Natural proteins can be versatile building blocks for multimeric, self-assembling structures. Yet, creating protein-based assemblies with specific geometries and chemical properties remains challenging. Highly porous materials represent particularly interesting targets for designed assembly. Here, we utilize a strategy of fusing two natural protein oligomers using a continuous alpha-helical linker to design a novel protein that self assembles into a 750 kDa, 225 Å diameter, cube-shaped cage with large openings into a 130 Å diameter inner cavity. A crystal structure of the cage showed atomic-level agreement with the designed model, while electron microscopy, native mass spectrometry and small angle X-ray scattering revealed alternative assembly forms in solution. These studies show that accurate design of large porous assemblies with specific shapes is feasible, while further specificity improvements will probably require limiting flexibility to select against alternative forms. These results provide a foundation for the design of advanced materials with applications in bionanotechnology, nanomedicine and material sciences.

Design and Build: Self-assembling biomolecules are attractive building blocks in the development of functional materials. Sophisticated DNA-based materials have been developed; however, progress in designing protein-based materials has been slower. King et al. (p. 1171 ) describe a general computational method in which protein building blocks are first symmetrically docked onto a target architecture, and then binding interfaces that drive self-assembly of the building blocks are designed. As a proof of principle, trimeric building blocks were used to design self-assembling 12-subunit complexes with tetrahedral symmetry and 24-subunit complexes with octahedral symmetry. Lai et al. (p. 1129 ) were able to build a 12-subunit tetrahedral protein cage from fused oligomeric protein domains.

Drug-resistant bacteria have caused serious medical problems in recent years, and the need for new antibacterial agents is undisputed. Transglycosylase, a multidomain membrane protein essential for cell wall synthesis, is an excellent target for the development of new antibiotics. Here, we determined the X-ray crystal structure of the bifunctional transglycosylase penicillin-binding protein 1b (PBP1b) from Escherichia coli in complex with its inhibitor moenomycin to 2.16-Å resolution. In addition to the transglycosylase and transpeptidase domains, our structure provides a complete visualization of this important antibacterial target, and reveals a domain for protein–protein interaction and a transmembrane helix domain essential for substrate binding, enzymatic activity, and membrane orientation.

An interactive structure-based approach was used to improve a vaccine antigen against respiratory syncytium virus (RSV), thus leading to immunogens with higher stability that elicit higher neutralizing titers in mice. Structure-based design of vaccines, particularly the iterative optimization used so successfully in the structure-based design of drugs, has been a long-sought goal. We previously developed a first-generation vaccine antigen called DS-Cav1, comprising a prefusion-stabilized form of the fusion (F) glycoprotein, which elicits high-titer protective responses against respiratory syncytial virus (RSV) in mice and macaques. Here we report the improvement of DS-Cav1 through iterative cycles of structure-based design that significantly increased the titer of RSV-protective responses. The resultant second-generation 'DS2'-stabilized immunogens have their F subunits genetically linked, their fusion peptides deleted and their interprotomer movements stabilized by an additional disulfide bond. These DS2 immunogens are promising vaccine candidates with superior attributes, such as their lack of a requirement for furin cleavage and their increased antigenic stability against heat inactivation. The iterative structure-based improvement described here may have utility in the optimization of other vaccine antigens.

Crystal structures of the HIV-1 Env trimer with cell-entry inhibitor antiviral drug leads BMS-378806 and BMS-626529 along with biophysical data define allosteric and competitive mechanisms to inhibit CD4-induced structural changes in Env. The HIV-1 envelope (Env) spike is a conformational machine that transitions between prefusion (closed, CD4- and CCR5-bound) and postfusion states to facilitate HIV-1 entry into cells. Although the prefusion closed conformation is a potential target for inhibition, development of small-molecule leads has been stymied by difficulties in obtaining structural information. Here, we report crystal structures at 3.8-Å resolution of an HIV-1-Env trimer with BMS-378806 and a derivative BMS-626529 for which a prodrug version is currently in Phase III clinical trials. Both lead candidates recognized an induced binding pocket that was mostly excluded from solvent and comprised of Env elements from a conserved helix and the β20–21 hairpin. In both structures, the β20–21 region assumed a conformation distinct from prefusion-closed and CD4-bound states. Together with biophysical and antigenicity characterizations, the structures illuminate the allosteric and competitive mechanisms by which these small-molecule leads inhibit CD4-induced structural changes in Env.