Rational conversion of chromophore selectivity of cyanobacteriochromes to accept mammalian intrinsic biliverdin

Abstract

Because cyanobacteriochrome photoreceptors need only a single compact domain for chromophore incorporation and for absorption of visible spectra including the long-wavelength far-red region, these molecules have been paid much attention for application to bioimaging and optogenetics. Most cyanobacteriochromes, however, have a drawback to incorporate phycocyanobilin that is not available in the mammalian cells. In this study, we focused on biliverdin (BV) that is a mammalian intrinsic chromophore and absorbs the far-red region and revealed that replacement of only four residues was enough for conversion from BV-rejective cyanobacteriochromes into BV-acceptable molecules. We succeeded in determining the crystal structure of one of such engineered molecules, AnPixJg2_BV4, at 1.6 Å resolution. This structure identified unusual covalent bond linkage, which resulted in deep BV insertion into the protein pocket. The four mutated residues contributed to reducing steric hindrances derived from the deeper insertion. We introduced these residues into other domains, and one of them, NpF2164g5_BV4, produced bright near-infrared fluorescence from mammalian liver in vivo. Collectively, this study provides not only molecular basis to incorporate BV by the cyanobacteriochromes but also rational strategy to open the door for application of cyanobacteriochromes to visualization and regulation of deep mammalian tissues.

Molecular Basis of Far-red Sensing in Cyanobacteriochrome

“…FR-CBCRs have evolved from green/red CBCRs as part of a Greater Green/Red (GGR) lineage, diversification comparable to that seen in the XRG (extended red/green) lineage (35,40,41). Owing to their small size and spectral overlap with the therapeutic window of optimum tissue penetrance (700-800 nm) (42)(43)(44)(45)(46), FR-CBCRs provide a tantalizing scaffold for development of FR-responsive optogenetic reagents for biomedical research and clinical applications (45,(47)(48)(49)(50).…”

Protein Engineering of Dual-Cys Cyanobacteriochrome AM1_1186g2 for Biliverdin Incorporation and Far-Red/Blue Reversible Photoconversion

“…Based on a sequence comparison between the BV-binding and non-BV-binding ones, we predicted residues involved in BV incorporation. Site-directed mutagenesis and structural analyses elucidated that a cooperative rearrangement of four replaced residues (BV4) well escapes steric hindrance to ring C [38]. Although we succeeded in BV incorporation of AnPixJg2, AnPixJg4, AM1_1870g3, and NpF2164g5 variants by introducing these BV4 residues, we failed to obtain a BV-binding AM1_1186g2 variant in a previous study.…”

“…Some typical XRG CBCRs detected from the cyanobacterium, Acaryochloris marina , covalently bind not only PCB but also biliverdin (BV), which is a precursor of PCB in the biosynthetic pathway from heme [36,37,38]. Since BV has a longer π-electron conjugated system than PCB, BV-binding ones show reversible photoconversion between a far-red-absorbing 15 Z dark state (Pfr) and an orange-absorbing 15 E photoproduct (Po).…”

Unveiling of a novel bifunctional photoreceptor, Dualchrome1, isolated from a cosmopolitan green alga

“…Homogenates were centrifuged at 165,000 g for 30 min and then the supernatants were filtered through a 0.8 µm cellulose acetate membrane before loading on to a nickel-affinity HisTrap HP column (GE Healthcare) using the ÄKTA pure 25 (GE Healthcare) system. His-tagged PpPHY incorporated with each chromophore was purified as described in previous studies 39,40 . The purified proteins were dialyzed against lysis buffer A containing 1 mM dithiothreitol (DTT).…”