp.) luciferases available in our laboratory (13-16). The results showed that the residues 227Y(F,V)GN(T)229 have a major influence on bioluminescence color determination and pH-sensitivity. We also investigated the effect of the substitutions at position 260, which in L. cruciata luciferase structure complexed with DLSA, is hydrogen bond with N230.