Tomohide Saio

Two-point anchoring of a lanthanide-binding peptide to a target protein enhances the paramagnetic anisotropic effect

Paramagnetic lanthanide ions fixed in a protein frame induce several paramagnetic effects such as pseudo-contact shifts and residual dipolar couplings. These effects provide long-range distance and angular information for proteins and, therefore, are valuable in protein structural analysis. However, until recently this approach had been restricted to metal-binding proteins, but now it has become applicable to non-metalloproteins through the use of a lanthanide-binding tag. Here we report a lanthanide-binding peptide tag anchored via two points to the target proteins. Compared to conventional single-point attached tags, the two-point linked tag provides two to threefold stronger anisotropic effects. Though there is slight residual mobility of the lanthanide-binding tag, the present tag provides a higher anisotropic paramagnetic effect.

Convenient method for resolving degeneracies due to symmetry of the magnetic susceptibility tensor and its application to pseudo contact shift-based protein–protein complex structure determination

Pseudo contact shifts (PCSs) induced by paramagnetic lanthanide ions fixed in a protein frame provide long-range distance and angular information, and are valuable for the structure determination of protein–protein and protein–ligand complexes. We have been developing a lanthanide-binding peptide tag (hereafter LBT) anchored at two points via a peptide bond and a disulfide bond to the target proteins. However, the magnetic susceptibility tensor displays symmetry, which can cause multiple degenerated solutions in a structure calculation based solely on PCSs. Here we show a convenient method for resolving this degeneracy by changing the spacer length between the LBT and target protein. We applied this approach to PCS-based rigid body docking between the FKBP12-rapamycin complex and the mTOR FRB domain, and demonstrated that degeneracy could be resolved using the PCS restraints obtained from two-point anchored LBT with two different spacer lengths. The present strategy will markedly increase the usefulness of two-point anchored LBT for protein complex structure determination.

Conformational Disorder of the Most Immature Cu,Zn-Superoxide Dismutase Leading to Amyotrophic Lateral Sclerosis

Misfolding of Cu,Zn-superoxide dismutase (SOD1) is a pathological change in the familial form of amyotrophic lateral sclerosis caused by mutations in the SOD1 gene. SOD1 is an enzyme that matures through the binding of copper and zinc ions and the formation of an intramolecular disulfide bond. Pathogenic mutations are proposed to retard the post-translational maturation, decrease the structural stability, and hence trigger the misfolding of SOD1 proteins. Despite this, a misfolded and potentially pathogenic conformation of immature SOD1 remains obscure. Here, we show significant and distinct conformational changes of apoSOD1 that occur only upon reduction of the intramolecular disulfide bond in solution. In particular, loop regions in SOD1 lose their restraint and become significantly disordered upon dissociation of metal ions and reduction of the disulfide bond. Such drastic changes in the solution structure of SOD1 may trigger misfolding and fibrillar aggregation observed as pathological changes in the familial form of amyotrophic lateral sclerosis.

The NMR structure of the p62 PB1 domain, a key protein in autophagy and NF-kappaB signaling pathway

In eukaryotic cells, proteins are degraded via two main pathways; One is the ubiquitin-proteasome system that degrades short-lived proteins, and the other is autophagy that degrades long-lived proteins and damaged organelles (Noda et al. 2009). The p62, also called ZIP (PKC-finteracting protein) or sequestosome 1, plays a crucial role in these protein degradation pathways (Sumimoto et al. 2007). In autophagy, polyubiquitinated aggregated proteins and damaged organelles are enclosed by the isolation membrane, eventually enwrapped by the autophagosome. The autophagosome is fused with the vacuole/lysosome and its inner content is delivered and then degraded. The p62, initially identified as a protein that binds to the SH2 domain of the tyrosine kinase Lck, functions as a receptor protein for aberrant proteins. It contains a PB1 domain at its N terminus, followed by a ZZ-type zinc-finger motif, a LC3 interacting region, and a UBA domain at its C-terminus (Geetha and Wooten 2002). The p62 interacts with ubiquitin through the UBA domain, and self-assembles through the PB1 domain to form large protein aggregates (Bjorkoy et al. 2005). The p62 also binds the autophagy adaptor LC3 through the WXXL motif in the LC3 interacting region (Noda et al. 2008). Defects in autophagy cause accumulation of protein aggregates that contain ubiquitin and p62, leading to severe liver damage such as steatohepatitis and hepatocellular carcinomas, and neurodegenerative diseases such as Parkinson’s disease, Alzheimer disease, and Huntington’s disease (Zatloukal et al. 2002). A recent study indicated that the oligomerization through the PB1 domain was important not only in the assembly of the targets, but also in the interaction with LC3 (Bjorkoy et al. 2005). In the ubiqutin-proteasome system, p62 acts as a shuttling factor that transports ubiquitinated proteins to the proteasome, by interaction through the PB1 domain with the proteasome subunits, S5a and Rpt1 (Seibenhener et al. 2004; Geetha et al. 2008). The p62 also works as a key factor in cell signal transduction. In the NF-jB signaling pathway, p62 controls osteoclastogenesis, T-cell differentiation, and tumor progression, via the PB1–PB1 interaction with PKCf (Geetha and Wooten 2002). Further, p62 controls adipogenesis and obesity via the interaction with ERK (Moscat et al. 2006), and apoptosis via ubiquitinated Caspase 8 (Jin et al. 2009). The p62 PB1 domain plays a variety of physiological roles, both through the PB1–PB1 interaction, and through ‘‘non-canonical’’ PB1 mediated interactions with proteins lacking the PB1 domain, such as S5a, Rpt1, ERK, and LCK. Thus, p62 PB1 can be expected to have characteristic features not common to other PB1 domains whose structures have been solved. The PB1 domain is classified into three types, type I, type II, and type I/II (Hirano et al. 2004). Type I contains a motif of 28 amino acid residues with highly conserved acidic and hydrophobic residues named the OPCA motif. Type II contains a conserved lysine residue on the side opposite to the OPCA motif. Type I/II contains the OPCA motif and the conserved lysine residue, and thus can selfinteract in a front-to-back topology. The p62 PB1 contains both the OPCA motif and the conserved lysine residue and T. Saio Graduate School of Life Science, Hokkaido University, Sapporo 001-0021, Japan

