Takashi Saitoh

Tom20 recognizes mitochondrial presequences through dynamic equilibrium among multiple bound states.

Most mitochondrial proteins are synthesized in the cytosol and imported into mitochondria. The N‐terminal presequences of mitochondrial‐precursor proteins contain a diverse consensus motif (ϕχχϕϕ, ϕ is hydrophobic and χ is any amino acid), which is recognized by the Tom20 protein on the mitochondrial surface. To reveal the structural basis of the broad selectivity of Tom20, the Tom20–presequence complex was crystallized. Tethering a presequence peptide to Tom20 through a disulfide bond was essential for crystallization. Unexpectedly, the two crystals with different linker designs provided unique relative orientations of the presequence with respect to Tom20, and neither configuration could fully account for the hydrophobic preference at the three hydrophobic positions of the consensus motif. We propose the existence of a dynamic equilibrium in solution among multiple states including the two bound states. In accordance, NMR 15N relaxation analyses suggested motion on a sub‐millisecond timescale at the Tom20–presequence interface. We suggest that the dynamic, multiple‐mode interaction is the molecular mechanism facilitating the broadly selective specificity of the Tom20 receptor toward diverse mitochondrial presequences.

Blockade of Inflammatory Responses by a Small-Molecule Inhibitor of the Rac Activator DOCK2

Tissue infiltration of activated lymphocytes isxa0a hallmark of transplant rejection and organ-specific autoimmune diseases. Migration and activation of lymphocytes depend on DOCK2, an atypical Rac activator predominantly expressed in hematopoietic cells. Although DOCK2 does not contain Dbl homology domain typically found in guanine nucleotide exchange factors, DOCK2 mediates the GTP-GDP exchange reaction for Rac through its DHR-2 domain. Here, we have identified 4-[3-(2″-chlorophenyl)-2-propen-1-ylidene]-1-phenyl-3,5-pyrazolidinedione (CPYPP) as a small-molecule inhibitor of DOCK2. CPYPP bound to DOCK2 DHR-2 domain inxa0a reversible manner and inhibited its catalytic activity inxa0vitro. When lymphocytes were treated with CPYPP, both chemokine receptor- and antigen receptor-mediated Rac activation were blocked, resulting in marked reduction of chemotactic response and Txa0cell activation. These results provide a rational of and a chemical scaffold for development of the DOCK2-targeting immunosuppressant.

Crystallographic and NMR Evidence for Flexibility in Oligosaccharyltransferases and Its Catalytic Significance

Oligosaccharyltransferase (OST) is a membrane-bound enzyme that catalyzes the transfer of an oligosaccharide to an asparagine residue in glycoproteins. It possesses a binding pocket that recognizes Ser and Thr residues at thexa0+2 position in thexa0N-glycosylation consensus, Asn-X-Ser/Thr. We determined the crystal structures of the C-terminal globular domains of the catalytic subunits of two archaeal OSTs. A comparison with previously determined structures identified a segment with remarkable conformational plasticity, induced by crystal contact effects. We characterized its dynamic properties in solution by (15)N NMR relaxation analyses. Intriguingly, the mobile region contains thexa0+2 Ser/Thr-binding pocket. In agreement, the flexibility restriction forced by an engineered disulfide crosslink abolished the enzymatic activity, and its cleavage fully restored activity. These results suggest the necessity of multiple conformational states in the reaction. The dynamic nature of the Ser/Thr pocket could facilitate the efficient scanning of N-glycosylation sequons along nascent polypeptide chains.

Crystallographic snapshots of tom20-mitochondrial presequence interactions with disulfide-stabilized peptides.

