6.1.1 GIRK as an effector of G-proteins

GIRK ion channel is a member of the inwardly rectifying potassium ion channel (Kir) family, regulating heart rate, neuronal excitability, and other physiological events (Hibino et al. Reference Hibino, Inanobe, Furutani, Murakami, Findlay and Kurachi2010). GIRK functions as a tetramer, in which a long, 88 Å K⁺ permeation pathway is formed by the transmembrane (TM) and cytoplasmic pore (CP) regions (Whorton & MacKinnon, Reference Whorton and Mackinnon2011). The crystal structure of GIRK revealed two K⁺-ion gates: the helix bundle crossing on the cytoplasmic side of the TM region and the loops on the membrane side of the CP region, referred to as G-loops. Although the gating of Kir family proteins is generally regulated by phosphatidylinositol 4,5-bisphosphate, the opening of the GIRK gate is also triggered by the direct binding of the Gβγ. The stimulation of G-protein-coupled receptors (GPCRs) causes the exchange of GDP with GTP on Gα, which decreases the affinity of Gα for Gβγ. Gβγ then dissociates from Gα in the GTP-bound state (Gα(GTP)) and associates with GIRK, resulting in the opening of the GIRK gate (Hibino et al. Reference Hibino, Inanobe, Furutani, Murakami, Findlay and Kurachi2010). When the GPCR stimulation ends, the GTP on Gα is hydrolyzed to GDP, and then Gα in the GDP-bound state (Gα(GDP)) removes Gβγ from GIRK, resulting in the closure of the GIRK gate. Although Gβγ directly binds to and activates GIRK, Gα also plays important roles in regulating GIRK. In particular, Gα(GTP) reportedly interacts with GIRK directly, and accelerates the deactivation of GIRK (Benians et al. Reference Benians, Leaney, Milligan and Tinker2003; Berlin et al. Reference Berlin, Tsemakhovich, Castel, Ivanina, Dessauer, Keren-Raifman and Dascal2011). Altogether, it is now evident that Gα not only functions as the donor and acceptor of Gβγ but also modulates the gating properties of GIRK.

6.1.2 The Gβγ binding and conformational rearrangements of GIRK

To reveal the structural mechanism by which Gβγ-binding activates GIRK, we investigated the direct interaction between the CP region of GIRK1 (GIRK_CP; residues 41–63 and 190–371 as a single polypeptide) and Gβγ  (Yokogawa et al. Reference Yokogawa, Osawa, Takeuchi, Mase and Shimada2011). To identify the Gβγ-binding site on GIRK_CP, TCS experiments were performed. The residues affected by the rf-irradiation were mapped on a single subunit of GIRK_CP (Fig. 9a). Although the mapped residues are dispersed on a single subunit of GIRK_CP, these residues formed two adjacent contiguous surfaces in the two neighboring subunits of the GIRK_CP tetramer (Fig. 9b), suggesting that the two surfaces could form a single Gβγ-binding site.

Fig. 9.

Gβγ-binding interface on GIRK_CP. (a) Mapping of the affected residues in the TCS experiment on a single subunit of the GIRK_CP structure (PDB code 1N9P). The residues with large intensity reduction ratios and small but significant intensity reduction ratios are colored red and orange, respectively. In the surface representations (center and right), proline residues and residues with no data are colored black and gray, respectively, because they lacked CS data. (b) Mapping of the affected residues in the TCS experiment on two adjacent subunits of a GIRK_CP tetramer (white and cyan), viewed from the outside (left) and the inside (right) of the pore, with the same color code as in (a). (c) Important residues for the interaction between GIRK_CP and Gβγ. (Upper) Surface representations of Gα (right) and Gβγ (left), viewed from the contacting surfaces in the crystal structure of the Gαβγ complex (PDB code 1GOT). The residues involved in the intermolecular interaction between Gα and Gβγ, within a distance of 4 Å in the Gαβγ complex structure, are colored according to their side chain properties: hydrophobic, hydrophilic, acidic, and basic residues are green, yellow, red, and blue, respectively. The interacting residues between the N-terminal helix of Gα and Gβγ are labeled in gray. The important residues of Gβγ for GIRK binding, identified by mutational analyses, are labeled with asterisks. The region of the Gβγ-binding site on Gα (the Gβγ-binding motif), composed of a hydrophobic area flanked by two acidic residues, and the interacting surface on Gβγ are enclosed in circles. (Lower) The Gβγ-binding surface of GIRK, determined by TCS experiments, on the GIRK_CP structure, colored according to the side chain properties. The similar Gβγ-binding motif to that found on the Gα surface in the upper panel is circled and enlarged, where the residues without TCS information are also labeled in black and blue, for the residues from one subunit colored in white and the other subunit, colored cyan, respectively.

On the other hand, the mutational analyses suggested several residues of Gβγ that are important for GIRK activation. It should be noted that most of these residues are located in the Gα-binding site, in the crystal structure of the heterotrimeric Gαβγ complex (Fig. 9c). In addition, the Gβγ-binding sites on GIRK possess surface properties similar to those on Gα. Therefore, it is tempting to speculate that GIRK binds to Gβγ in a similar manner to Gα in the crystal structure of the heterotrimeric Gαβγ complex.

The Gβγ-binding effects on GIRK_CP were also investigated by chemical shift perturbation experiments, which reflect the effects of the direct binding of Gβγ as well as the conformational change of GIRK_CP induced by Gβγ binding. Upon the addition of 50, 100, 200, and 400 μM Gβγ to 50 μM uniformly ²H,¹⁵N-labeled GIRK_CP tetramer, no signals exhibited normalized chemical shift changes (Mulder et al. Reference Mulder, Schipper, Bott and Boelens1999; Osawa et al. Reference Osawa, Yokogawa, Muramatsu, Kimura, Mase and Shimada2009) larger than 0.05 ppm, but significant intensity reductions were observed for a number of signals.

The observed intensity reductions of the NMR signals are caused by slower tumbling, due to the increased average molecular weight of the complex of GIRK_CP and Gβγ. Furthermore, the intensity reductions are induced by the chemical shift differences of GIRK_CP between the free and bound states (Matsuo et al. Reference Matsuo, Walters, Teruya, Tanaka, Gassner, Lippard, Kyogoku and Wagner1999). The differences reflect the changes of the microenvironments on the Gβγ-binding site on GIRK_CP, including the disruption of the fourfold symmetry of the GIRK_CP tetramer, as well as the concomitant conformational changes of remote regions from the binding site.

