Search

Self-assembling cyclic protein homo-oligomers play important roles in biology, and the ability to generate custom homo-oligomeric structures could enable new approaches to probe biological function. Here we report a general approach to design cyclic homo-oligomers that employs a new residue-pair-transform method to assess the designability of a protein-protein interface. This method is sufficiently rapid to enable the systematic enumeration of cyclically docked arrangements of a monomer followed by sequence design of the newly formed interfaces. We use this method to design interfaces onto idealized repeat proteins that direct their assembly into complexes that possess cyclic symmetry. Of 96 designs that were characterized experimentally, 21 were found to form stable monodisperse homo-oligomers in solution, and 15 (four homodimers, six homotrimers, six homotetramers and one homopentamer) had solution small-angle X-ray scattering data consistent with the design models. X-ray crystal structures were obtained for five of the designs and each is very close to their corresponding computational model.

Small molecules that bind to tubulin exert powerful effects on cell division and apoptosis (programmed cell death). Cell-based high-throughput screening combined with chemo/bioinformatic and biochemical analyses recently revealed a novel compound MI-181 as a potent mitotic inhibitor with heightened activity towards melanomas. MI-181 causes tubulin depolymerization, activates the spindle assembly checkpoint arresting cells in mitosis, and induces apoptotic cell death. C2 is an unrelated compound previously shown to have lethal effects on microtubules in tumorigenic cell lines. We report 2.60 Å and 3.75 Å resolution structures of MI-181 and C2, respectively, bound to a ternary complex of αβ-tubulin, the tubulin-binding protein stathmin, and tubulin tyrosine ligase. In the first of these structures, our crystallographic results reveal a unique binding mode for MI-181 extending unusually deep into the well-studied colchicine-binding site on β-tubulin. In the second structure the C2 compound occupies the colchicine-binding site on β-tubulin with two chemical moieties recapitulating contacts made by colchicine, in combination with another system of atomic contacts. These insights reveal the source of the observed effects of MI-181 and C2 on microtubules, mitosis, and cultured cancer cell lines. The structural details of the interaction between tubulin and the described compounds may guide the development of improved derivative compounds as therapeutic candidates or molecular probes to study cancer cell division.

The self-assembly of proteins into highly ordered nanoscale architectures is a hallmark of biological systems. The sophisticated functions of these molecular machines have inspired the development of methods to engineer self- assembling protein nanostructures; however, the design of multi-component protein nanomaterials with high accuracy remains an outstanding challenge. Here we report a computational method for designing protein nanomaterials in which multiple copies of two distinct subunits co-assemble into a specific architecture. We use the method to design five 24- subunit cage-like protein nanomaterials in two distinct symmetric architectures and experimentally demonstrate that their structures are in close agreement with the computational design models. The accuracy of the method and the number and variety of two-component materials that it makes accessible suggest a route to the construction of functional protein nanomaterials tailored to specific applications., The unique functional opportunities afforded by protein self-assembly range from the dynamic cellular scaffolding provided by cytoskeletal proteins to the encapsulation, protection and delivery of viral genomes to new host [...]

Tral, a bifunctional enzyme containing relaxase and helicase activities, initiates and drives the conjugative transfer of the Escherichia coli F plasmid. Here, we examined the structure and function of the Tral helicase. We show that Tral binds to single- stranded DNA (ssDNA) with a site size of ∼25 nucleotides, which is significantly longer than the site size of other known superfamily! helicases. Low cooperativity was observed with the binding of Ira! to ssDNA, and a double-stranded DNA-binding site was identified within the N-terminal region of Tral 1-858, outside the core helicase motifs of Tral. We have revealed that the affinity of Tra! for DNA is negatively correlated with the ionic strength of the solution. The binding of AMPPNP or ADP results in a 3-fold increase in the affinity of Tra! for ssDNA. Moreover, Tral prefers to bind ssDNA oligomers containing a single type of base. Finally, we elucidated the solution structure of Ira! using small angle x-ray scattering. Tral exhibits an ellipsoidal shape in solution with four domains aligning along one axis. Taken together, these data result in the assembly of a model for the multidomain helicase activity of Tra!. [ABSTRACT FROM AUTHOR]