Ligand-driven conformational changes of MurD visualized by paramagnetic NMR

Proteins, especially multi-domain proteins, often undergo drastic conformational changes upon binding to ligands or by post-translational modifications, which is a key step to regulate their function. However, the detailed mechanisms of such dynamic regulation of the functional processes are poorly understood because of the lack of an efficient tool. We here demonstrate detailed characterization of conformational changes of MurD, a 47 kDa protein enzyme consisting of three domains, by the use of solution NMR equipped with paramagnetic lanthanide probe. Quantitative analysis of pseudocontact shifts has identified a novel conformational state of MurD, named semi-closed conformation, which is found to be the key to understand how MurD regulates the binding of the ligands. The modulation of the affinity coupled with conformational changes accentuates the importance of conformational state to be evaluated in drug design.

Practical applications of hydrostatic pressure to refold proteins from inclusion bodies for NMR structural studies

Recently, the hydrostatic pressure refolding method was reported as a practical tool for solubilizing and refolding proteins from inclusion bodies; however, there have been only a few applications for protein structural studies. Here, we report the successful applications of the hydrostatic pressure refolding method to refold proteins, including the MOE-2 tandem zinc-finger, the p62 PB1 domain, the GCN2 RWD domain, and the mTOR FRB domain. Moreover, the absence of aggregation and the correct folding of solubilized protein samples were evaluated with size exclusion chromatography and NMR experiments. The analyses of NMR spectra for MOE-2 tandem zinc-finger and GCN2 RWD further led to the determination of tertiary structures, which are consistent with those from soluble fractions. Overall, our results indicate that the hydrostatic pressure method is effective for preparing samples for NMR structural studies.

Energetic Mechanism of Cytochrome c-Cytochrome c Oxidase Electron Transfer Complex Formation under Turnover Conditions Revealed by Mutational Effects and Docking Simulation.

Based on the mutational effects on the steady-state kinetics of the electron transfer reaction and our NMR analysis of the interaction site (Sakamoto, K., Kamiya, M., Imai, M., Shinzawa-Itoh, K., Uchida, T., Kawano, K., Yoshikawa, S., and Ishimori, K. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 12271–12276), we determined the structure of the electron transfer complex between cytochrome c (Cyt c) and cytochrome c oxidase (CcO) under turnover conditions and energetically characterized the interactions essential for complex formation. The complex structures predicted by the protein docking simulation were computationally selected and validated by the experimental kinetic data for mutant Cyt c in the electron transfer reaction to CcO. The interaction analysis using the selected Cyt c-CcO complex structure revealed the electrostatic and hydrophobic contributions of each amino acid residue to the free energy required for complex formation. Several charged residues showed large unfavorable (desolvation) electrostatic interactions that were almost cancelled out by large favorable (Columbic) electrostatic interactions but resulted in the destabilization of the complex. The residual destabilizing free energy is compensated by the van der Waals interactions mediated by hydrophobic amino acid residues to give the stabilized complex. Thus, hydrophobic interactions are the primary factors that promote complex formation between Cyt c and CcO under turnover conditions, whereas the change in the electrostatic destabilization free energy provides the variance of the binding free energy in the mutants. The distribution of favorable and unfavorable electrostatic interactions in the interaction site determines the orientation of the binding of Cyt c on CcO.

Investigation of the redox-dependent modulation of structure and dynamics in human cytochrome c.

Redox-dependent changes in the structure and dynamics of human cytochrome c (Cyt c) were investigated by solution NMR. We found significant structural changes in several regions, including residues 23-28 (loop 3), which were further corroborated by chemical shift differences between the reduced and oxidized states of Cyt c. These differences are essential for discriminating redox states in Cyt c by cytochrome c oxidase (CcO) during electron transfer reactions. Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments identified that the region around His33 undergoes conformational exchanges on the μs-ms timescale, indicating significant redox-dependent structural changes. Because His33 is not part of the interaction site for CcO, our data suggest that the dynamic properties of the region, which is far from the interaction site for CcO, contribute to conformational changes during electron transfer to CcO.