The residues with large intensity reduction ratios seemed to be distributed widely on the molecular surface of the single subunit of GIRK_CP (Fig. 10a). However, mapping on the neighboring two subunits revealed that the affected residues are clustered at the subunit interface and its surrounding regions on both the inside and the outside of the pore (Fig. 10b). The residues located on the outside of the pore reside in the Gβγ-binding site (Fig. 9b) and its proximity. The other residues are located on the inside of the pore, at the membrane side, and at other distal sites (Fig. 10b, right). In particular, the significant chemical shift perturbations observed for the residues located at the subunit–subunit interface away from the Gβγ-binding site (Fig. 10b, right) strongly suggested that the rearrangements of the four subunits are induced by Gβγ binding. Indeed, two distinct conformations of the CP region by rigid body subunit rearrangements were observed in the crystal structure of a chimeric channel of KirBac1.3, KirBac3.1, and GIRK1 (PDB code 2QKS). As shown in Fig. 10, significant chemical shift perturbations upon Gβγ binding were observed for Gly³⁰⁷ and Met³⁰⁸ on the G-loop, suggesting that the relative positions between the loops of the four subunits change upon Gβγ binding, which could result in the opening of the K⁺ ion gate.

Fig. 10.

Binding mode of Gβγ-and GIRK. (a) The residues with large intensity reductions and small but significant reductions upon addition of Gβγ in the chemical shift perturbation experiments, mapped on the surface of a single subunit of the GIRK_CP structure and colored red and orange, respectively. Proline residues and residues with no data are colored black and gray, respectively. (b) Mapping of the residues with significant intensity reductions clustered in the interface on the outside surface of the two neighboring subunits of GIRK_CP (left), and mapping of the other residues, which are not exposed to the outside surface, on two neighboring subunits of GIRK_CP viewed from the inside of the pore (right), with the same color scheme as in (a). The buried residues are labeled with dashed lines. (c) Interacting residues between the CP and TM regions of the chimeric channel. (Left) Backbone structure of the chimeric channel of KirBac1.3 and GIRK1 (PDB code 2QKS). Two neighboring subunits are shown from the outside, and are colored gray and cyan, respectively. The regions lacking electron density are depicted by dashed lines. The interface between the CP and TM regions is boxed, and the residues in G-loop (the loop between the H and I strands of one subunit), proximal to those in the TM region of the same and adjacent subunits within 4 Å, are depicted by stick models. (Right, upper) Enlarged view of the interface between the CP and TM regions boxed in the left panel. The residue numbers are labeled according to those of GIRK1. (Right, lower) Schematic drawing of the interface between the CP and TM regions of the chimeric channel, shown in (b). The residue pairs within 4 Å of each other are connected by green lines. The following are the shortest distances between the inter- and intra-subunit residues: intersubunit, 4.0 Å (Phe⁷² (CE2) –Thr³⁰⁵ (CG2)), 4.0 Å (Thr⁷³ (OG1) –Thr³⁰⁵ (OG1)), 3.7 Å (Ile¹⁸² (CG2) –Thr³⁰⁵ (O)), and 2.6 Å (Gln¹⁸⁶ (NE2)–Thr³⁰⁵ (O)); and intrasubunit, 3.4 Å (Ser¹⁸⁵ (O)–Thr³⁰⁶ (OG1)), 3.1 Å (Ser¹⁸⁵ (O)–Met³⁰⁸ (CG)), 3.7 Å (Gln¹⁸⁶ (NE2)–Met³⁰⁸ (CE)), 3.6 Å (Pro¹⁸⁷ (CB)–Val³⁰³ (CG2)), and 3.8 Å (Pro¹⁸⁷ (CG)–Cys³¹⁰ (CB)). The positions of the Cα atoms are depicted as red dots, except for those of the residues with significant chemical shift perturbations, which are indicated as yellow dots.

6.1.3 Structural basis for the modulation of the gating properties of GIRK by Gα

In order to reveal the underlying mechanism by which Gα modulates GIRK, we performed NMR analyses of the interaction between the CP region of GIRK1 (GIRK_CP-L; residues 41–63 and 190–386) and Giα _i3(GTP), which accelerates the deactivation of GIRK (Mase et al. Reference Mase, Yokogawa, Osawa and Shimada2012). To identify the GIRK_CP-L -binding site on Gα _i3, TCS experiments were performed. The residues with large intensity reductions are located on the helical domain and the GTPase domain. The mapping of these residues on the structure of Gα revealed that the residues identified on the GTPase domain of Gα _i3 are mostly clustered, indicating that this site (hereafter, referred to as the ‘α2/α3 site’) mainly contributes to the GIRK_CP-L binding (Fig. 11a).

Fig. 11.

Direct interactions between GIRK_CP-L and Gα _i3, revealed by TCS. (a) Mapping of the affected residues in the TCS experiment from GIRK_CP-L to Gα _i3 is shown on the Gα structure (PDB code 1GIA). The affected residues are colored red on the Gα structure, whereas residues with no data, including proline residues, are colored black. The αA, αB, α2, and α3 helices are colored green. (b) Mapping of the affected residues in the TCS experiment from Gα _i3 to GIRK_CP-L on a single subunit of the GIRK_CP-L tetramer. Residues 371–386 are depicted by gray tubes. The affected residues are colored red on the GIRK_CP-L structure, whereas residues with no data, including proline residues, are colored black. The αA helix is colored green in the ribbon diagrams. (c) Mapping of the affected residues (colored red) in the TCS experiment on two adjacent subunits of a GIRK_CP-L tetramer (white and gray), viewed from the outside (left) and the inside (right) of the tetramer.

The residues identified on the helical domain, which are distant from the α2/α3 site, do not form a contiguous surface but are located on the same side of the protein surface as the α2/α3 site, suggesting that they transiently contact GIRK_CP-L. We further investigated the interactions of the helical domain with GIRK_CP-L (see below).

Conversely, to identify the Gα _i3-binding site on GIRK_CP-L, TCS experiments observing GIRK_CP-L were performed. The affected residues were mapped on a single subunit (Fig. 11b) and two adjacent subunits (Fig. 11c) of GIRK_CP (PDB code 1N9P). Almost all of these residues are located on the C-terminal region, around the αA helix of GIRK_CP-L (Fig. 11). Therefore, we concluded that these residues are the Gα _i3-binding residues.

The interaction between the helical domain of Gα _i3 and GIRK_CP-L was further investigated by paramagnetic relaxation enhancement (PRE) experiments. PRE arises from magnetic dipolar interactions between the nuclear spin and the unpaired electron spin of the paramagnetic center, which enhances the relaxation of the nuclear spins, leading to the line-broadening and thus the intensity reduction of the NMR signals of the residues within about 20 Å of the spin label (Battiste & Wagner, Reference Battiste and Wagner2000; Clore & Iwahara, Reference Clore and Iwahara2009). In addition, PRE can detect a transient interaction, because the PRE effect occurs within 250–500 μs (Clore, Reference Clore2011), which is a much shorter duration than that of the CS (the effective saturation time of 200–300 ms for the TCS experiments in this study) (Matsumoto et al. Reference Matsumoto, Ueda and Shimada2010).

