Structure of the frequency-interacting RNA helicase: a protein interaction hub for the circadian clock.

In the Neurospora crassa circadian clock, a protein complex of frequency (FRQ), casein kinase 1a (CK1a), and the FRQ-interacting RNA Helicase (FRH) rhythmically represses gene expression by the white-collar complex (WCC). FRH crystal structures in several conformations and bound to ADP/RNA reveal differences between FRH and the yeast homolog Mtr4 that clarify the distinct role of FRH in the clock. The FRQ-interacting region at the FRH N-terminus has variable structure in the absence of FRQ A known mutation that disrupts circadian rhythms (R806H) resides in a positively charged surface of the KOW domain, far removed from the helicase core. We show that changes to other similarly located residues modulate interactions with the WCC and FRQ A V142G substitution near the N-terminus also alters FRQ and WCC binding to FRH, but produces an unusual short clock period. These data support the assertion that FRH helicase activity does not play an essential role in the clock, but rather FRH acts to mediate contacts among FRQ, CK1a and the WCC through interactions involving its N-terminus and KOW module.

Photochemistry of flavoprotein light sensors.

Three major classes of flavin photosensors, light oxygen voltage (LOV) domains, blue light sensor using FAD (BLUF) proteins and cryptochromes (CRYs), 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 remains highly controversial. For CRYs, 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.

Spectroscopic and computational studies of cobalamin species with variable lower axial ligation: implications for the mechanism of Co-C bond activation by class I cobalamin-dependent isomerases.

5'-deoxyadenosylcobalamin (coenzyme B12, AdoCbl) serves as the cofactor for several enzymes that play important roles in fermentation and catabolism. All of these enzymes initiate catalysis by promoting homolytic cleavage of the cofactor's Co-C bond in response to substrate binding to their active sites. Despite considerable research efforts, the role of the lower axial ligand in facilitating Co-C bond homolysis remains incompletely understood. In the present study, we characterized several derivatives of AdoCbl and its one-electron reduced form, Co(II)Cbl, by using electronic absorption and magnetic circular dichroism spectroscopies. To complement our experimental data, we performed computations on these species, as well as additional Co(II)Cbl analogues. The geometries of all species investigated were optimized using a quantum mechanics/molecular mechanics method, and the optimized geometries were used to compute absorption spectra with time-dependent density functional theory. Collectively, our results indicate that a reduction in the basicity of the lower axial ligand causes changes to the cofactor's electronic structure in the Co(II) state that replicate the effects seen upon binding of Co(II)Cbl to Class I isomerases, which replace the lower axial dimethylbenzimidazole ligand of AdoCbl with a protein-derived histidine (His) residue. Such a reduction of the basicity of the His ligand in the enzyme active site may be achieved through proton uptake by the catalytic triad of conserved residues, DXHXGXK, during Co-C bond homolysis.

Light-induced subunit dissociation by a light-oxygen-voltage domain photoreceptor from Rhodobacter sphaeroides.

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 anti-sigma 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 domain protein from Rhodobacter sphaeroides (RsLOV). RsLOV exhibits N- 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 the oligomeric state. Size exclusion chromatography, multiangle 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 to be useful for engineering molecular associations in controlled cellular settings.

Combined spectroscopic/computational studies of vitamin B12 precursors: geometric and electronic structures of cobinamides.

Vitamin B(12) (cyanocobalamin) and its biologically active derivatives, methylcobalamin and adenosylcobalamin, are members of the family of corrinoids, which also includes cobinamides. As biological precursors to cobalamins, cobinamides possess the same structural core, consisting of a low-spin Co(3+) ion that is ligated equatorially by the four nitrogens of a highly substituted tetrapyrrole macrocycle (the corrin ring), but differ with respect to the lower axial ligation. Specifically, cobinamides possess a water molecule instead of the nucleotide loop that coordinates axially to Co(3+)cobalamins via its dimethylbenzimidazole (DMB) base. Compared to the cobalamin species, cobinamides have proven much more difficult to study experimentally, thus far eluding characterization by X-ray crystallography. In this study, we have utilized combined quantum mechanics/molecular mechanics (QM/MM) computations to generate complete structural models of a representative set of cobinamide species with varying upper axial ligands. To validate the use of this approach, analogous QM/MM geometry optimizations were carried out on entire models of the cobalamin counterparts for which high-resolution X-ray structural data are available. The accuracy of the cobinamide structures was assessed further by comparing electronic absorption spectra computed using time-dependent density functional theory to those obtained experimentally. Collectively, the results obtained in this study indicate that the DMB → H(2)O lower axial ligand switch primarily affects the energies of the Co 3d(z(2))-based molecular orbital (MO) and, to a lesser extent, the other Co 3d-based MOs as well as the corrin π-based highest energy MO. Thus, while the energy of the lowest-energy electronic transition of cobalamins changes considerably as a function of the upper axial ligand, it is nearly invariant for the cobinamides.

Spectroscopic and computational studies of glutathionylcobalamin: nature of Co-S bonding and comparison to Co-C bonding in coenzyme B12.

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.

Lysosomal integral membrane protein-2 as a phospholipid receptor revealed by biophysical and cellular studies.

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.