Most mitochondrial proteins are synthesized in the cytosol and imported into mitochondria. The Tom20 protein, residing on the mitochondrial surface, recognizes the N-terminal presequences of precursor proteins. We previously determined the crystal structures of the Tom20-presequence complex. The successful crystallization involved tethering the presequence to Tom20 through an intermolecular disulfide bond with an optimized linker. In this work, we assessed the tethering method. The intermolecular disulfide bond was cleaved in crystal with a reducing agent. The pose (i.e., conformation and position) of the presequence was identical to the previously determined pose. In another experiment, a longer linker than the optimized length was used for the tethering. The perturbation of the tether changed the pose slightly, but the interaction mode was preserved. These results argue against the forced interaction of the presequence by its covalent attachment to Tom20. Second, as an alternative method referred to as molecular stiffening, we introduced a disulfide bond within the presequence peptide to restrict the freedom of the peptide in the unbound states. One presequence analogue exhibited over 100-fold higher affinity than its linear counterpart and generated cocrystals with Tom20. One of the two crystallographic snapshots revealed a known pose previously determined by the tethering method, and the other snapshot depicted a new pose. These results confirmed and extended the dynamic, multiple bound state model of the Tom20-presequence interactions and also demonstrated the validity of the molecular tethering and stiffening techniques in studies of transient protein-peptide interactions.

Energetics of the Presequence-Binding Poses in Mitochondrial Protein Import Through Tom20

Tom20 is located at the outer membrane of mitochondria and functions as a receptor for the N-terminal presequence of mitochondrial-precursor proteins. Recently, three atomic structures of the Tom20-presequence complex were determined using X-ray crystallography and classified into A-, M-, and Y-poses in terms of their presequence-binding modes. Combined with biochemical and NMR data, a dynamic-equilibrium model between the three poses has been proposed. To investigate this mechanism in further detail, we performed all-atom molecular dynamics (MD) simulations and replica-exchange MD (REMD) simulations of the Tom20-presequence complex in explicit water. In the REMD simulations, one major distribution and another minor one were observed in the converged free-energy landscape at 300 K. In the major distribution, structures similar to A- and M-poses exist, whereas those similar to Y-pose are located in the minor one, suggesting that A-pose in solution is more stable than Y-pose. A k-means clustering algorithm revealed a new pose not yet obtained by X-ray crystallography. This structure has double salt bridges between Arg14 in the presequence and Glu78 or Glu79 in Tom20 and can explain the binding affinity of the complex in previous pull-down assay experiments. Structural clustering and analyses of contacts between Tom20 and the presequence suggest smooth conformational changes from Y- to A-poses through low activation barriers. M-pose lies between Y- and A-poses as a metastable state. The REMD simulations thus provide insights into the energetics of the multiple-binding forms and help to detail the progressive conformational states in the dynamic-equilibrium model based on the experimental data.

Structural basis for the binding of the membrane-proximal C-terminal region of chemokine receptor CCR2 with the cytosolic regulator FROUNT.

The membrane‐proximal C‐terminal region (Pro‐C) is important for the regulation of G‐protein‐coupled receptors (GPCRs), but the binding of the Pro‐C region to a cytosolic regulator has not been structurally analyzed. The chemokine receptor CCR2 is a member of the GPCR superfamily, and the Pro‐C region of CCR2 binds to the cytosolic regulator FROUNT. Studying the interaction between CCR2 Pro‐C and FROUNT at an atomic level provides a basis for understanding the signal transduction mechanism via GPCRs. NOE‐based NMR experiments showed that, when bound to FROUNT, CCR2 Pro‐C adopted a helical conformation, as well as when embedded in dodecylphosphocholine micelles. A comparison of two types of cross‐saturation‐based NMR experiments, applied to a three‐component mixture of Pro‐C, FROUNT and micelles or a two‐component mixture of Pro‐C and micelles, revealed that the hydrophobic binding surface on Pro‐C for FROUNT mostly overlapped with the binding site for micelles, suggesting competitive binding of Pro‐C between FROUNT and micelles. Leu316 was important for both FROUNT and micelle binding. Phe319 was newly identified to be crucial for FROUNT binding, by NMR and mutational analyses. The association and dissociation rates of CCR2 Pro‐C for lipid bilayer biomembranes were faster than those for FROUNT. We previously reported that FROUNT binding to CCR2 is detectable even in unstimulated cells and increases in response to chemokine stimulation. Taken together, these results support a model of CCR2 equilibrium: chemokine binding changes the conformational equilibrium of CCR2 toward the active state, and Pro‐C switches its binding partner from the membrane to FROUNT.