Figure 12a shows that the PRE effects from Hexa III-I82C-MTSL, in which the six natively existing cysteine residues that are exposed and reactive were substituted with other residues, and Ile⁸² was substituted by Cys and chemically modified by MTSL, were observed for 13 signals from residues within the C-terminal region of GIRK_CP-L. It should be noted that most of the other residues in the C-terminal region could not be analyzed due to signal overlapping, and thus these residues might also be affected by MTSL. On the other hand, only Cys⁵³ was detected for Hexa III-S153C-MTSL (Fig. 12b), which might be caused by the partial MTSL modification of Cys⁵³. Since the spin label was introduced at I82C, which is at the center of the three residues identified by TCS, we concluded that the Ile⁸² side of the helical domain can approach within 20 Å of the C-terminal region of GIRK_CP.

Fig. 12.

PRE results of the interaction between GIRK_CP-L and Gα _i3. Mapping of the affected residues in the PRE experiment using HexaIII-I82C-MTSL (a, left) and HexaIII-S153C-MTSL (b, left), respectively, on a single subunit of the GIRK_CP-L structure. The affected residues are colored red. Proline residues and residues with no data are colored black. (Right) Mapping of the spin-labeled site of Gα _i3 on the structure of Gα.

To build a model of the Gα _i3-GIRK_CP-L complex satisfying the NMR-derived structural information, rigid body docking was performed by using the HADDOCK program. The residues in the α2/α3 site of Gα and in the αA helix of GIRK_CP-L, identified by the TCS experiments, were specified as the ‘active residues’ in the program, so that they formed the interface of the complex. Although the information about the residues on the helical domain derived from the TCS and PRE experiments was not used for the calculation, the helical domain of Gα was calculated to be proximal to the αA helix in a neighboring subunit of GIRK_CP-L, probably due to the restraint of Glu²⁴² on the subunit interface of GIRK_CP-L, as determined from the TCS experiments (Fig. 13a). In this binding mode, Glu²⁴² of GIRK_CP-L at a distance from the αA helix, approaches the side chain of Glu¹⁸⁶ of Gα _i3, which accounts for its intensity reduction in the TCS experiment. Altogether, we concluded that the α2/α3 site of Gα _i3 mainly recognizes the αA helix of GIRK_CP-L in one subunit of the tetramer, whereas the helical domain of Gα _i3 transiently approaches the αA helix in another neighboring subunit.

Fig. 13.

Proposed model of Gα(GTP) and the cytoplasmic region of the GIRK complex. (a, Left) Two adjacent subunits of a GIRK_CP-L tetramer, viewed from the inside of the tetramer, are depicted by a ribbon diagram, and a homology model of Gα _i3 is shown in a surface representation. The nitrogen atoms of the GIRK_CP-L residues identified in the TCS experiments, and the residues with PRE effects from the MTSL modification of I82C on the helical domain of Gα _i3, are shown as red and magenta balls, respectively. The α2/α3 residues of Gα _i3 identified in the TCS experiments are colored blue. (Center and right) Binding model of Gα _i3(GTP) and the cytoplasmic region of GIRK1 (PDB code 1N9P), obtained with the HADDOCK software. Only the two adjacent subunits of the GIRK tetramer are shown, from the inside of the tetramer. (b) Model of the Gα-GIRK-Gβγ ternary complex. (Left) The nitrogen atoms of the Gβγ-binding residues on GIRK1 are mapped as magenta balls on the complex model of Gα and the GIRK tetramer. (Right) Ternary complex model of Gα-GIRK-Gβγ. Gβ and Gγ are colored magenta and violet, respectively. The residues involved in the intermolecular interactions between Gα and Gβγ, within a distance of 4 Å in the Gα _i1(GDP)β ₁γ ₂ (PDB code 1GP2) structure, are colored orange. (c) GIRK- and RGS-binding sites on Gα. The GIRK-binding residues identified in this study are colored red, and the residues involved in the intermolecular interaction between Gα and RGS, within a distance of 4 Å in the Gα _i1(GDP/Mg²⁺/AlF₄⁻) – RGS4 complex structure (PDB code 1AGR), are colored green. The overlapping residues, Lys²⁰⁹ and His²¹³, are colored blue.

The mapping of the Gβγ-binding residues on the structure of the Gα-GIRK complex indicated that the binding sites of Gα _i3(GTP) and Gβγ on GIRK do not overlap with each other, and therefore, Gα _i3 (GTP) and Gβγ can simultaneously bind to GIRK (Fig. 13b). Furthermore, the mapping of the RGS-binding site on the structure of Gα suggested that regulator of G-protein signaling (RGS) can bind to Gα _i3 in the complex with GIRK (Fig. 13c). Therefore, the Gα _i/o-GIRKGβγ ternary complex model obtained here provides the structural basis for the rapid closure of the GIRK channel upon signal termination, through the efficient removal of the proximal Gβγ from GIRK.

6.2.1 Oligomerization of chemokines

Chemokines are an important class of signaling molecules that activate leukocytes. Chemokine concentration gradients on endothelial layers and in the surrounding tissue provide directional cues to guide cell movement. One factor that may be vital to the establishment and maintenance of a gradient is the chemokines’ ability to oligomerize (Appay et al. Reference Appay, Brown, Cribbes, Randle and Czaplewski1999). Several studies using non-oligomerizing variants have shown that the induction of migration in vivo is dependent on oligomerization (Campanella et al. Reference Campanella, Grimm, Manice, Colvin, Medoff, Wojtkiewicz, Weissleder and Luster2006), although monomeric chemokines are capable of causing cell migration in vitro (Laurence et al. Reference Laurence, Blanpain, Burgner, Parmentier and Liwang2000).

Therefore, structural knowledge of chemokine oligomers could provide insights into the molecular mechanisms by which chemokines facilitate the recruitment and activation of leukocytes in vivo.

An oligomer interface of the CCL5 chemokine was investigated by the CS method (Wang et al. Reference Wang, Watson, Sharp, Handel and Prestegard2011). NMR and SAXS analyses revealed that the CCL5 primarily form a tetramer at pH 4.5. In order to observe the homooligomer-binding interface, ²H, ¹⁵N-labeled CCL5 was mixed with unlabeled CCL5 in a 1:3 ratio. To distinguish the monomer-monomer from dimer–dimer interfaces, the same experiment was performed for an E66S mutant, which forms the dimer exclusively. For WT CCL5, the intensities of N-terminal residues 6–8 (red in Fig. 14), 10, 15–17, 23, 26–29, 33–34, and 49–50 were highly reduced. Additional intensity changes were seen for residues 40–43 and 64–68. The decreased intensities for the N-terminal residue signals are clearly due to the interactions at the monomer–monomer interface in the dimer units, because the same residues are perturbed in the spectra of the E66S mutant. Residues 26–29, 33 and 34, and 64–68 were perturbed in the WT data only (orange area in Fig. 14), suggesting that they are in the dimer–dimer interface. On the whole, the binding interface defined by the CS experiment agreed with the tetramer model found through the SAXS/RDC grid search procedure (Wang et al. Reference Wang, Watson, Sharp, Handel and Prestegard2011). The perturbation of residues 40–43 and 48–51 can be explained by the fact that these residues are in the vicinity of the monomer–monomer interface, and may be indirectly saturated by their proximity to the interfacial residues.

