Three major classes of flavin photosensors, LOV domains, BLUF proteins and cryptochromes regulate diverse biological activities in response to blue-light. Recent studies of structure, spectroscopy and chemical mechanism have provided unprecedented insight into how each family operates at the molecular level. In general, the photoexcitation of the flavin cofactor leads to changes in redox and protonation states that ultimately remodel protein conformation and molecular interactions. For LOV domains, issues remain regarding early photochemical events, but common themes in conformational propagation have emerged across a diverse family of proteins. For BLUF proteins, photoinduced electron transfer reactions critical to light conversion are defined, but the subsequent rearrangement of hydrogen bonding networks key for signaling remain highly controversial. For cryptochromes, the relevant photocycles are actively debated, but mechanistic and functional studies are converging. Despite these challenges, our current understanding has enabled the engineering of flavoprotein photosensors for control of signaling processes within cells.

Light-oxygen-voltage (LOV) domains bind a flavin chromophore to serve as blue light sensors in a wide range of eukaryotic and prokaryotic proteins. LOV domains are associated with a variable effector domain or a separate protein signaling partner to execute a wide variety of functions that include regulation of kinases, generation of antisigma-factor antagonists, and regulation of circadian clocks. Here we present the crystal structure, photocycle kinetics, association properties, and spectroscopic features of a full-length LOV protein from Rhodobacter sphaeroides (RsLOV). RsLOV exhibits N-terminal and C-terminal helical extensions that form an unusual helical bundle at its dimer interface with some resemblance to the helical transducer of sensory rhodopsin II. The blue light-induced conformational changes of RsLOV revealed from a comparison of light and dark state crystal structures support a shared signaling mechanism of LOV domain proteins that originates with the light-induced formation of a flavin-cysteinyl photoadduct. Adduct formation disrupts hydrogen bonding in the active site and propagates structural changes through the LOV domain core to the N- and C-terminal extensions. Single residue variants in the active site and dimer interface of RsLOV alter photoadduct lifetimes and induce structural changes that perturb oligomeric state. Size exclusion chromatography, multi-angle light scattering, small-angle X-ray scattering and cross-linking studies indicate that RsLOV dimerizes in the dark, but upon light excitation, dissociates into monomers. This light-induced switch in oligomeric state may prove useful for engineering molecular associations in controlled cellular settings.

No abstract

Lysosomal integral membrane protein-2 (LIMP-2/SCARB2) contributes to endosomal and lysosomal function. LIMP-2 deficiency is associated with neurological abnormalities and kidney failure and, as an acid glucocerebrosidase receptor, impacts Gaucher and Parkinson’s diseases. Here we report a crystal structure of a LIMP-2 luminal domain dimer with bound cholesterol and phosphatidylcholine. Binding of these lipids alters LIMP-2 from functioning as a glucocerebrosidase-binding monomer toward a dimeric state that preferentially binds anionic phosphatidylserine over neutral phosphatidylcholine. In cellular uptake experiments, LIMP-2 facilitates transport of phospholipids into murine fibroblasts, with a strong substrate preference for phosphatidylserine. Taken together, these biophysical and cellular studies define the structural basis and functional importance of a form of LIMP-2 for lipid trafficking. We propose a model whereby switching between monomeric and dimeric forms allows LIMP-2 to engage distinct binding partners, a mechanism that may be shared by SR-BI and CD36, scavenger receptor proteins highly homologous to LIMP-2.

Glutathionylcobalamin (GSCbl) is a unique, biologically relevant cobalamin featuring an axial Co-S bond that distinguishes it from the enzymatically active forms of vitamin B(12), which possess axial Co-C bonds. GSCbl has been proposed to serve as an intermediate in cobalamin processing and, more recently, as a therapeutic for neurological disorders associated with oxidative stress. In this study, GSCbl and its close relative cysteinylcobalamin (CysCbl) were investigated using electronic absorption, circular dichroism, magnetic circular dichroism, and resonance Raman spectroscopies. The spectroscopic data were analyzed in the framework of density functional theory (DFT) and time-dependent DFT computations to generate experimentally validated electronic structure descriptions. Although the change in the upper axial ligand from an alkyl to a thiol group represents a major perturbation in terms of the size, basicity, and polarizability of the coordinating atom, our spectroscopic and computational results reveal striking similarities in electronic structure between methylcobalamin (MeCbl) and GSCbl, especially with regard to the σ donation from the alkyl/thiol ligand and the extent of mixing between the cobalt 3d and the ligand frontier orbitals. A detailed comparison of Co-C and Co-S bonding in MeCbl and GSCbl, respectively, is presented, and the implications of our results with respect to the proposed biological roles of GSCbl are discussed.