Molecular architecture of the Spire-actin nucleus and its implication for actin filament assembly.

The Spire protein is a multifunctional regulator of actin assembly. We studied the structures and properties of Spire-actin complexes by X-ray scattering, X-ray crystallography, total internal reflection fluorescence microscopy, and actin polymerization assays. We show that Spire-actin complexes in solution assume a unique, longitudinal-like shape, in which Wiskott-Aldrich syndrome protein homology 2 domains (WH2), in an extended configuration, line up actins along the long axis of the core of the Spire-actin particle. In the complex, the kinase noncatalytic C-lobe domain is positioned at the side of the first N-terminal Spire-actin module. In addition, we find that preformed, isolated Spire-actin complexes are very efficient nucleators of polymerization and afterward dissociate from the growing filament. However, under certain conditions, all Spire constructs--even a single WH2 repeat--sequester actin and disrupt existing filaments. This molecular and structural mechanism of actin polymerization by Spire should apply to other actin-binding proteins that contain WH2 domains in tandem.

Solution structure of the soluble receptor for advanced glycation end products (sRAGE).

The receptor for advanced glycation end products (RAGE) is a multiligand cell surface receptor involved in various human diseases, as it binds to numerous molecules and proteins that modulate the activity of other proteins. Elucidating the three-dimensional structure of this receptor is therefore most important for understanding its function during activation and cellular signaling. The major alternative splice product of RAGE comprises its extracellular region that occurs as a soluble protein (sRAGE). Although the structures of sRAGE domains were available, their assembly into the functional full-length protein remained unknown. We observed that the protein has concentration-dependent oligomerization behavior, and this is also mediated by the presence of Ca(2+) ions. Moreover, using synchrotron small angle x-ray scattering, the solution structure of human sRAGE was determined in the monomeric and dimeric forms. The model for the monomer displays a J-like shape, whereas the dimer is formed through the association of the two N-terminal domains and has an elongated structure. These results provide insights into the assembly of the RAGE homodimer, which is essential for signal transduction, and the sRAGE:RAGE heterodimer that leads to blockage of the receptor signaling, paving the way for the design of therapeutic strategies for a large number of different pathologies.

Small-angle X-ray scattering study of a Rex family repressor: conformational response to NADH and NAD+ binding in solution.

The transcriptional repressor Rex is a sensor of the intracellular NADH/NAD(+) redox state through direct binding of NADH or NAD(+). Homodimeric Rex protein from Thermus aquaticus (T-Rex) and Bacillus subtilis (B-Rex) exists in several different conformations. In both organisms, Rex in complex with NADH has the DNA binding domains packed together at the dimer interface, whereas in the apo form of B-Rex the linkers connecting these domains to the core are flexible. The crystal structures of the apo forms of B-Rex and a mutated variant of T-Rex are radically different. We describe the solution structures of B-Rex in complex with NAD(+) or NADH and in its apo form, on the basis of small-angle X-ray scattering (SAXS) measurements. This study addresses to what extent the unusual orientation of the DNA recognition domains of the crystal structure of apo B-Rex is due to stabilization by crystal packing. Low-resolution ab initio solution structures were obtained for apo B-Rex, B-Rex:NADH and B-Rex:NAD(+). Models giving a more detailed picture of these three solution structures were obtained also by rigid body fitting of the crystallographic domains. The SAXS data confirm the elongated and flexible nature of apo-B-Rex and the existence of two distinct and more rigid conformations for the complexes with NADH and NAD(+). The models emerging from this study indicate a reaction mechanism for B-Rex in which the recognition domains are rotated upon binding to NADH.

Structure of chlorosomes from the green filamentous bacterium Chloroflexus aurantiacus.

The green filamentous bacterium Chloroflexus aurantiacus employs chlorosomes as photosynthetic antennae. Chlorosomes contain bacteriochlorophyll aggregates and are attached to the inner side of a plasma membrane via a protein baseplate. The structure of chlorosomes from C. aurantiacus was investigated by using a combination of cryo-electron microscopy and X-ray diffraction and compared with that of Chlorobi species. Cryo-electron tomography revealed thin chlorosomes for which a distinct crystalline baseplate lattice was visualized in high-resolution projections. The baseplate is present only on one side of the chlorosome, and the lattice dimensions suggest that a dimer of the CsmA protein is the building block. The bacteriochlorophyll aggregates inside the chlorosome are arranged in lamellae, but the spacing is much greater than that in Chlorobi species. A comparison of chlorosomes from different species suggested that the lamellar spacing is proportional to the chain length of the esterifying alcohols. C. aurantiacus chlorosomes accumulate larger quantities of carotenoids under high-light conditions, presumably to provide photoprotection. The wider lamellae allow accommodation of the additional carotenoids and lead to increased disorder within the lamellae.

Hexanol-induced order-disorder transitions in lamellar self-assembling aggregates of bacteriochlorophyll c in Chlorobium tepidum chlorosomes.

Chlorosomes are light-harvesting complexes of green photosynthetic bacteria. Chlorosomes contain bacteriochlorophyll (BChl) c, d, or e aggregates that exhibit strong excitonic coupling. The short-range order, which is responsible for the coupling, has been proposed to be augmented by pigment arrangement into undulated lamellar structures with spacing between 2 and 3 nm. Treatment of chlorosomes with hexanol reversibly converts the aggregated chlorosome chlorophylls into a form with spectral properties very similar to that of the monomer. Although this transition has been extensively studied, the structural basis remains unclear due to variability in the obtained morphologies. Here we investigated hexanol-induced structural changes in the lamellar organization of BChl c in chlorosomes from Chlorobium tepidum by a combination of X-ray scattering, electron cryomicroscopy, and optical spectroscopy. At a low hexanol/pigment ratio, the lamellae persisted in the presence of hexanol while the short-range order and exciton interactions between chlorin rings were effectively eliminated, producing a monomer-like absorption. The result suggested that hexanol hydroxyls solvated the chlorin rings while the aliphatic tail partitioned into the hydrophobic part of the lamellar structure. This partitioning extended the chlorosome along its long axis. Further increase of the hexanol/pigment ratio produced round pigment-hexanol droplets, which lost all lamellar order. After hexanol removal the spectral properties were restored. In the samples treated under the high hexanol/pigment ratio, lamellae reassembled in small domains after hexanol removal while the shape and long-range order were irreversibly lost. Thus, all the interactions required for establishing the short-range order by self-assembly are provided by BChl c molecules alone. However, the long-range order and overall shape are imposed by an external structure, e.g., the proteinaceous chlorosome baseplate.

Internal structure of chlorosomes from brown-colored chlorobium species and the role of carotenoids in their assembly.

Chlorosomes are the main light harvesting complexes of green photosynthetic bacteria. Recently, a lamellar model was proposed for the arrangement of pigment aggregates in Chlorobium tepidum chlorosomes, which contain bacteriochlorophyll (BChl) c as the main pigment. Here we demonstrate that the lamellar organization is also found in chlorosomes from two brown-colored species (Chl. phaeovibrioides and Chl. phaeobacteroides) containing BChl e as the main pigment. This suggests that the lamellar model is universal among green sulfur bacteria. In contrast to green-colored Chl. tepidum, chlorosomes from the brown-colored species often contain domains of lamellar aggregates that may help them to survive in extremely low light conditions. We suggest that carotenoids are localized between the lamellar planes and drive lamellar assembly by augmenting hydrophobic interactions. A model for chlorosome assembly, which accounts for the role of carotenoids and secondary BChl homologs, is presented.