Fig. 14.

Identification of the oligomer interface of CCL5 by CS experiments. Surface representation of the CCL5 tetramer model. The residues involved in the dimer–dimer contacts (residues 26–29, 33, 34, 66, and 67), and the monomer–monomer contacts (residues 6–10) are colored red and orange, respectively.

6.2.2 Formation of amyloid fibrils

β2-Microglobulin (β2m) is one of the most extensively investigated amyloidogenic proteins. In the body of a healthy individual, the β2m molecules released into the plasma are degraded by the kidneys. However, in patients on long-term hemodialysis, released β2m cannot be degraded because of the dysfunction of the kidneys, and the β2m concentration can reach 40 μg ml⁻¹. Such a high concentration is considered to be a risk factor for the formation of amyloid fibrils (Yamamoto et al. Reference Yamamoto, Hasegawa, Yamaguchi, Goto, Gejyo and Naiki2005).

Kinetic measurements of Trp fluorescence revealed that the seed-dependent fibril extension reaction of β2m proceeds by two steps (Chatani et al. Reference Chatani, Ohnishi, Konuma, Sakurai, Naiki and Goto2010) (Fig. 15a). The first step is the association of the free monomer with the seed to form a transient monomer–seed complex (hereafter referred to as TMSC). In the next step, the conformation of the monomer converts to the final, fibril structure. The conformational conversion from the TMSC to the fibril structure is thought to be the rate-limiting step of fibril extension.

Fig. 15.

Identification of the fibril seeds-binding site in β2-microglobulin. (a) Scheme of two-step fibril formation by β2m. (b) Profiles of TCS experiments performed in the presence (blue bars) and the absence (black diamonds) of fibril. The intensity ratios of signals with and without RF irradiation (I _sat/I _non-sat) are plotted. The red circle indicates the residues that showed significant R ₂ relaxation dispersion.

To determine which residues participate in the TMSC interactions, TCS experiments were performed, using a sample containing unlabeled fibrils and ²H,¹⁵N-labeled β2m monomers in 12.5 mM NaCl, to achieve equilibrium between the free and bound states (Yanagi et al. Reference Yanagi, Sakurai, Yoshimura, Konuma, Lee, Sugase, Ikegami, Naiki and Goto2012). The results of the TCS experiments in the presence of fibril revealed that most of the residues in the middle of the molecule, including the loop regions in the native structure, showed significant reductions in the relative intensity (I _sat/I _non-sat) (Fig. 15b, blue bar), indicating that these residues interact with the fibril, whereas the N-terminus is excluded from the binding site. On the other hand, the TCS experiment in the absence of fibril exhibited no significant reduction in the relative intensity (Fig. 15b, diamond). The residues with significant reduction in the TCS experiment correlated to those with significant R _ex values in the R ₂ dispersion experiments of β2m monomers, which reflect the exchange between partially structured and extensively unfolded forms (Fig. 15b, red circle). Considering these results and those from H/D exchange experiments in the presence of fibril, it was postulated that the extensively unfolded form is the precursor for the fibril (Yanagi et al. Reference Yanagi, Sakurai, Yoshimura, Konuma, Lee, Sugase, Ikegami, Naiki and Goto2012).

6.4.1 Reconstitution of CCR5 into rHDLs

CCR5, like CXCR4, belongs to the GPCR family, and the interactions between CCR5 and its chemokines, MIP-1α, MIP-1β, and RANTES (CCL5), induce chemotactic responses in CCR5-expressing leukocytes (Rajagopalan & Rajarathnam, Reference Rajagopalan and Rajarathnam2006). CCR5 is reportedly the major coreceptor for HIV-1, and its ligands are potent inhibitors of HIV infection (Berger et al. Reference Berger, Murphy and Farber1999). However, the structural basis for the interactions between CCR5 and its ligands remain poorly understood.

The methodology applied for CXCR4 was not directly applicable for CCR5, because CCR5 in detergent micelles was much less stable than CXCR4. Surface plasmon resonance (SPR) analyses revealed that the response of the conformation-dependent antibody 2D7 for CCR5 immobilized on the sensor chip was reduced to 50% after a 3 h incubation at 4°C (Fig. 17b), and was undetectable after a 24 h incubation (Fig. 17a, solid line).

Fig. 17.

Interaction between CCR5–rHDL and MIP-1α variant. (a and b) Stability of the solubilized CCR5. Normalized SPR responses for 2D7 antibody binding to CCR5 in DDM micelles, immediately after solubilization (dotted line) and after a 24 h incubation at 4°C (solid line) are shown in (a). Responses were normalized for the amount of captured CCR5. Relative values of the responses for 2D7 antibody binding of CCR5 in DDM micelles (immediately after solubilization=100%) over time are shown in (b). (c) Schematic representation of the preparation of CCR5–rHDL. CCR5 embedded in Sf9 cell membranes was solubilized, immediately reconstituted into rHDL, and subsequently purified using the His-tag and the 1D4-tag in MSP and CCR5, respectively. (d) Normalized SPR responses for the binding of the 2D7 antibody to CCR5-rHDL, immediately after preparation (dotted line) and after a 24 h incubation at 4°C (solid line). Responses were normalized for the amount of captured CCR5-rHDL. (e) SPR analyses of the interaction between the MIP-1α variant and CCR5-rHDL. Overlay plots of the sensorgrams obtained for the interaction between 0.33 and 13.2 μM of the MIP-1α variant and the immobilized CCR5-rHDL. (f) Determination of the CCR5 binding interface on MIP-1α by methyl-TCS experiments. Mapping of the affected residues in the TCS experiment with CCR5-rHDL are colored orange on the MIP-1α structure (PDB code 1B53). Glu⁵⁷, whose mutations were identified in SNPs involved in HIV-1 infection, is also colored red.

