Mariko Yokogawa

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.

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.

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.

Evidence for the Direct Interaction of Spermine with the Inwardly Rectifying Potassium Channel

The inwardly rectifying potassium channel (Kir) regulates resting membrane potential, K+ homeostasis, heart rate, and hormone secretion. The outward current is blocked in a voltage-dependent manner, upon the binding of intracellular polyamines or Mg2+ to the transmembrane pore domain. Meanwhile, electrophysiological studies have shown that mutations of several acidic residues in the intracellular regions affected the inward rectification. Although these acidic residues are assumed to bind polyamines, the functional role of the binding of polyamines and Mg2+ to the intracellular regions of Kirs remains unclear. Here, we report thermodynamic and structural studies of the interaction between polyamines and the cytoplasmic pore of mouse Kir3.1/GIRK1, which is gated by binding of G-protein βγ-subunit (Gβγ). ITC analyses showed that two spermine molecules bind to a tetramer of Kir3.1/GIRK1 with a dissociation constant of 26 μm, which is lower than other blockers. NMR analyses revealed that the spermine binding site is Asp-260 and its surrounding area. Small but significant chemical shift perturbations upon spermine binding were observed in the subunit-subunit interface of the tetramer, suggesting that spermine binding alters the relative orientations of the four subunits. Our ITC and NMR results postulated a spermine binding mode, where one spermine molecule bridges two Asp-260 side chains from adjacent subunits, with rearrangement of the subunit orientations. This suggests the functional roles of spermine binding to the cytoplasmic pore: stabilization of the resting state conformation of the channel, and instant translocation to the transmembrane pore upon activation through the Gβγ-induced conformational rearrangement.

NMR Method for Characterizing Microsecond-to-Millisecond Chemical Exchanges Utilizing Differential Multiple-Quantum Relaxation in High Molecular Weight Proteins

Chemical exchange processes of proteins on the order of microseconds (μs) to milliseconds (ms) play critical roles in biological functions. Developments in methyl-transverse relaxation optimized spectroscopy (methyl-TROSY), which observes the slowly relaxing multiple quantum (MQ) coherences, have enabled the studies of biologically important large proteins. However, the analyses of μs to ms chemical exchange processes based on the methyl-TROSY principle are still challenging, because the interpretation of the chemical exchange contributions to the MQ relaxation profiles is complicated, as significant chemical shift differences occur in both (1)H and (13)C nuclei. Here, we report a new methyl-based NMR method for characterizing chemical exchanges, utilizing differential MQ relaxation rates and a heteronuclear double resonance pulse technique. The method enables quantitative evaluations of the chemical exchange processes, in which significant chemical shift differences exist in both the (1)H and (13)C nuclei. The versatility of the method is demonstrated with the application to KirBac1.1, with an apparent molecular mass of 200 kDa.

Dynamic regulation of GDP binding to G proteins revealed by magnetic field-dependent NMR relaxation analyses

Heterotrimeric guanine-nucleotide-binding proteins (G proteins) serve as molecular switches in signalling pathways, by coupling the activation of cell surface receptors to intracellular responses. Mutations in the G protein α-subunit (Gα) that accelerate guanosine diphosphate (GDP) dissociation cause hyperactivation of the downstream effector proteins, leading to oncogenesis. However, the structural mechanism of the accelerated GDP dissociation has remained unclear. Here, we use magnetic field-dependent nuclear magnetic resonance relaxation analyses to investigate the structural and dynamic properties of GDP bound Gα on a microsecond timescale. We show that Gα rapidly exchanges between a ground-state conformation, which tightly binds to GDP and an excited conformation with reduced GDP affinity. The oncogenic D150N mutation accelerates GDP dissociation by shifting the equilibrium towards the excited conformation.

Structural basis for the ethanol action on G-protein–activated inwardly rectifying potassium channel 1 revealed by NMR spectroscopy

Significance Ethanol exerts various functions by acting on ion channels in neural systems. Despite its biological importance, molecular mechanisms underlying the ethanol action remained unknown, due to the weak binding affinity and the dynamic nature of the interaction. Here, by using solution NMR techniques, we investigated the molecular interaction of ethanol with G-protein–activated inwardly rectifying potassium channel (GIRK), which is a physiologically relevant target of ethanol, and revealed that ethanol activates GIRK by shifting the conformational equilibrium of GIRK to stabilize the open conformation of the cytoplasmic ion gate. These findings provide structural insights into mechanisms of the ethanol action for various types of ethanol-sensitive signaling molecules and would facilitate the developments of pharmaceutical compounds targeting ion channels. Ethanol consumption leads to a wide range of pharmacological effects by acting on the signaling proteins in the human nervous system, such as ion channels. Despite its familiarity and biological importance, very little is known about the molecular mechanisms underlying the ethanol action, due to extremely weak binding affinity and the dynamic nature of the ethanol interaction. In this research, we focused on the primary in vivo target of ethanol, G-protein–activated inwardly rectifying potassium channel (GIRK), which is responsible for the ethanol-induced analgesia. By utilizing solution NMR spectroscopy, we characterized the changes in the structure and dynamics of GIRK induced by ethanol binding. We demonstrated here that ethanol binds to GIRK with an apparent dissociation constant of 1.0 M and that the actual physiological binding site of ethanol is located on the cavity formed between the neighboring cytoplasmic regions of the GIRK tetramer. From the methyl-based NMR relaxation analyses, we revealed that ethanol activates GIRK by shifting the conformational equilibrium processes, which are responsible for the gating of GIRK, to stabilize an open conformation of the cytoplasmic ion gate. We suggest that the dynamic molecular mechanism of the ethanol-induced activation of GIRK represents a general model of the ethanol action on signaling proteins in the human nervous system.

Nuclear Magnetic Resonance Approaches for Characterizing Protein-Protein Interactions

The gating of potassium ion (K+) channels is regulated by various kinds of protein-protein interactions (PPIs). Structural investigations of these PPIs provide useful information not only for understanding the gating mechanisms of K+ channels, but also for developing the pharmaceutical compounds targeting K+ channels. Here, we describe a nuclear magnetic resonance spectroscopic method, termed the cross saturation (CS) method, to accurately determine the binding surfaces of protein complexes, and its application to the investigation of the interaction between a G protein-coupled inwardly rectifying K+ channel and a G protein α subunit.

CHAPTER 26:Large Protein Complexes Revealed by Solution-State NMR: G Proteins and G Protein-Activated Inwardly Rectifying Potassium Ion Channel

In contrast to solid-state NMR, which allows analyses of proteins with a theoretically unlimited molecular size, solution-state NMR techniques are limited by the molecular mass of proteins. However, recent progress, especially in the development of transverse relaxation-optimized spectroscopy (TROSY), has expanded the upper limit of the molecular mass to higher than 100 kDa. Furthermore, the binding interfaces of larger protein complexes can be identified by transferred cross-saturation (TCS), paramagnetic relaxation enhancement (PRE), etc. These solution-state NMR techniques for larger protein complexes were applied to GTP-binding proteins (G proteins) and the G protein-gated inwardly rectifying potassium channel (GIRK). The residues at the complex interface and those undergoing structural changes upon binding revealed the mechanism of GIRK channel regulation by G proteins.