Proteins represent the most sophisticated building blocks available to an organism or the laboratory chemist. Yet, in contrast to nearly all other types of molecular building blocks, the designed self-assembly of proteins has been largely inaccessible owing to the chemical and structural heterogeneity of protein surfaces. To circumvent the challenge of programming extensive non-covalent interactions for controlling protein self-assembly, we had previously exploited the directionality and strength of metal coordination interactions to guide the formation of closed, homoligomeric protein assemblies. Here, we extend this strategy to the generation of periodic protein arrays. We show that a monomeric protein with properly oriented coordination motifs on its surface can arrange upon metal binding into one-dimensional nanotubes, and two-or three-dimensional crystalline arrays whose dimensions collectively span nearly the entire nano- and micrometer length scale. The assembly of these arrays is predictably tuned by external stimuli, such as metal concentration and pH.

Gram-negative pathogens commonly exhibit adhesive pili on their surfaces that mediate specific attachment to the host. A major class of pili is assembled via the chaperone/usher pathway. Here, the structural basis for pilus fiber assembly and secretion performed by the outer membrane assembly platform--the usher--is revealed by the crystal structure of the translocation domain of the P pilus usher PapC and single particle cryo-electron microscopy imaging of the FimD usher bound to a translocating type 1 pilus assembly intermediate. These structures provide molecular snapshots of a twinned-pore translocation machinery in action. Unexpectedly, only one pore is used for secretion, while both usher protomers are used for chaperone-subunit complex recruitment. The translocating pore itself comprises 24 beta strands and is occluded by a folded plug domain, likely gated by a conformationally constrained beta-hairpin. These structures capture the secretion of a virulence factor across the outer membrane of gram-negative bacteria.

SUMMARY The postsynaptic density (PSD) is crucial for synaptic functions, but the molecular architecture retaining its structure and components remains elusive. Homer and Shank are among the most abundant scaffolding proteins in the PSD, working synergistically for maturation of dendritic spines. Here, we demonstrate that Homer and Shank, together, form a mesh-like matrix structure. Crystallographic analysis of this region revealed a pair of parallel dimeric coiled-coils intercalated in a tail-to-tail fashion to form a tetramer, giving rise to the unique configuration of a pair of amino-terminal EVH1 domains at each end of the coiled-coil. In neurons, the tetramerization is required for structural integrity of the dendritic spines and recruitment of proteins to synapses. We propose that the Homer-Shank complex serves as a structural framework and as an assembly platform for other PSD proteins.

Summary Proteasome-mediated protein turnover in all domains of life is an energy-dependent process that requires ATPase activity. Mycobacterium tuberculosis (Mtb) was recently shown to possess a ubiquitin-like proteasome pathway that plays an essential role in Mtb resistance to killing by products of host macrophages. Here we report our structural and biochemical investigation of Mpa, the presumptive Mtb proteasomal ATPase. We demonstrate that Mpa binds to the Mtb proteasome in the presence of ATPγS, providing the physical evidence that Mpa is the proteasomal ATPase. X-ray crystallographic determination of the conserved inter-domain showed a five-stranded double β-barrel structure containing a Greek key motif. The structure and mutagenesis indicate a major role of the inter-domain for Mpa hexamerization. Our mutational and functional studies further suggest that the central channel in the Mpa hexamer is involved in protein substrate translocation and degradation. These studies provide insights into how a bacterial proteasomal ATPase interacts with and facilitates protein degradation by the proteasome.

The origin recognition complex (ORC) is conserved in all eukaryotes. The six proteins of the Saccharomyces cerevisiae ORC that form a stable complex bind to origins of DNA replication and recruit prereplicative complex (pre-RC) proteins, one of which is Cdc6. To further understand the function of ORC we recently determined by single-particle reconstruction of electron micrographs a low-resolution, 3D structure of S. cerevisiae ORC and the ORC-Cdc6 complex. In this article, the spatial arrangement of the ORC subunits within the ORC structure is described. In one approach, a maltose binding protein (MBP) was systematically fused to the N or the C termini of the five largest ORC subunits, one subunit at a time, generating 10 MBP-fused ORCs, and the MBP density was localized in the averaged, 2D EM images of the MBP-fused ORC particles. Determining the Orc1-5 structure and comparing it with the native ORC structure localized the Orc6 subunit near Orc2 and Orc3. Finally, subunit-subunit interactions were determined by immunoprecipitation of ORC subunits synthesized in vitro. Based on the derived ORC architecture and existing structures of archaeal Orc1-DNA structures, we propose a model for ORC and suggest how ORC interacts with origin DNA and Cdc6. The studies provide a basis for understanding the overall structure of the pre-RC.electron microscopy ͉ structure ͉ ATPase I n Saccharomyces cerevisiae origins of DNA replication contain conserved A, B1, and B2 elements, where the A element and part of B1 define the binding sequence for the origin recognition complex (ORC) (1-3). ORC binds to origin DNA in an ATPdependent manner and recruits other essential proteins, such as the initiation factors Cdc6, Cdt1, and the presumptive DNA helicase MCM, to the autonomously replicating sequence (ARS) to form a prereplicative complex (pre-RC) before the initiation of DNA replication that occurs in S phase (4-7). ORC consists of six proteins named in the descending order of their relative mass: Orc1 (120 kDa), Orc2 (71 kDa), Orc3 (62 kDa), Orc4 (56 kDa), Orc5 (53 kDa), and Orc6 (50 kDa). The calculated mass of ORC is Ϸ412 kDa. Only two of the ORC subunits (Orc1 and Orc5) are known to bind ATP (8), although the largest five subunits are predicted to contain an AAAϩ fold and a DNAbinding winged helix domain (WHD) within their C-terminal halves (9, 10).The prokaryotic origin recognition proteins consist of a single polypeptide that can form oligomeric structures (11-17), which raises the question of why in eukaryotes ORC has six subunits and why Cdc6 also contributes to origin recognition (18,19). Structural studies of the replication initiator proteins have begun to shed light on the mechanism of origin recognition, but how the individual initiator proteins cooperate to promote initiation of DNA replication is not clear. The eubacterial DnaA structure suggested that the origin DNA might wrap around a super helical assembly of multiple subunits of this replication initiator (11,12). In contrast, the recent structures of archaeal Orc1/C...