Membrane proteins maintain their native conformations more stably in lipid bilayers than in detergent micelles. It has recently been reported that rHDLs, also known as nanodiscs, can accommodate membrane proteins within a 10-nm-diamter disc-shaped lipid bilayer, in a detergent-free solution (Bayburt et al. Reference Bayburt, Grinkova and Sligar2002; Nath et al. Reference Nath, Atkins and Sligar2007). rHDLs are composed of phospholipid bilayers, containing embedded membrane proteins, and membrane scaffold proteins (MSPs), which stabilize the discoidal bilayer and control its size. rHDLs are a promising resource for solution NMR studies, because of their water solubility and monodisperse character (Glueck et al. Reference Glueck, Wittlich, Feuerstein, Hoffmann, Willbold and Koenig2009; Lyukmanova et al. Reference Lyukmanova, Shenkarev, Paramonov, Sobol, Ovchinnikova, Chupin, Kirpichnikov, Blommers and Arseniev2008; Raschle et al. Reference Raschle, Hiller, Yu, Rice, Walz and Wagner2009). Therefore, it is important to reconstitute CCR5 into a lipid bilayer by using rHDLs for NMR analyses of the interaction between CCR5 and its ligand.

Due to the extreme instability of the detergent-solubilized CCR5, it was difficult to apply the general rHDL reconstitution procedure, in which the membrane proteins are first purified in detergents, and then the rHDLs are reconstructed by adding lipids and MSPs. Therefore, a novel rHDL reconstitution procedure was employed for the preparation of functional CCR5 in rHDLs (CCR5-rHDLs), as follows (Fig. 17c). The plasma membrane of the CCR5-expressing Sf9 cells was partially purified by sucrose gradient ultracentrifugation. CCR5 was solubilized by detergents and then immediately reconstituted into rHDLs, by adding MSP and removing the detergents. CCR5-rHDLs were purified by sequential affinity chromatographies, utilizing the His-tag and the 1D4 epitope-tag on MSP and CCR5, respectively. This method minimized the period during which CCR5 is in the detergent-solubilized state to as short as 90 min, by reconstituting CCR5 directly from the lysate of Sf9 cells before purification.

To examine the stability of CCR5-rHDL, we analyzed the time course of the binding activity to 2D7 by SPR (Fig. 17d). About 85% of the initial 2D7 antibody binding was retained after a 24 h incubation at 4°C.

We applied SPR to examine the binding between the monomeric P8A/F13Y/E67Q variant of MIP-1α, which was utilized for the TCS study, and CCR5-rHDLs. As a result, the responses were observed upon the addition of the MIP-1α variant to CCR5-rHDLs immobilized on the sensor chip, and the estimated dissociation constant was ∼5 μM (Fig. 17e). In addition, the activation of G proteins by CCR5-rHDLs with the MIP-1α variant was revealed by a GDP–GTP exchange assay.

The methyl-TCS experiments were performed with CCR5-rHDL and the MIP-1α variant. These results revealed that the methyl resonances derived from Val⁵⁹ and Val⁶³ are significantly affected by the rf-irradiation (Fig. 17f), suggesting that the region including MIP-1α, Val⁵⁹, and Val⁶³, which has not been investigated in previous structure–function studies, is in close proximity to CCR5 in the complex.

Several single nucleotide polymorphisms (SNPs) with non-synonymous amino-acid changes were recently identified within the MIP-1α gene, and one of the SNPs, with the E57D mutation, reportedly has a protective genetic influence on HIV-1 infection (Modi et al. Reference Modi, Lautenberger, An, Scott, Goedert, Kirk, Buchbinder, Phair, Donfield, O'BRIEN and Winkler2006). However, it was unknown how these SNPs affect the MIP-1α function. The present TCS study suggested that the protective influence on HIV-1 infection of the SNP may be due to the change in the interaction mode between CCR5 and MIP-1α, which causes the increased affinity, because Glu⁵⁷ is in close proximity to Val⁵⁹ and Val⁶³. In addition, another SNP containing the V63F mutation was also reported (Paximadis et al. Reference Paximadis, Mohanlal, Gray, Kuhn and Tiemessen2009). Although this SNP has not been well characterized, it may also have a genetic influence on HIV-1 infection.

6.4.2 Interaction between photosynthetic membrane protein complexes and an electron transport protein

In photosynthetic light reactions, ATP and NADPH are produced by light-driven electron transfer from water to NADP⁺, mediated by proteins such as cytochrome b ₆f (cyt b ₆f) and photosystem I (PSI), which are thylakoid membrane-embedded protein complexes, and plastocyanin (Pc). Pc is a soluble electron carrier protein that shuttles between cyt b ₆f and PSI. The heme molecules in the cytochrome f (cyt f) subunits of cyt b ₆f donate electrons to the copper ions in Pc, and the chlorophyll molecules, P700, in the PsaA and PsaB subunits of PSI accept electrons from Pc. Pc rapidly cycles through the following steps: binding to cyt b ₆f, accepting an electron from cyt b ₆f, dissociating from cyt b ₆f, binding to PSI, donating an electron to the photo-excited PSI, and then dissociating from PSI. These electron transport reactions are important in plant growth (Chida et al. Reference Chida, Nakazawa, Akazaki, Hirano, Suruga, Ogawa, Satoh, Kadokura, Yamada, Hakamata, Isobe, Ito, Ishii, Nishio, Sonoike and Oku2007).

Pc forms separate electron transfer complexes with PSI and cyt b ₆f, and the distances between their reaction centers are sufficiently short to accomplish their electron transfer reactions in <10⁻⁴ s (Hope, Reference Hope2000). In addition, the Pc–PSI and Pc–cyt b ₆f electron transfer complexes rapidly associate and dissociate, at rates of ∼10⁸ M⁻¹ s⁻¹ and ∼10³ s⁻¹, respectively. The formation of such rapidly associating and dissociating electron transfer complexes facilitates efficient electron transport (Busch & Hippler, Reference Busch and Hippler2011).

Mutational analyses of Pc revealed that the residues in the hydrophobic patch that surrounds the copper ion, and those in the acidic patch located adjacent to the hydrophobic patch, are reportedly important for Pc–PSI and Pc–cyt b ₆f electron transport in eukaryotic plants (Fig. 18a) (Hope, Reference Hope2000). PSI and cyt b ₆f from eukaryotic plants contain several hydrophobic residues surrounding P700 and the heme in cyt f, respectively, and clusters of basic residues located adjacent to the hydrophobic residues. These residues in PSI and cyt b ₆f also participate in electron transport, based on mutational analyses (Busch & Hippler, Reference Busch and Hippler2011; Hope, Reference Hope2000). However, the roles of these residues in the rapid turnover of Pc remain unknown.

Fig. 18.

