Methods and Results for Semi-automated Cloning Using Integrated Robotics

The Joint Center for Structural Genomics (JCSG) has emphasized automation and parallel processing approaches. Here, we describe automated methods used across the cloning process with results from JCSG projects. The protocols for PCR, restriction digests and ligations, as well as for gel electrophoresis and microtiter plate assays have all been automated. The system has the capacity to routinely process 384 clones a week. This throughput can adequately supply our expression and purification pipeline with expression-ready clones, including novel targets and truncations. The utility of our system is demonstrated by our results from three diverse projects. In summary, 94% of the PCR amplicons generated to date have been successfully cloned and verified by sequencing (83% of the total attempted targets). Our results demonstrate the capabilities of this robotic platform to provide an avenue to high-throughput cloning which requires little manpower and is rapid and cost-effective while providing insights for method optimization.     

Crystal structure of a putative quorum sensing‐regulated protein (PA3611) from the Pseudomonas‐specific DUF4146 family

Pseudomonas aeruginosa is an opportunistic pathogen commonly found in humans and other organisms and is an important cause of infection especially in patients with compromised immune defense mechanisms. The PA3611 gene of P. aeruginosa PAO1 encodes a secreted protein of unknown function, which has been recently classified into a small Pseudomonas‐specific protein family called DUF4146. As part of our effort to extend structural coverage of novel protein space and provide a structure‐based functional insight into new protein families, we report the crystal structure of PA3611, the first structural representative of the DUF4146 protein family. Proteins 2014; 82:1086–1092. © 2013 Wiley Periodicals, Inc.     

Structures of a Bifunctional Cell Wall Hydrolase CwlT Containing a Novel Bacterial Lysozyme and an NlpC/P60 dl-Endopeptidase

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).     

Comparative structural analysis of a novel glutathioneS-transferase (ATU5508) from Agrobacterium tumefaciens at 2.0 A resolution

Glutathione S-transferases (GSTs) comprise a diverse superfamily of enzymes found in organisms from all kingdoms of life. GSTs are involved in diverse processes, notably small-molecule biosynthesis or detoxification, and are frequently also used in protein engineering studies or as biotechnology tools. Here, we report the high-resolution X-ray structure of Atu5508 from the pathogenic soil bacterium Agrobacterium tumefaciens (atGST1). Through use of comparative sequence and structural analysis of the GST superfamily, we identified local sequence and structural signatures, which allowed us to distinguish between different GST classes. This approach enables GST classification based on structure, without requiring additional biochemical or immunological data. Consequently, analysis of the atGST1 crystal structure suggests a new GST class, distinct from previously characterized GSTs, which would make it an attractive target for further biochemical studies.     

Bacterial Pleckstrin Homology Domains: A Prokaryotic Origin for the PH Domain

Pleckstrin homology (PH) domains have been identified only in eukaryotic proteins to date. We have determined crystal structures for three members of an uncharacterized protein family (Pfam PF08000), which provide compelling evidence for the existence of PH-like domains in bacteria (PHb). The first two structures contain a single PHb domain that forms a dome-shaped, oligomeric ring with C₅ symmetry. The third structure has an additional helical hairpin attached at the C-terminus and forms a similar but much larger ring with C₁₂ symmetry. Thus, both molecular assemblies exhibit rare, higher-order, cyclic symmetry but preserve a similar arrangement of their PHb domains, which gives rise to a conserved hydrophilic surface at the intersection of the β-strands of adjacent protomers that likely mediates protein-protein interactions. As a result of these structures, additional families of PHb domains were identified, suggesting that PH domains are much more widespread than originally anticipated. Thus, rather than being a eukaryotic innovation, the PH domain superfamily appears to have existed before prokaryotes and eukaryotes diverged.