Structural engineering of a phage lysin that targets gram-negative pathogens.

Bacterial pathogens are becoming increasingly resistant to antibiotics. As an alternative therapeutic strategy, phage therapy reagents containing purified viral lysins have been developed against gram-positive organisms but not against gram-negative organisms due to the inability of these types of drugs to cross the bacterial outer membrane. We solved the crystal structures of a Yersinia pestis outer membrane transporter called FyuA and a bacterial toxin called pesticin that targets this transporter. FyuA is a ß-barrel membrane protein belonging to the family of TonB dependent transporters, whereas pesticin is a soluble protein with two domains, one that binds to FyuA and another that is structurally similar to phage T4 lysozyme. The structure of pesticin allowed us to design a phage therapy reagent comprised of the FyuA binding domain of pesticin fused to the N-terminus of T4 lysozyme. This hybrid toxin kills specific Yersinia and pathogenic E. coli strains and, importantly, can evade the pesticin immunity protein (Pim) giving it a distinct advantage over pesticin. Furthermore, because FyuA is required for virulence and is more common in pathogenic bacteria, the hybrid toxin also has the advantage of targeting primarily disease-causing bacteria rather than indiscriminately eliminating natural gut flora.

A ten-year retrospective analysis of the distribution, use and phenotypic characteristics of the LAD2 human mast cell line.

BACKGROUND: In 2003, this laboratory published an account of the human mast cell line LAD2 (Laboratory of Allergic Diseases 2) that expressed FcεRI, responded to recombinant human stem cell factor (rhSCF) and resembled CD34+-derived human mast cells. LAD2 cells have now been distributed worldwide. To study the impact of this transfer, we analyzed the number of investigators receiving LAD2 cells and resulting publications. METHODS: Records maintained in our laboratory, the Technology Transfer and Intellectual Property Office and Office of Technology Transfer, were reviewed for material transfer agreements (MTAs) and licensing agreements (LAs). Journals and impact factors were obtained from PubMed.gov by cross-referencing LAD2 and human mast cells from 2003 through November 2013. RESULTS: Over 300 MTAs and 40 LAs were approved. LAD2 cells were shipped to over 30 countries. More than 80 papers have been published in journals with impact factors from 1.31 to 13.21. Intended uses include the study of receptors, degranulation, and cell signaling. LAD2 cells continue to express described markers and have consistent FcεR1-mediated degranulation. CONCLUSIONS: Success of the LAD2 line reflects the demand for a human mast cell line in research, the uniqueness of this cell line, and that it continues to exhibit minimal variation from its original description. We hope that the awareness of the impact of this cell line on mast cell research will encourage others to develop and distribute other similar cell lines with additional characteristics so as to address the limitations of depending on the study of cultured human mast cells from tissues.

Electron paramagnetic resonance reveals a large-scale conformational change in the cytoplasmic domain of phospholamban upon binding to the sarcoplasmic reticulum Ca-ATPase.

We used EPR spectroscopy to probe directly the interaction between phospholamban (PLB) and its regulatory target, the sarcoplasmic reticulum Ca-ATPase (SERCA). Synthetic monomeric PLB was prepared with a single cytoplasmic cysteine at residue 11, which was then spin labeled. PLB was reconstituted into membranes in the presence or absence of SERCA, and spin label mobility and accessibility were measured. The spin label was quite rotationally mobile in the absence of SERCA, but became more restricted in the presence of SERCA. SERCA also decreased the dependence of spin label mobility on PLB concentration in the membrane, indicating that SERCA reduces PLB-PLB interactions. The spin label MTSSL, attached to Cys11 on PLB by a disulfide bond, was stable at position 11 in the absence of SERCA. In the presence of SERCA, the spin label was released and a covalent bond was formed between PLB and SERCA, indicating direct interaction of one or more SERCA cysteine residues with Cys11 on PLB. The accessibility of the PLB-bound spin label IPSL to paramagnetic agents, localized in different phases of the membrane, indicates that SERCA greatly reduces the level of interaction of the spin label with the membrane surface. We propose that the cytoplasmic domain of PLB associates with the lipid surface, and that association with SERCA induces a major conformational change in PLB in which the cytoplasmic domain is drawn away from the lipid surface by SERCA.

Phospholamban structural dynamics in lipid bilayers probed by a spin label rigidly coupled to the peptide backbone.

We have used chemical synthesis and electron paramagnetic resonance to probe the structural dynamics of phospholamban (PLB) in lipid bilayers. Derivatives of monomeric PLB were synthesized, each of which contained a single spin-labeled 2,2,6,6,-Tetramethyl-piperidine-N-oxyl-4-amino-4-carboxylic acid amino acid, with the nitroxide-containing ring covalently and rigidly attached to the alpha-carbon, providing direct insight into the conformational dynamics of the peptide backbone. 2,2,6,6,-tetramethyl-piperidine-N-oxyl-4-amino-4-carboxylic acid was attached at positions 0, 11, and 24 in the cytoplasmic domain or at position 46 in the transmembrane domain. The electron paramagnetic resonance spectrum of the transmembrane domain site (position 46) indicates a single spectral component corresponding to strong immobilization of the probe, consistent with the presence of a stable and highly ordered transmembrane helix. In contrast, each of the three cytoplasmic domain probes has two clearly resolved spectral components (conformational states), one of which indicates nearly isotropic nanosecond dynamic disorder. For the probe at position 11, an N-terminal lipid anchor shifts the equilibrium toward the restricted component, whereas Mg(2+) shifts it in the opposite direction. Relaxation enhancement, due to Ni(2+) ions chelated to lipid head-groups, provides further information about the membrane topology of PLB, allowing us to confirm and refine a structural model based on previous NMR data. We conclude that the cytoplasmic domain of PLB is in a dynamic equilibrium between an ordered conformation, which is in direct contact with the membrane surface, and a dynamically disordered form, which is detached from the membrane and poised to interact with its regulatory target.

Overexpression, purification, and characterization of recombinant Ca-ATPase regulators for high-resolution solution and solid-state NMR studies.

Phospholamban (PLB) and Sarcolipin (SLN) are integral membrane proteins that regulate muscle contractility via direct interaction with the Ca-ATPase in cardiac and skeletal muscle, respectively. The molecular details of these protein-protein interactions are as yet undetermined. Solution and solid-state NMR spectroscopies have proven to be effective tools for deciphering such regulatory mechanisms to a high degree of resolution; however, large quantities of pure recombinant protein are required for these studies. Thus, recombinant PLB and SLN production in Escherichia coli was optimized for use in NMR experiments. Fusions of PLB and SLN to maltose binding protein (MBP) were constructed and optimal conditions for protein expression and purification were screened. This facilitated the large-scale production of highly pure protein. To confirm their functionality, the biological activities of recombinant PLB and SLN were compared to those of their synthetic counterparts. The regulation of Ca-ATPase activity by recombinant PLB and SLN was indistinguishable from the regulation by synthetic proteins, demonstrating the functional integrity of the recombinant constructs and ensuring the biological relevance of our future structural studies. Finally, NMR spectroscopic conditions were established and optimized for use in investigations of the mechanism of Ca-ATPase regulation by PLB and SLN.