Determination of the interface residues of Pc for cyt b ₆f or PSI by TCS. (a) Distribution of the hydrophobic and acidic patches in spinach Pc (PDB code 1YLB). The surface of Pc is transparent, and the copper atom and the ribbon diagram are simultaneously displayed. The residues in the hydrophobic and acidic patches that were proposed to be responsible for the electron transport in previous mutational analyses are labeled, and colored green and red, respectively. (b,g) TCS experiments with excess amounts of Pc relative to (b,c) PSI and cyt b ₆f embedded in thylakoid vesicles, (d,e) solubilized PSI, or (f,g) solubilized b ₆f. Panels (b), (d), and (f) are schematic diagrams of the experiments. Panels (c), (e), and (g) are maps of the residues affected by the rf-irradiation in the TCS experiments. The residues with signal intensity reduction ratios >0.3 and within the 0.2–0.3 range are colored red and pink, respectively. Proline residues and residues with intensity reductions that were not determined, because of low signal intensity or spectral overlap, are white. The surface of Pc is transparent, and the copper atom and the ribbon diagram are simultaneously displayed. The molecular diagrams were generated with Web Lab Viewer Pro (Molecular Simulations, Inc.).

Therefore, we performed TCS experiments to identify the residues of Pc in close proximity to PSI and/or cyt b ₆f embedded in the thylakoid vesicles, in which the Pc-binding sites of PSI and cyt b ₆f are exposed to the solvent (Ueda et al. Reference Ueda, Nomoto, Koga, Ogasa, Ogawa, Matsumoto, Stampoulis, Sode, Terasawa and Shimada2012). Based on the spectra with and without irradiation, we calculated the reduction ratios of the peak intensities. The affected residues formed a continuous surface on the hydrophobic patch of Pc (Fig. 18b and c).

In order to observe the Pc–PSI and Pc–cyt b ₆f interactions separately, we also performed the TCS experiments using detergent-solubilized PSI and cyt b ₆f from spinach thylakoid membranes. These results showed that the affected residues formed a continuous surface on the hydrophobic patch of Pc (Fig. 18d–g).

These TCS experiments revealed that the acidic patch of Pc is not close to either PSI or cyt b ₆f in the electron transfer complexes. In the TCS experiments using vesicles, no detergents were employed, and therefore no decomposition of the subunits of PSI and/or cyt b ₆f caused by detergents occurred.

According to the classification proposed by Kumar and Nussinov, an ion pair is defined as a salt bridge if the side-chain charged group centroids and at least one pair of side-chain nitrogen and oxygen atoms are within 4 Å (Kumar & Nussinov, Reference Kumar and Nussinov2002). The definition of a stable salt bridge is based on the distance between the side-chain atoms, whereas the distances between the main chain amide protons of Pc and the protons of PSI or cyt b ₆f are reflected in the TCS experiments. However, our statistical analyses of the salt bridge structures revealed that most of the stable salt bridges could be identified by the TCS experiments, and thus the acidic patch residues of Pc do not form stable salt bridges with either PSI or cyt b ₆f in the electron transfer complexes.

The previously reported mutational analyses, cross-linking studies, and ion strength dependencies of the Pc–PSI and Pc–cyt b ₆f electron transport rates showed that the acidic patch residues of Pc and the basic residues of PSI and cyt b ₆f are responsible for the Pc–PSI and Pc–cyt b ₆f electron transport reactions (Busch & Hippler, Reference Busch and Hippler2011; Hope, Reference Hope2000). Therefore, the acidic patch of Pc may increase its local concentration around PSI and cyt b ₆f, thus facilitating the rapid formation of the electron transfer complex by occasionally forming dynamic salt bridges or long distance ion pairs (Fig. 19a–d). Most of these loose electrostatic interactions would not contribute to the complex stabilization or be observable in the present TCS experiments. The absence of stable salt bridges in the acidic patch is in agreement with the rapid dissociation of the complexes.

Fig. 19.

Schematic views of the interaction modes in the Pc–PSI and Pc–cyt b ₆f complexes.

6.5.1 Interaction of the ceramide transporter (CERT) PH domain with PI4P-embedded membrane

In mammalian cells, a de novo synthesized ceramide in ER membranes is transported to the Golgi in a non-vesicular manner by an ER-to-Golgi-specific CERT (Hanada et al. Reference Hanada, Kumagai, Yasuda, Miura, Kawano, Fukasawa and Nishijima2003). CERT is a cytoplasmic 68-kDa protein, and its N-terminal PH domain specifically recognizes PI4P in membranes. Since PI4P is the most abundant and preferentially distributed PI in trans-Golgi membranes, it is assumed that the PH domain is required for the efficient association of CERT with the Golgi membrane (Hanada et al. Reference Hanada, Kumagai, Yasuda, Miura, Kawano, Fukasawa and Nishijima2003; Kawano et al. Reference Kawano, Kumagai, Nishijima and Hanada2006). However, the detailed mechanism by which the CERT PH domain interacts with PI4P in the Golgi membrane, to exert the transportation activity, remain unclear. To clarify the structural basis of the PI4P interaction by the CERT PH domain in the Golgi membrane, we determined the structure of the CERT PH domain, and separately identified the interaction sites for the PI4P head group and the Golgi membrane in the CERT PH domain (Sugiki et al. Reference Sugiki, Takeuchi, Yamaji, Takano, Tokunaga, Kumagai, Hanada, Takahashi and Shimada2012).

The determined structure of the CERT PH domain (residues 23–117) revealed that the basic residues are clustered in the middle of the molecule, forming a basic groove around the protruding part of the β1/β2 region on the surface of the CERT PH domain (Fig. 20a and b). The site that interacts with the head group of PI4P on the CERT PH domain was mapped onto the tertiary structure of the CERT PH domain by chemical shift perturbation experiments, using a soluble form of PI4P (diC₄–PI4P). The mapping clearly defined a phosphatidylinositol phosphate-binding pocket in the CERT PH domain (Fig. 20b), which shares the common phosphatidylinositol phosphate-binding site with the other PH domains (Lemmon, Reference Lemmon2008). However, the affinity of the CERT PH domain for the soluble form of PI4P was quite weak (K _D: ∼0.76 mM), as compared to its affinity for the PI4P-embedded Golgi-mimetic liposomes (K _D: ∼3 μM). This indicated that the additional interactions between the CERT PH domain and the PI4P-embedded membrane are essential to maintain sufficient affinity for the membrane trafficking function of CERT.

Fig. 20.

Interaction of CERT PH domain with PI4P-embedded membrane. (a) Ribbon representation of the CERT PH domain, with magnification of the basic groove. The side chains of the basic and aromatic residues in the basic groove and the protruding part of the β1/β2 region are displayed as blue and green sticks, respectively. (b) Electrostatic potential surface of the CERT PH domain. Negative and positive electrostatic surfaces are colored red and blue, respectively. (c) Mapping of the residues that were significantly perturbed by the titration of diC₄–PI4P on the surface representation of the CERT PH domain structure. The side chains of the residues with Δδ>0.2 and 0.1–0.2 ppm chemical shift perturbation are indicated by red and orange surfaces, respectively. (d) Schematic representation of the TCS experiment using PI4P-embedded liposomes. The proton resonances originating from the lipid head group were saturated by radiofrequency irradiation, and this saturation was transferred to the residues in the CERT PH domain that were proximal to the PI4P-embedded liposome. (e) Mapping of the residues affected by the TCS on the surface of the CERT PH domain. The side chains of the residues with signal intensity reduction ratios >0.4 and 0.3–0.4 are indicated by red and orange surfaces, respectively. (f) Proposed mechanism of PH domain-mediated CERT translocation to the Golgi apparatus. I, Unbound state of the CERT PH domain. II, Weakly bound CERT PH domain. A non-specific electrostatic interaction occurs between the basic groove in the CERT PH domain and the surface of the phospholipid membrane. III, Orientation of the CERT PH domain for high affinity interaction. The specific high affinity interaction with the PI4P-embedded membrane is shown.

