A covalent linker allows for membrane targeting of an oxylipin biosynthetic complex

A naturally occurring bifunctional protein from Plexaura homomalla links sequential catalytic activities in an oxylipin biosynthetic pathway. The C-terminal lipoxygenase (LOX) portion of the molecule catalyzes the transformation of arachidonic acid (AA) to the corresponding 8R-hydroperoxide, and the N-terminal allene oxide synthase (AOS) domain promotes the conversion of the hydroperoxide intermediate to the product allene oxide (AO). Small-angle X-ray scattering data indicate that in the absence of a covalent linkage the two catalytic domains that transform AA to AO associate to form a complex that recapitulates the structure of the bifunctional protein. The SAXS data also support a model for LOX and AOS domain orientation in the fusion protein inferred from a low-resolution crystal structure. However, results of membrane binding experiments indicate that covalent linkage of the domains is required for Ca2+-dependent membrane targeting of the sequential activities, despite the noncovalent domain association. Furthermore, membrane targeting is accompanied by a conformational change as monitored by specific proteolysis of the linker that joins the AOS and LOX domains. Our data are consistent with a model in which Ca2+-dependent membrane binding relieves the noncovalent interactions between the AOS and LOX domains and suggests that the C2-like domain of LOX mediates both protein-protein and protein-membrane interactions. © 2008 American Chemical Society

Detection of intermediates and kinetic control during assembly of bacteriophage P22 procapsid

Bacteriophage P22 serves as a model for the assembly and maturation of other icosahedral double-stranded DNA viruses. P22 coat and scaffolding proteins assemble in vitro into an icosahedral procapsid, which then expands during DNA packaging (maturation). Efficient in vitro assembly makes this system suitable for design and production of monodisperse spherical nanoparticles (diameter ≈50 nm). In this work we explore the possibility of controlling the outcome of assembly by scaffolding protein engineering. The scaffolding protein exists in monomer-dimer-tetramer equilibrium. We address the role of monomers and dimers in assembly by using three different scaffolding proteins with altered monomer-dimer equilibrium (weak dimer, covalent dimer, monomer). The progress and outcome of assembly was monitored by time-resolved X-ray scattering which allowed us to distinguish between closed shells and incomplete assembly intermediates. Binding of scaffolding monomer activates the coat protein for assembly. Excess dimeric scaffolding protein resulted in rapid nucleation and kinetic trapping yielding incomplete shells. Addition of monomeric wild type scaffold with excess coat protein completed these metastable shells. Thus, the monomeric scaffolding protein plays an essential role in the elongation phase by activating the coat and effectively lowering its critical concentration for assembly

Structures of the Signal Recognition Particle Receptor from the Archaeon Pyrococcus furiosus: Implications for the Targeting Step at the Membrane

In all organisms, a ribonucleoprotein called the signal recognition particle (SRP) and its receptor (SR) target nascent proteins from the ribosome to the translocon for secretion or membrane insertion. We present the first X-ray structures of an archeal FtsY, the receptor from the hyper-thermophile Pyrococcus furiosus (Pfu), in its free and GDP•magnesium-bound forms. The highly charged N-terminal domain of Pfu-FtsY is distinguished by a long N-terminal helix. The basic charges on the surface of this helix are likely to regulate interactions at the membrane. A peripheral GDP bound near a regulatory motif could indicate a site of interaction between the receptor and ribosomal or SRP RNAs. Small angle X-ray scattering and analytical ultracentrifugation indicate that the crystal structure of Pfu-FtsY correlates well with the average conformation in solution. Based on previous structures of two sub-complexes, we propose a model of the core of archeal and eukaryotic SRP•SR targeting complexes

Balanced Electrostatic and Structural Forces Guide the Large Conformational Change Associated with Maturation of T = 4 Virus

Nudaurelia capensis omega virus has a well-characterized T = 4 capsid that undergoes a pH-dependent large conformational changes (LCC) and associated auto-catalytic cleavage of the subunit. We examined previously the particle size at different pH values and showed that maturation occurred at pH 5.5. We now characterized the LCC with time-resolved small-angle x-ray scattering and showed that there were three kinetic stages initiated with an incremental drop in pH: 1), a rapid (<10 ms) collapse to an incrementally smaller particle; 2), a continuous size reduction over the next 5 s; and 3), a smaller final transition occurring in 2–3 min. Equilibrium measurements similar to those reported previously, but now more precise, showed that the particle dimension between pH 5.5 and 5 requires the autocatalytic cleavage to achieve its final compact size. A balance of electrostatic and structural forces shapes the energy landscape of the LCC with the latter requiring annealing of portions of the subunit. Equilibrium experiments showed that many intermediate states could be populated with a homogeneous ensemble of particles by carefully controlling the pH. A titration curve for the LCC was generated that showed that the virtual pKa (i.e., the composite of all titratable residues that contribute to the LCC) is 5.8

Structures of the signal recognition particle receptor from the archaeon Pyrococcus furiosus: implications for the targeting step at the membrane.

In all organisms, a ribonucleoprotein called the signal recognition particle (SRP) and its receptor (SR) target nascent proteins from the ribosome to the translocon for secretion or membrane insertion. We present the first X-ray structures of an archeal FtsY, the receptor from the hyper-thermophile Pyrococcus furiosus (Pfu), in its free and GDP*magnesium-bound forms. The highly charged N-terminal domain of Pfu-FtsY is distinguished by a long N-terminal helix. The basic charges on the surface of this helix are likely to regulate interactions at the membrane. A peripheral GDP bound near a regulatory motif could indicate a site of interaction between the receptor and ribosomal or SRP RNAs. Small angle X-ray scattering and analytical ultracentrifugation indicate that the crystal structure of Pfu-FtsY correlates well with the average conformation in solution. Based on previous structures of two sub-complexes, we propose a model of the core of archeal and eukaryotic SRP*SR targeting complexes

Available online at www.sciencedirect.com Integration of Small-Angle X-Ray Scattering Data into Structural Modeling of Proteins and Their Assemblies

A major challenge in structural biology is to determine the configuration of domains and proteins in multidomain proteins and assemblies, respectively. All available data should be considered to maximize the accuracy and precision of these models. Small-angle X-ray scattering (SAXS) efficiently provides low-resolution experimental data about the shapes of proteins and their assemblies. Thus, we integrated SAXS profiles into our software for modeling proteins and their assemblies by satisfaction of spatial restraints. Specifically, we modeled the quaternary structures of multidomain proteins with structurally defined rigid domains as well as quaternary structures of binary complexes of structurally defined rigid proteins. In addition to SAXS profiles and the component structures, we used stereochemical restraints and an atomic distance-dependent statistical potential. The scoring function is optimized by a biased Monte Carlo protocol, including quasi-Newton and simulated annealing schemes. The final prediction corresponds to the best scoring solution in the largest cluste