Masanori Osawa

A novel target recognition revealed by calmodulin in complex with Ca2+-calmodulin-dependent kinase kinase.

The structure of calcium-bound calmodulin (Ca2+/CaM) complexed with a 26-residue peptide, corresponding to the CaM-binding domain of rat Ca2+/CaM-dependent protein kinase kinase (CaMKK), has been determined by NMR spectroscopy. In this complex, the CaMKK peptide forms a fold comprising an α-helix and a hairpin-like loop whose C-terminus folds back on itself. The binding orientation of this CaMKK peptide by the two CaM domains is opposite to that observed in all other CaM–target complexes determined so far. The N- and C-terminal hydrophobic pockets of Ca2+/CaM anchor Trp 444 and Phe 459 of the CaMKK peptide, respectively. This 14-residue separation between two key hydrophobic groups is also unique among previously determined CaM complexes. The present structure represents a new and distinct class of Ca2+/CaM target recognition that may be shared by other Ca2+/CaM-stimulated proteins.

Structural basis of the collagen-binding mode of discoidin domain receptor 2

Discoidin domain receptor (DDR) is a cell‐surface receptor tyrosine kinase activated by the binding of its discoidin (DS) domain to fibrillar collagen. Here, we have determined the NMR structure of the DS domain in DDR2 (DDR2‐DS domain), and identified the binding site to fibrillar collagen by transferred cross‐saturation experiments. The DDR2‐DS domain structure adopts a distorted jellyroll fold, consisting of eight β‐strands. The collagen‐binding site is formed at the interloop trench, consisting of charged residues surrounded by hydrophobic residues. The surface profile of the collagen‐binding site suggests that the DDR2‐DS domain recognizes specific sites on fibrillar collagen. This study provides a molecular basis for the collagen‐binding mode of the DDR2‐DS domain.

Structural basis underlying the dual gate properties of KcsA

KcsA is a prokaryotic pH-dependent potassium (K) channel. Its activation, by a decrease in the intracellular pH, is coupled with its subsequent inactivation, but the underlying mechanisms remain elusive. Here, we have investigated the conformational changes and equilibrium of KcsA by using solution NMR spectroscopy. Controlling the temperature and pH of KcsA samples produced three distinct methyl-TROSY and NOESY spectra, corresponding to the resting, activated, and inactivated states. The pH-dependence of the signals from the extracellular side was affected by the mutation of H25 on the intracellular side, indicating the coupled conformational changes of the extracellular and intracellular gates. K+ titration and NOE experiments revealed that the inactivated state was obtained by the replacement of K+ with H2O, which may interfere with the K+-permeation. This structural basis of the activation-coupled inactivation is closely related to the C-type inactivation of other K channels.

Structural Insights into Activation of Phosphatidylinositol 4-Kinase (Pik1) by Yeast Frequenin (Frq1)

Yeast frequenin (Frq1), a small N-myristoylated EF-hand protein, activates phosphatidylinositol 4-kinase Pik1. The NMR structure of Ca2+-bound Frq1 complexed to an N-terminal Pik1 fragment (residues 121-174) was determined. The Frq1 main chain is similar to that in free Frq1 and related proteins in the same branch of the calmodulin superfamily. The myristoyl group and first eight residues of Frq1 are solvent-exposed, and Ca2+ binds the second, third, and fourth EF-hands, which associate to create a groove with two pockets. The Pik1 peptide forms two helices (125-135 and 156-169) connected by a 20-residue loop. Side chains in the Pik1 N-terminal helix (Val-127, Ala-128, Val-131, Leu-132, and Leu-135) interact with solvent-exposed residues in the Frq1 C-terminal pocket (Leu-101, Trp-103, Val-125, Leu-138, Ile-152, and Leu-155); side chains in the Pik1 C-terminal helix (Ala-157, Ala-159, Leu-160, Val-161, Met-165, and Met-167) contact solvent-exposed residues in the Frq1 N-terminal pocket (Trp-30, Phe-34, Phe-48, Ile-51, Tyr-52, Phe-55, Phe-85, and Leu-89). This defined complex confirms that residues in Pik1 pinpointed as necessary for Frq1 binding by site-directed mutagenesis are indeed sufficient for binding. Removal of the Pik1 N-terminal region (residues 8-760) from its catalytic domain (residues 792-1066) abolishes lipid kinase activity, inconsistent with Frq1 binding simply relieving an autoinhibitory constraint. Deletion of the lipid kinase unique motif (residues 35-110) also eliminates Pik1 activity. In the complex, binding of Ca2+-bound Frq1 forces the Pik1 chain into a U-turn. Frq1 may activate Pik1 by facilitating membrane targeting via the exposed N-myristoyl group and by imposing a structural transition that promotes association of the lipid kinase unique motif with the kinase domain.