Therefore, we performed TCS experiments with CERT PH domain and the PI4P embedded Golgi-mimetic liposomes, to identify the additional membrane-interaction site in the CERT PH domain (Fig. 20c). The mapping of the residues affected by the rf-irradiation indicated that the membrane interaction surface of the CERT PH domain extends through the entire region of the basic groove, including the PI4P-binding site (Fig. 20d). Based on these observations, we proposed a model that describes the PH domain-mediated Golgi association of the CERT protein (Fig. 20e). The initial membrane association of the CERT PH domain might be driven by a non-specific electrostatic interaction between the basic groove and the phospholipid membrane. The interaction using the basic groove would optimally orient the protruding membrane insertion β1/β2 loop of the CERT PH domain toward the bilayer normal, which would be suitable for the efficient recognition of PI4P in the membrane. In addition, the protruding membrane insertion β1/β2 loop contains multiple aromatic/hydrophobic residues (Fig. 20a), and thus, it would be optimally located deeper in the membrane. Unlike other PH domains that show greater contributions of specific PI recognition in the binding, the specific PI4P-binding and non-specific phospholipid membrane interactions contribute almost equally to the binding of the CERT PH domain to PI4P-embedded liposomes. Indeed, immunofluorescence microscopy experiments using HeLa cells verified the Golgi mislocalization of the CERT with a mutation in the PI4P-binding pocket or the non-specific basic groove. Thus, the multistep interaction model revealed by the TCS methods is crucial for the membrane trafficking activity of CERT, and clearly confirms the methodological advantage of this NMR method.

6.5.2 Atypical membrane-embedded PI(3,4)P₂-binding site on p47^phox PX domain

An interaction analysis of the PI-embedded lipid-bilayer membrane by TCS also revealed the unique PI-binding site in the p47^phox PX domain (Stampoulis et al. Reference Stampoulis, Ueda, Matsumoto, Terasawa, Miyano, Sumimoto and Shimada2012). Both p47^phox and p40^phox are pivotal components of the phagocyte NADPH oxidase, which is a multicomponent enzyme that produces reactive oxygen species (ROS) as a defense against invading microorganisms. p47^phox and p40^phox contain PX domains, which are crucial for the recruitment of cytosolic components and the activation of NADPH oxidase (Ago et al. Reference Ago, Takeya, Hiroaki, Kuribayashi, Ito, Kohda and Sumimoto2001, Reference Ago, Kuribayashi, Hiroaki, Takeya, Ito, Kohda and Sumimoto2003). While the p40^phox PX domain specifically binds to PI3P, the p47^phox PX domain reportedly binds to PI(3,4)P₂, PA, and PtdSer (Sato et al. Reference Sato, Overduin and Emr2001).

The crystal structure of p40^phox in complex with diC₄–PI3P has been solved (Bravo et al. Reference Bravo, Karathanassis, Pacold, Pacold, Ellson, Anderson, Butler, Lavenir, Perisic, Hawkins, Stephens and Williams2001) (Fig. 21a), and the 3D structures of p47^phox PX alone have been determined by solution NMR (Hiroaki et al. Reference Hiroaki, Ago, Ito, Sumimoto and Kohda2001) and X-ray crystallography (Karathanassis et al. Reference Karathanassis, Stahelin, Bravo, Perisic, Pacold, Cho and Williams2002). However, the structure of p47^phox PX in complex with its ligand has not been reported. The crystal structure of its free form revealed two sulfate molecules bound in two distinct sites (Karathanassis et al. Reference Karathanassis, Stahelin, Bravo, Perisic, Pacold, Cho and Williams2002) (Fig. 21b). One of the sulfates is at the C-terminal end of the β3 strand (Fig. 21b), which coincides with the three-phosphate of the bound PI3P in the crystal structure of p40^phox PX (Fig. 21a). The second sulfate is on the surface composed of the α1 helix and the adjacent loop that leads to the distorted left-handed PP_II helix. Based on these results and SPR-based mutational analyses, the first sulfate-binding site was proposed to be the PI(3,4)P₂-binding site, and the second sulfate-binding site was considered to be the PA-binding site. However, there are no direct structural data that have specified the PI(3,4)P₂-binding site as well as the PA and PtdSer-binding sites in p47^phox PX. Thus, it was unclear whether these sulfate-binding sites of p47^phox PX are the actual ligand-binding sites, and the structural basis of the broad ligand specificity of p47^phox PX remained enigmatic.

Fig. 21.

Interaction of p47^phox PX domain with PI(3,4)P₂-embedded membrane. (a) Ribbon representation of the p47^phox PX domain. The bound diC₄–PI3P₂ is displayed in a ball-and-stick representation. (b) Left: Ribbon representation of the p47^phox PX domain, with mapping of residues displaying strong intensity reductions in the TCS experiment with PI(3,4)P₂-containing liposomes. Right: Mapping of residues displaying strong intensity reductions in the TCS experiment with PI(3,4)P₂-containing liposomes on the surface of the crystal structure of p47^phox PX. Residues are color-coded according to their intensity reduction (IR), with red for strong (IR>0.3) and orange for moderate (0.25<IR<0.3) reduction. The bound sulfates are shown in a ball-and-stick representation. (c) Schematic representation of the TCS experiment using PI(3,4)P₂-embedded liposomes. The proton resonances originating from the lipid head group were saturated by radiofrequency irradiation, and this saturation was transferred to the residues in the p47^phox PX domain that were proximal to the PI(3,4)P₂-embedded liposome. (d) Mapping of residues displaying strong diC₄–PI(3,4)P₂-induced chemical shift perturbations onto a ribbon representation of the crystal structure of the p47^phox PX domain. Normalized chemical shift changes were calculated based on the equation Δδ=((ΔδH)²+(ΔδN/5)²)^1/2. Residues that showed strong (Δδ>0.2 ppm) and moderate (0.12 ppm<Δδ<0.2 ppm) normalized chemical shift changes are colored red and orange, respectively.

