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Current Research Summaries

  • [Article] Conformational changes of JNK3 splice variants upon ATP binding

    c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase (MAPK) family that regulate apoptosis, inflammation, cytokine production, and metabolism. JNKs have alternative splicing at the C-terminus, resulting in long and short variants. For example, JNK3 has splice variants called JNK3α1 and JNK3α2, the short and long forms, respectively. Functional or conformational differences between the short and long splice variants have been poorly investigated. Here, we analyzed the conformational differences between JNK3α1 and JNK3α2 upon ATP-binding by using hydrogen/deuterium exchange mass spectrometry (HDX-MS). HDX-MS showed ATP-binding affect the regions near the ATP-binding pocket in both JNK3α1 and JNK3α2 as expected. Interestingly JNK3α1 showed an additional conformational change in αL16 upon ATP-binding.

  • [Article] Assembly model of the heavy metal efflux pump CusBAC based on the protein-protein interaction

    The CusBAC heavy metal efflux pump of Escherichia coli consists of three essential components spanning the inner membrane, periplasmic space, and outer membrane. The periplasmic adaptor protein CusB connects the inner membrane transporter CusA to the outer membrane factor CusC. The structural studies revealed that the periplasmic component CusB has a unique structure in the CusC interaction region. Recent advances in cryo-electron microscopy have revealed the intermeshing cogwheel interaction between the periplasmic adaptor protein and the outer membrane factor in other types of tripartite efflux pumps, such as AcrAB-TolC and MacAB-TolC. In this study, we built a model of the full complex of the CusBAC pump, where the inner α-helices in CusB are twisted inward to make a similar cogwheel structure to that of AcrA. Binding assay results support the finding that the CusB and CusC binding mode is shared with AcrA and TolC in AcrAB-TolC. Our results give structural insight into a common mechanism for how to seal these proteins to prevent leaks in tripartite efflux pumps.

  • [Article] Antibody reformatting vector system (AbVec): a platform for converting single-chain variable fragment format to other ones using shared restriction enzyme sites

    Single-chain antibody variable fragment (scFv) is widely used at screening stage. However, the scFv format often proves to be inefficient in downstream stages such as crystallization and in vivo applications. Conversion of a scFv format to other ones such as a fragment antigen binding (Fab), a scFv-fragment crystallizable region (scFv-Fc), a scFv-constant heavy chain (scFv-CH) and an immunoglobulin (IgG) formats can be cumbersome due to different sets of restriction enzyme sites in multiple vectors. To facilitate antibody format conversion in laboratory settings, we developed a cloning platform, called “AbVec”, with the common restriction enzyme sites across various forms of vectors. In the AbVec platform, a scFv clone selected from phage display is modularized into VH and VL regions and transferred to vectors for Fab, scFv-Fc, scFv- CH and IgG production using the same restriction enzyme sites. Using scFv F9, specifically recognizing a pneumococcal secreted peptide, we demonstrated the AbVec platform yielded the expression vectors within a week. Different formats of the antibodies were successfully expressed and purified homogenously, supporting the usefulness of the AbVec platform. The AbVec platform will facilitate otherwise time-consuming conversion process in laboratory settings.

  • [Crystallization] Bacillus amyloliquefaciens NAD+-dependent protein deacetylase: purification, crystallization and X-ray crystallographic analysis

    Sirtuins are NAD+-dependent deacetylase that are broadly conserved throughout bacteria, archaea, and eukaryotes. The members of sirtuins are important in regulating diverse biological pathways, including gene silencing, DNA repair, genome stability, longevity, metabolism, and cell physiology. Sirtuin from Bacillus amyloliquefaciens (BaSrtN) is a particularly interesting bacterial Sir2 homologue. In this study, to further understand the function and mechanisms of this protein, BaSrtN was successfully expressed and purified using Ni-NTA affinity, Q anion-exchange, and gel-filtration chromatography. Purified BaSrtN was crystallized and diffracted to the resolution of 1.45 Å. The preliminary crystallographic analysis suggested that BaSrtN crystal belongs to the trigonal space group P31 or P32, with unit-cell parameters of a = b = 90.115 and c = 86.306 Å. Size-exclusion chromatography suggested that BaSrtN prefer to exit as monomers in solution.

  • [Crystallization] Purification, crystallization and X-ray crystallographic analysis of nicotinamidase Pnc1 from Kluyveromyces lactis

    Pnc1 converts nicotinamide to nicotinic acid to generate NAD+ through the Preiss-Handler pathway that is one of the NAD+-salvage pathway. By reducing levels of nicotinamide, an inhibitor of the NAD+-dependent histone deacetylase Sir2, yeast Pnc1 contributes gene silencing. In this study, to understand the structural features and molecular mechanism of nicotinamidase Pnc1, we overexpressed, purified, and crystallized the N-terminally His6-tagged Pnc1 protein from Kluyveromyces lactis and obtained X-ray diffraction data at a resolution of 2.2 Å. The crystals of the K. lactis Pnc1 (KlPnc1) belonged to space group P212121 with unit cell parameters a=38.5, b=77.3, c=83.3, and α=β=γ= 90º. There is one molecule in the asymmetric unit.