The NIH Protein Structure Initiative centers, such as the Joint Center for Structural Genomics (JCSG), have developed highly efficient technological platforms that are capable of experimentally determining the three-dimensional structures of hundreds of proteins per year. However, the overwhelming majority of the almost 5000 protein structures determined by these centers have yet to be described in the peer-reviewed literature. In a high-throughput structural genomics environment, the process of structure determination occurs independently of any associated experimental characterization of function, which creates a challenge for the annotation and analysis of structures and the publication of these results. This challenge has been addressed by developing TOPSAN (`The Open Protein Structure Annotation Network'), which enables the generation of knowledge via collaborations among globally distributed contributors supported by automated amalgamation of available information. TOPSAN currently provides annotations for all protein structures determined by the JCSG in addition to preliminary annotations on a large number of structures from the other PSI production centers. TOPSAN-enabled collaborations have resulted in insightful structure-function analysis for many proteins and have led to numerous peer-reviewed publications, as exemplified by the articles included in this issue of Acta Crystallographica Section F.

The Joint Center for Structural Genomics high-throughput structural biology pipeline has delivered more than 1000 structures to the community over the past ten years. The JCSG has made a significant contribution to the overall goal of the NIH Protein Structure Initiative (PSI) of expanding structural coverage of the protein universe, as well as making substantial inroads into structural coverage of an entire organism. Targets are processed through an extensive combination of bioinformatics and biophysical analyses to efficiently characterize and optimize each target prior to selection for structure determination. The pipeline uses parallel processing methods at almost every step in the process and can adapt to a wide range of protein targets from bacterial to human. The construction, expansion and optimization of the JCSG gene-to-structure pipeline over the years have resulted in many technological and methodological advances and developments. The vast number of targets and the enormous amounts of associated data processed through the multiple stages of the experimental pipeline required the development of variety of valuable resources that, wherever feasible, have been converted to free-access web-based tools and applications.

Clan CD cysteine peptidases, a structurally related group of peptidases that include mammalian caspases, exhibit a wide range of important functions, along with a variety of specificities and activation mechanisms. However, for the clostripain family (denoted C11), little is currently known. Here, we describe the first crystal structure of a C11 protein from the human gut bacterium, Parabacteroides merdae (PmC11), determined to 1.7-Å resolution. PmC11 is a monomeric cysteine peptidase that comprises an extended caspase-like α/β/α sandwich and an unusual C-terminal domain. It shares core structural elements with clan CD cysteine peptidases but otherwise structurally differs from the other families in the clan. These studies also revealed a well ordered break in the polypeptide chain at Lys(147), resulting in a large conformational rearrangement close to the active site. Biochemical and kinetic analysis revealed Lys(147) to be an intramolecular processing site at which cleavage is required for full activation of the enzyme, suggesting an autoinhibitory mechanism for self-preservation. PmC11 has an acidic binding pocket and a preference for basic substrates, and accepts substrates with Arg and Lys in P1 and does not require Ca(2+) for activity. Collectively, these data provide insights into the mechanism and activity of PmC11 and a detailed framework for studies on C11 peptidases from other phylogenetic kingdoms.

Tn916-like conjugative transposons carrying antibiotic resistance genes are found in a diverse range of bacteria. Orf14 within the conjugation module encodes a bifunctional cell wall hydrolase CwlT that consists of an N-terminal bacterial lysozyme domain (N-acetylmuramidase, bLysG) and a C-terminal NlpC/P60 domain (γ-d-glutamyl-l-diamino acid endopeptidase) and is expected to play an important role in the spread of the transposons. We determined the crystal structures of CwlT from two pathogens, Staphylococcus aureus Mu50 (SaCwlT) and Clostridium difficile 630 (CdCwlT). These structures reveal that NlpC/P60 and LysG domains are compact and conserved modules, connected by a short flexible linker. The LysG domain represents a novel family of widely distributed bacterial lysozymes. The overall structure and the active site of bLysG bear significant similarity to other members of the glycoside hydrolase family 23 (GH23), such as the g-type lysozyme (LysG) and Escherichia coli lytic transglycosylase MltE. The active site of bLysG contains a unique structural and sequence signature (DxxQSSES+S) that is important for coordinating a catalytic water. Molecular modeling suggests that the bLysG domain may recognize glycan in a similar manner to MltE. The C-terminal NlpC/P60 domain contains a conserved active site (Cys-His-His-Tyr) that appears to be specific to murein tetrapeptide. Access to the active site is likely regulated by isomerism of a side chain atop the catalytic cysteine, allowing substrate entry or product release (open state), or catalysis (closed state).

The Dlx5 homeodomain is a transcription factor related to the Drosophila distal-less gene that is associated with breast and lung cancer, lymphoma, Rett syndrome and osteoporosis in humans. Mutations in the DLX5 gene have been linked to deficiencies in craniofacial and limb development in higher eukaryotes, including split hand and foot malformation 1 in humans. Our characterization of a Dlx5 homeodomain:(CGACTAATTAGTCG)2 complex by NMR spectroscopy paved the way for determination of its crystal structure at 1.85Å resolution that enabled rationalization of the effects of disease-related mutations on the protein function. A Q186H mutation linked to split hand and foot malformation 1 likely affects affinity of DNA binding by disrupting water-mediated interactions with the DNA major groove. A more subtle effect is implicated for the Q178P mutation, which is not in direct contact with the DNA. Our data indicate that these mutations diminish the ability of the Dlx5 homeodomain to recognize and bind target DNAs, and they likely destabilize the formation of functional complexes.