We applied the TCS experiments to identify the interacting interface of the p47^phox PX domain to PI(3,4)P₂-embedded liposomes (Stampoulis et al. Reference Stampoulis, Ueda, Matsumoto, Terasawa, Miyano, Sumimoto and Shimada2012) (Fig. 21c). The residues affected by the rf-irradiation coexisted in a well-defined area on the surface of the p47^phox PX domain (Fig. 21b). This area is outlined by helix α1, helix α1’, and the following loop that leads to the distorted, left-handed PP_II helix, which clearly indicated the interacting interface of the p47^phox PX domain with the PI(3,4)P₂-embadded membrane near the second sulfate-binding site. In vitro and in vivo mutational analyses corroborated these results. The interface determined for PI differs from those of all of the PX domains elucidated so far, including the p40^phox PX domain.

It is notable that the chemical shift perturbation area on p47^phox PX, caused by the addition of diC₄-PI(3,4)P₂, is larger than the PI-binding site determined by the TCS experiments, and includes residues in the site that corresponds to the PI3P-binding site of p40^phox PX (Fig. 21d). Considering that chemical shift perturbation is induced by both the direct binding effects and conformational changes (Takeda et al. Reference Takeda, Terasawa, Sakakura, Yamaguchi, Kajiwara, Kawashima, Miyasaka and Shimada2003), the perturbation of the residues in the site that corresponds to the PI3P-binding site of p40^phox PX would be a secondary effect, due to a conformational change. Thus, these results confirmed the significant advantage of the TCS experiments, which led to the correct identification of the p47^phox PX domain interaction with membrane-embedded PI(3,4)P₂.

6.5.3 Remarks for structural analyses of membrane-associating proteins by TCS

We have used liposomes, which provide the physiological characteristic of a cellular membrane, for our NMR studies. However, liposomes are not always amenable for conventional solution NMR studies because they can be quite unstable and precipitate, depending on the lipid composition. In this regard, Yokogawa et al. (Reference Yokogawa, Kobashigawa, Yoshida, Ogura, Harada and Inagaki2012), utilized rHDLs, which have been proved to be useful for the stable reconstitution of membrane proteins by various groups (Gluck et al. Reference Gluck, Wittlich, Feuerstein, Hoffmann, Willbold and Koenig2009; Hagn et al. Reference Hagn, Etzkorn, Raschle and Wagner2013; Raschle et al. Reference Raschle, Hiller, Yu, Rice, Walz and Wagner2009; Ritchie et al. Reference Ritchie, Grinkova, Bayburt, Denisov, Zolnerciks, Atkins and Sligar2009; Shenkarev et al. Reference Shenkarev, Lyukmanova, Paramonov, Shingarova, Chupin, Kirpichnikov, Blommers and Arseniev2010; Yoshiura et al. Reference Yoshiura, Kofuku, Ueda, Yokogawa, Osawa, Terashima, Matsushima and Shimada2010), for their NMR study of the interaction between the FYVE domain of early endosome antigen 1 (EEA1), denoted as EEA1 FYVE domain, and PI3P-embedded in rHDL (Fig. 22a). As discussed above, rHDLs are composed of a lipid bilayer in which the hydrophobic edge is sealed by a pair of engineered human apolipoprotein A-I (apoA-I). Due to their high solubility in water, variability in lipid composition, and lipid bilayer characteristics, rHDLs are also suited to structural studies of membrane–protein interactions in water.

Fig. 22.

Interaction of EEA1 FYVE domain with PI3P-embedded rHDL. (a) Schematic representation of the TCS experiment using PI3P-embedded rHDL. The proton resonances originating from the lipids were saturated by radiofrequency irradiation, and the saturation was transferred to the residues in the EEA1 FYVE domain that were proximal to the PI3P-embedded rHDL. (b) Mapping of the residues affected by the TCS on the surface of the EEA1 FYVE domain. Residues with differences in the reduction ratio (ΔRR) values larger than 0.10, and within the 0.06–0.10 range are indicated by red and orange surfaces, respectively. The ΔRR value was calculated by the following equation: ΔRR=RR_PI3P–rHDL−RR_{PI-free–rHDL}, where RR_PI3P–rHDL and RR_{PI-free–rHDL} were obtained from (I _unsat−I _sat)/I _unsat in the presence of PI3P–rHDL and PI-free rHDL. (c) Model for the complex of EEA1 FYVE domain bound to PI3P-embedded in the membrane. The residues on the EEA1 FYVE domain affected in both the TCS and PRE (green), only in TCS (blue), and only in PRE (yellow) experiments are mapped on the surface of EEA1 FYVE domain. The Ins(1,3)P₂ bound to EEA1 FYVE is depicted by a stick model, and the residues that were not analyzed in either TCS or PRE experiments are colored black. Lipid molecules within the membrane are schematically shown, with the phosphate and choline head groups depicted as orange and blue spheres, respectively.

In the TCS experiment using the PI3P-rHDLs, the residues affected by the rf-irradiation were mostly located on a specific side of EEA1 FYVE domain (Fig. 22b). Based on the results of TCS analyses, the model of EEA1 FYVE bound to a PI3P-embedded membrane was constructed (Fig. 22c). The authors also used PI3P-rHDLs containing 1,2-dipalmitoyl-sn-glycero-3-phopho(tempo)choline (TEMPO-PC) for a PRE study. The results from the TCS and PRE experiments are largely consistent with each other, and thus PRE can be used as an orthogonal method to confirm the TCS results. The PRE results would be sensitive to the position of TEMPO introduced with the membrane, and it should be carefully noted that TEMPO-PC can unevenly distributed in the rHDLs, due to its rather bulky head group. Nevertheless, it is evident that rHDL can be used as an alternative membrane system for analyses of membrane-protein interactions by the TCS method.

The different patterns of PI recognition between p47^phox PX and p40^phox PX, despite their similar overall structures, underscore the feat that substitutions of the key residues on the surface of the PI-binding domain can significantly alter its function. It was also shown that a few mutations can completely change the specificity and binding modes of the p47^phox PX domain (Stampoulis et al. Reference Stampoulis, Ueda, Matsumoto, Terasawa, Miyano, Sumimoto and Shimada2012), and thus the direct determination of the binding interfaces by structural biological methods is crucial for understanding the function of PI-binding proteins. In addition, the functional interactions of the PI-binding domains are not solely dependent on specific PI-binding. Non-specific phospholipid membrane interactions were equally important for functional PI-binding by the CERT PH domain (Sugiki et al. Reference Sugiki, Takeuchi, Yamaji, Takano, Tokunaga, Kumagai, Hanada, Takahashi and Shimada2012). This might be true for other membrane-associating proteins as well. Currently, NMR techniques are the only structural method to access these interactions at the membrane interface with atomic-resolution. For these purposes, TCS would be the method of choice, due to its accuracy in determining the interfaces of inhomogeneous systems.