NMR Analyses of the Interaction between CCR5 and Its Ligand Using Functional Reconstitution of CCR5 in Lipid Bilayers

CC-chemokine receptor 5 (CCR5) belongs to the G protein-coupled receptor (GPCR) family and plays important roles in the inflammatory response. In addition, its ligands inhibit the HIV infection. Structural analyses of CCR5 have been hampered by its instability in the detergent-solubilized form. Here, CCR5 was reconstituted into high density lipoprotein (rHDL), which enabled CCR5 to maintain its functions for >24 h and to be suitable for structural analyses. By applying the methyl-directed transferred cross-saturation (TCS) method to the complex between rHDL-reconstituted CCR5 and its ligand MIP-1alpha, we demonstrated that valine 59 and valine 63 of MIP-1alpha are in close proximity to CCR5 in the complex. Furthermore, these results suggest that the protective influence on HIV-1 infection of a SNP of MIP-1alpha is due to its change of affinity for CCR5. This method will be useful for investigating the various and complex signaling mediated by GPCR, and will also provide structural information about the interactions of other GPCRs with lipids, ligands, G-proteins, and effector molecules.

Evidence for calmodulin inter-domain compaction in solution induced by W-7 binding

Small‐angle X‐ray scattering and nuclear magnetic resonance were used to investigate the structural change of calcium‐bound calmodulin (Ca2+/CaM) in solution upon binding to its antagonist, N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide (W‐7). The radius of gyration was 17.4±0.3 Å for Ca2+/CaM‐W‐7 with a molar ratio of 1:5 and 20.3±0.7 Å for Ca2+/CaM. Comparison of the radius of gyration and the pair distance distribution function of the Ca2+/CaM‐W‐7 complex with those of other complexes indicates that binding of two W‐7 molecules induces a globular shape for Ca2+/CaM, probably caused by an inter‐domain compaction. The results suggest a tendency for Ca2+/CaM to form a globular structure in solution, which is inducible by a small compound like W‐7.

Molecular Interactions of Yeast Frequenin (Frq1) with the Phosphatidylinositol 4-Kinase Isoform, Pik1

Frq1, a 190-residue N-myristoylated calcium-binding protein, associates tightly with the N terminus of Pik1, a 1066-residue phosphatidylinositol 4-kinase. Deletion analysis of an Frq1-binding fragment, Pik1-(10–192), showed that residues within 80–192 are necessary and sufficient for Frq1 association in vitro. A synthetic peptide (residues 151–199) competed for binding of [35S]Pik1-(10–192) to bead-immobilized Frq1, whereas shorter peptides (164–199 and 174–199) did not. Correspondingly, a deletion mutant, Pik1(Δ152–191), did not co-immunoprecipitate efficiently with Frq1 and did not support growth at elevated temperature. Site-directed mutagenesis of Pik1-(10–192) suggested that recognition determinants lie over an extended region. Titration calorimetry demonstrated that binding of an 83-residue fragment, Pik1-(110–192), or the 151–199 peptide to Frq1 shows high affinity (K d ∼100 nm) and is largely entropic, consistent with hydrophobic interaction. Stoichiometry of Pik1-(110–192) binding to Frq1 was 1:1, as judged by titration calorimetry, by changes in NMR spectrum and intrinsic tryptophan fluorescence, and by light scattering. In cell extracts, Pik1 and Frq1 exist mainly in a heterodimeric complex, as shown by size exclusion chromatography. Cys-15 in Frq1 is notS-palmitoylated, as assessed by mass spectrometry; a Frq1(C15A) mutant and even a non-myristoylated Frq1(G2A,C15A) double mutant rescued the inviability of frq1Δ cells. This study defines the segment of Pik1 required for high affinity binding of Frq1.

