Masashi Yokochi

Structural basis for the transforming activity of human cancer-related signaling adaptor protein CRK

CRKI (SH2-SH3) and CRKII (SH2-SH3-SH3) are splicing isoforms of the oncoprotein CRK that regulate transcription and cytoskeletal reorganization for cell growth and motility by linking tyrosine kinases to small G proteins. CRKI shows substantial transforming activity, whereas the activity of CRKII is low, and phosphorylated CRKII has no biological activity whatsoever. The molecular mechanisms underlying the distinct biological activities of the CRK proteins remain elusive. We determined the solution structures of CRKI, CRKII and phosphorylated CRKII by NMR and identified the molecular mechanism that gives rise to their activities. Results from mutational analysis using rodent 3Y1 fibroblasts were consistent with those from the structural studies. Together, these data suggest that the linker region modulates the binding of CRKII to its targets, thus regulating cell growth and motility.

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.

The PB1 domain and the PC motif‐containing region are structurally similar protein binding modules

The PC motif is evolutionarily conserved together with the PB1 domain, a binding partner of the PC motif‐containing protein. For interaction with the PB1 domain, the PC motif‐containing region (PCCR) comprising the PC motif and its flanking regions is required. Because the PB1 domain and the PCCR are novel binding modules found in a variety of signaling proteins, their structural and functional characterization is crucial. Bem1p and Cdc24p interact through the PB1–PCCR interaction and regulate cell polarization in budding yeast. Here, we determined a tertiary structure of the PCCR of Cdc24p by NMR. The tertiary structure of the PCCR is similar to that of the PB1 domain of Bem1p, which is classified into a ubiquitin fold. The PC motif portion takes a compact ββα‐fold, presented on the ubiquitin scaffold. Mutational studies indicate that the PB1–PCCR interaction is mainly electrostatic. Based on the structural information, we group the PB1 domains and the PCCRs into a novel family, named the PB1 family. Thus, the PB1 family proteins form a specific dimer with each other.

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.

Solution structure of the Grb2 SH2 domain complexed with a high-affinity inhibitor

The solution structure of the growth factor receptor-bound protein 2 (Grb2) SH2 domain complexed with a high-affinity inhibitor containing a non-phosphorus phosphate mimetic within a macrocyclic platform was determined by nuclear magnetic resonance (NMR) spectroscopy. Unambiguous assignments of the bound inhibitor and intermolecular NOEs between the Grb2 SH2 domain and the inhibitor was accomplished using perdeuterated Grb2 SH2 protein. The well-defined solution structure of the complex was obtained and compared to those by X-ray crystallography. Since the crystal structure of the Grb2 SH2 domain formed a domain-swapped dimer and several inhibitors were bound to a hinge region, there were appreciable differences between the solution and crystal structures. Based on the binding interactions between the inhibitor and the Grb2 SH2 domain in solution, we proposed a design of second-generation inhibitors that could be expected to have higher affinity.

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.

An evaluation tool for FKBP12-dependent and -independent mTOR inhibitors using a combination of FKBP-mTOR fusion protein, DSC and NMR

Mammalian target of rapamycin (mTOR), a large multidomain protein kinase, regulates cell growth and metabolism in response to environmental signals. The FKBP rapamycin-binding (FRB) domain of mTOR is a validated therapeutic target for the development of immunosuppressant and anticancer drugs but is labile and insoluble. Here we designed a fusion protein between FKBP12 and the FRB domain of mTOR. The fusion protein was successfully expressed in Escherichia coli as a soluble form, and was purified by a simple two-step chromatographic procedure. The fusion protein exhibited increased solubility and stability compared with the isolated FRB domain, and facilitated the analysis of rapamycin and FK506 binding using differential scanning calorimetry (DSC) and solution nuclear magnetic resonance (NMR). DSC enabled the rapid observation of protein–drug interactions at the domain level, while NMR gave insights into the protein–drug interactions at the residue level. The use of the FKBP12–FRB fusion protein combined with DSC and NMR provides a useful tool for the efficient screening of FKBP12-dependent as well as -independent inhibitors of the mTOR FRB domain.