Toshihiko Sugiki

Structural Basis for the Golgi Association by the Pleckstrin Homology Domain of the Ceramide Trafficking Protein (CERT)

Background: The CERT PH domain is indispensable for the ER-to-Golgi ceramide transport. Results: The three-dimensional structure and interaction study revealed the Golgi recognition mode of the CERT PH domain. Conclusion: The basic groove in the CERT PH domain plays a critical role in the Golgi recognition. Significance: Conservation of the basic groove within lipid transporters uncovers functional significance of the structural motif. Ceramide transport from the endoplasmic reticulum to the Golgi apparatus is crucial in sphingolipid biosynthesis, and the process relies on the ceramide trafficking protein (CERT), which contains pleckstrin homology (PH) and StAR-related lipid transfer domains. The CERT PH domain specifically recognizes phosphatidylinositol 4-monophosphate (PtdIns(4)P), a characteristic phosphoinositide in the Golgi membrane, and is indispensable for the endoplasmic reticulum-to-Golgi transport of ceramide by CERT. In this study, we determined the three-dimensional structure of the CERT PH domain by using solution NMR techniques. The structure revealed the presence of a characteristic basic groove near the canonical PtdIns(4)P recognition site. An extensive interaction study using NMR and other biophysical techniques revealed that the basic groove coordinates the CERT PH domain for efficient PtdIns(4)P recognition and localization in the Golgi apparatus. The notion was also supported by Golgi mislocalization of the CERT mutants in living cells. The distinctive binding modes reflect the functions of PH domains, as the basic groove is conserved only in the PH domains involved with the PtdIns(4)P-dependent lipid transport activity but not in those with the signal transduction activity.

Isotopic Labeling of Heterologous Proteins in the Yeast Pichia pastoris and Kluyveromyces lactis

Several protein expression systems are available for the preparation of stable isotope-labeled recombinant proteins for NMR studies. Yeast expression systems have several advantages over prokaryotic systems, such as the widely used Escherichia coli expression system. Protein expression using the methylotrophic yeast Pichia pastoris is commonly employed for the preparation of isotope-labeled proteins. Recently, the hemiascomycete yeast Kluyveromyces lactis expression system was reported as being useful for preparing proteins for NMR studies. Since each yeast expression system has different features, their applications have increased in number. In this chapter, we describe procedures for the efficient production of uniformly isotope-labeled proteins using the P. pastoris and the K. lactis yeast expression systems.

Real-time assay method of lipid extraction activity

Intracellular lipid translocation is mediated by lipid transfer proteins and their functional impairments cause severe disorder in lipid metabolism. However, molecular mechanisms of protein-mediated lipid transfer remain unclear since conventional assay methods could not observe elementary processes in the lipid transfer reaction, such as lipid bilayer binding and lipid uptake. In this study, we found that ceramide extraction mediated by a ceramide trafficking protein (CERT) could be detected as decreasing the response of surface plasmon resonance (SPR). Based on this finding, we developed a novel real-time assay method that enables quantitative evaluation of the ceramide extraction activity of CERT, using the SPR technique. Performing this SPR-based assay using ceramide-embedded and ceramide-free lipid bilayers as ligands allows for the exclusive investigation of ceramide uptake processes, differentiating them from other CERT-membrane binding events. Furthermore, mutagenesis experiments of CERT using this SPR-based assay clearly elucidated whether an amino acid residue plays a role in the ceramide uptake process or the lipid bilayer binding process. This SPR-based assay method can separately evaluate the lipid extraction activity and lipid bilayer binding activity of the lipid transfer proteins, and provide more detailed information about lipid transfer phenomena.

Stable isotope labeling of protein by Kluyveromyces lactis for NMR study

Stable isotope labeling for proteins of interest is an important technique in structural analyses of proteins by NMR spectroscopy. Escherichia coli is one of the most useful protein expression systems for stable isotope labeling because of its high-level protein expression and low costs for isotope-labeling. However, for the expression of proteins with numerous disulfide-bonds and/or post-translational modifications, E. coli systems are not necessarily appropriate. Instead, eukaryotic cells, such as yeast Pichia pastoris, have great potential for successful production of these proteins. The hemiascomycete yeast Kluyveromyces lactis is superior to the methylotrophic yeast P. pastoris in some respects: simple and rapid transformation, good reproducibility of protein expression induction and easy scale-up of culture. In the present study, we established a protein expression system using K. lactis, which enabled the preparation of labeled proteins using glucose and ammonium chloride as a stable isotope source.

High-throughput screening of optimal solution conditions for structural biological studies by fluorescence correlation spectroscopy.

Protein aggregation is an essential molecular event in a wide variety of biological situations, and is a causal factor in several degenerative diseases. The aggregation of proteins also frequently hampers structural biological analyses, such as solution NMR studies. Therefore, precise detection and characterization of protein aggregation are of crucial importance for various research fields. In this study, we demonstrate that fluorescence correlation spectroscopy (FCS) using a single‐molecule fluorescence detection system enables the detection of otherwise invisible aggregation of proteins at higher protein concentrations, which are suitable for structural biological experiments, and consumes relatively small amounts of protein over a short measurement time. Furthermore, utilizing FCS, we established a method for high‐throughput screening of protein aggregation and optimal solution conditions for structural biological experiments.

Transient antibody-antigen interactions mediate the strain-specific recognition of a conserved malaria epitope

Transient interactions in which binding partners retain substantial conformational disorder play an essential role in regulating biological networks, challenging the expectation that specificity demands structurally defined and unambiguous molecular interactions. The monoclonal antibody 6D8 recognises a completely conserved continuous nine-residue epitope within the intrinsically disordered malaria antigen, MSP2, yet it has different affinities for the two allelic forms of this antigen. NMR chemical shift perturbations, relaxation rates and paramagnetic relaxation enhancements reveal the presence of transient interactions involving polymorphic residues immediately C-terminal to the structurally defined epitope. A combination of these experimental data with molecular dynamics simulations shows clearly that the polymorphic C-terminal extension engages in multiple transient interactions distributed across much of the accessible antibody surface. These interactions are determined more by topographical features of the antibody surface than by sequence-specific interactions. Thus, specificity arises as a consequence of subtle differences in what are highly dynamic and essentially non-specific interactions.Krishnarjuna et al. show that multiple transient interactions mediate monoclonal antibody recognition of an epitope within a disordered malaria antigen, MSP2. These results explain the antibody’s differential affinities for two allelic forms of the antigen.