NMR Analyses of the Gβγ Binding and Conformational Rearrangements of the Cytoplasmic Pore of G Protein-activated Inwardly Rectifying Potassium Channel 1 (GIRK1)

G protein-activated inwardly rectifying potassium channel (GIRK) plays crucial roles in regulating heart rate and neuronal excitability in eukaryotic cells. GIRK is activated by the direct binding of heterotrimeric G protein βγ subunits (Gβγ) upon stimulation of G protein-coupled receptors, such as M2 acetylcholine receptor. The binding of Gβγ to the cytoplasmic pore (CP) region of GIRK causes structural rearrangements, which are assumed to open the transmembrane ion gate. However, the crucial residues involved in the Gβγ binding and the structural mechanism of GIRK gating have not been fully elucidated. Here, we have characterized the interaction between the CP region of GIRK and Gβγ, by ITC and NMR. The ITC analyses indicated that four Gβγ molecules bind to a tetramer of the CP region of GIRK with a dissociation constant of 250 μm. The NMR analyses revealed that the Gβγ binding site spans two neighboring subunits of the GIRK tetramer, which causes conformational rearrangements between subunits. A possible binding mode and mechanism of GIRK gating are proposed.

Functional Equilibrium of the KcsA Structure Revealed by NMR

Background: The selectivity filter of KcsA undergoes an equilibrium between permeable and impermeable conformations under acidic conditions. Results: Truncation of the intracellular region or addition of 2,2,2-trifluoroethanol modulates the equilibrium. Conclusion: Membrane environments affect dynamics of KcsA. Significance: This is the first evidence that a structural equilibrium in the membrane is related to the inactivation of a potassium channel. KcsA is a tetrameric K+ channel that is activated by acidic pH. Under open conditions of the helix bundle crossing, the selectivity filter undergoes an equilibrium between permeable and impermeable conformations. Here we report that the population of the permeable conformation (pperm) positively correlates with the tetrameric stability and that the population in reconstituted high density lipoprotein, where KcsA is surrounded by the lipid bilayer, is lower than that in detergent micelles, indicating that dynamic properties of KcsA are different in these two media. Perturbation of the membrane environment by the addition of 1–3% 2,2,2-trifluoroethanol increases pperm and the open probability, revealed by NMR and single-channel recording analyses. These results demonstrate that KcsA inactivation is determined not only by the protein itself but also by the surrounding membrane environments.

Structural Basis for Modulation of Gating Property of G Protein-gated Inwardly Rectifying Potassium Ion Channel (GIRK) by i/o-family G Protein α Subunit (Gαi/o)

Background: Although Gβγ is known to activate GIRK, Gαi/o also modulates GIRK gating. Results: The α2/α3 helices of Gαi3 in the GTP-bound state directly bind to the αA helix of GIRK. Conclusion: The complex model explains how Gαi/o sequesters Gβγ efficiently from GIRK upon GTP hydrolysis. Significance: The structural basis for the rapid closure of GIRK by Gαi/o is provided. G protein-gated inwardly rectifying potassium channel (GIRK) plays a crucial role in regulating heart rate and neuronal excitability. The gating of GIRK is regulated by the association and dissociation of G protein βγ subunits (Gβγ), which are released from pertussis toxin-sensitive G protein α subunit (Gαi/o) upon GPCR activation in vivo. Several lines of evidence indicate that Gαi/o also interacts directly with GIRK, playing functional roles in the signaling efficiency and the modulation of the channel activity. However, the underlying mechanism for GIRK regulation by Gαi/o remains to be elucidated. Here, we performed NMR analyses of the interaction between the cytoplasmic region of GIRK1 and Gαi3 in the GTP-bound state. The NMR spectral changes of Gα upon the addition of GIRK as well as the transferred cross-saturation (TCS) results indicated their direct binding mode, where the Kd value was estimated as ∼1 mm. The TCS experiments identified the direct binding sites on Gα and GIRK as the α2/α3 helices on the GTPase domain of Gα and the αA helix of GIRK. In addition, the TCS and paramagnetic relaxation enhancement results suggested that the helical domain of Gα transiently interacts with the αA helix of GIRK. Based on these results, we built a docking model of Gα and GIRK, suggesting the molecular basis for efficient GIRK deactivation by Gαi/o.