Free-standing single-layer β-sheets are extremely rare in naturally occurring proteins, even though β-sheet motifs are ubiquitous. Here we report the crystal structures of three homologous, single-layer, anti-parallel β-sheet proteins, comprised of three or four twisted β-hairpin repeats. The structures reveal that, in addition to the hydrogen bond network characteristic of β-sheets, additional hydrophobic interactions mediated by small clusters of residues adjacent to the turns likely play a significant role in the structural stability and compensate for the lack of a compact hydrophobic core. These structures enabled identification of a family of secreted proteins that are broadly distributed in bacteria from the human gut microbiome and are putatively involved in the metabolism of complex carbohydrates. A conserved surface patch, rich in solvent-exposed tyrosine residues, was identified on the concave surface of the β-sheet. These new modular single-layer β-sheet proteins may serve as a new model system for studying folding and design of β-rich proteins.

Flavodoxins in combination with the flavin mononucleotide (FMN) cofactor play important roles for electron transport in prokaryotes. Here, novel insights into the FMN-binding mechanism to flavodoxins-4 were obtained from the NMR structures of the apo-protein from Lactobacillus acidophilus (YP_193882.1) and comparison of its complex with FMN. Extensive reversible conformational changes were observed upon FMN binding and release. The NMR structure of the FMN complex is in agreement with the crystal structure (PDB ID: 3EDO) and exhibits the characteristic flavodoxin fold, with a central five-stranded parallel β-sheet and five α-helices forming an α/β-sandwich architecture. The structure differs from other flavoproteins in that helix α2 is oriented perpendicular to the β-sheet and covers the FMN-binding site. This helix reversibly unfolds upon removal of the FMN ligand, which represents a unique structural rearrangement among flavodoxins.

Approximately 50% of cell wall peptidoglycan in Gram-negative bacteria is recycled with each generation. The primary substrates used for peptidoglycan biosynthesis and recycling in the cytoplasm are GlcNAc-MurNAc(anhydro)-tetrapeptide and its degradation product, the free tetrapeptide. This complex process involves ∼15 proteins, among which the cytoplasmic enzyme ld-carboxypeptidase A (LdcA) catabolizes the bond between the last two l- and d-amino acid residues in the tetrapeptide to form the tripeptide, which is then utilized as a substrate by murein peptide ligase (Mpl). LdcA has been proposed as an antibacterial target. The crystal structure of Novosphingobium aromaticivorans DSM 12444 LdcA (NaLdcA) was determined at 1.89-Å resolution. The enzyme was biochemically characterized and its interactions with the substrate modeled, identifying residues potentially involved in substrate binding. Unaccounted electron density at the dimer interface in the crystal suggested a potential site for disrupting protein-protein interactions should a dimer be required to perform its function in bacteria. Our analysis extends the identification of functional residues to several other homologs, which include enzymes from bacteria that are involved in hydrocarbon degradation and destruction of coral reefs. The NaLdcA crystal structure provides an alternate system for investigating the structure-function relationships of LdcA and increases the structural coverage of the protagonists in bacterial cell wall recycling.

NlpC/P60 superfamily papain-like enzymes play important roles in all kingdoms of life. Two members of this superfamily, LRAT-like and YaeF/YiiX-like families, were predicted to contain a catalytic domain that is circularly permuted such that the catalytic cysteine is located near the C-terminus, instead of at the N-terminus. These permuted enzymes are widespread in virus, pathogenic bacteria, and eukaryotes. We determined the crystal structure of a member of the YaeF/YiiX-like family from Bacillus cereus in complex with lysine. The structure, which adopts a ligand-induced, "closed" conformation, confirms the circular permutation of catalytic residues. A comparative analysis of other related protein structures within the NlpC/P60 superfamily is presented. Permutated NlpC/P60 enzymes contain a similar conserved core and arrangement of catalytic residues, including a Cys/His-containing triad and an additional conserved tyrosine. More surprisingly, permuted enzymes have a hydrophobic S1 binding pocket that is distinct from previously characterized enzymes in the family, indicative of novel substrate specificity. Further analysis of a structural homolog, YiiX (PDB 2if6) identified a fatty acid in the conserved hydrophobic pocket, thus providing additional insights into possible function of these novel enzymes.

GlcNAc-1,6-anhydro-MurNAc-tetrapeptide is a major peptidoglycan degradation intermediate and a cytotoxin. It is generated by lytic transglycosylases and further degraded and recycled by various enzymes. We have identified and characterized a highly specific N-acetylmuramoyl-L-alanine amidase (AmiA) from Bacteroides uniformis, a member of the DUF1460 protein family, that hydrolyzes GlcNAc-1,6-anhydro-MurNAc-peptide into disaccharide and stem peptide. The high-resolution apo structure at 1.15 Å resolution shows that AmiA is related to NlpC/P60 γ-D-Glu-meso-diaminopimelic acid amidases and shares a common catalytic core and cysteine peptidase-like active site. AmiA has evolved structural adaptations that reconfigure the substrate recognition site. The preferred substrates for AmiA were predicted in silico based on structural and bioinformatics data, and subsequently were characterized experimentally. Further crystal structures of AmiA in complexes with GlcNAc-1,6-anhydro-MurNAc and GlcNAc have enabled us to elucidate substrate recognition and specificity. DUF1460 is highly conserved in structure and defines another amidase family.