Ichio Shimada

A novel NMR method for determining the interfaces of large protein-protein complexes

Identification of the interfaces of large (Mr > 50,000) protein–protein complexes in solution by high resolution NMR has typically been achieved using experiments involving chemical shift perturbation and/or hydrogen-deuterium exchange of the main chain amide groups of the proteins. Interfaces identified using these techniques, however, are not always identical to those revealed using X-ray crystallography. In order to identify the contact residues in a large protein–protein complex more accurately, we developed a novel NMR method that uses cross-saturation phenomena in combination with TROSY detection in an optimally deuterium labeled system.

Structural Basis of the Interaction between Chemokine Stromal Cell-derived Factor-1/CXCL12 and Its G-protein-coupled Receptor CXCR4

The chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) and its G-protein-coupled receptor (GPCR) CXCR4 play fundamental roles in many physiological processes, and CXCR4 is a drug target for various diseases such as cancer metastasis and human immunodeficiency virus, type 1, infection. However, almost no structural information about the SDF-1-CXCR4 interaction is available, mainly because of the difficulties in expression, purification, and crystallization of CXCR4. In this study, an extensive investigation of the preparation of CXCR4 and optimization of the experimental conditions enables NMR analyses of the interaction between the full-length CXCR4 and SDF-1. We demonstrated that the binding of an extended surface on the SDF-1 β-sheet, 50-s loop, and N-loop to the CXCR4 extracellular region and that of the SDF-1 N terminus to the CXCR4 transmembrane region, which is critical for G-protein signaling, take place independently by methyl-utilizing transferred cross-saturation experiments along with the usage of the CXCR4-selective antagonist AMD3100. Furthermore, based upon the data, we conclude that the highly dynamic SDF-1 N terminus in the 1st step bound state plays a crucial role in efficiently searching the deeply buried binding pocket in the CXCR4 transmembrane region by the “fly-casting” mechanism. This is the first structural analyses of the interaction between a full-length GPCR and its chemokine, and our methodology would be applicable to other GPCR-ligand systems, for which the structural studies are still challenging.

1H nuclear magnetic resonance study of the solution conformation of an antibacterial protein, sapecin.

The solution conformation of an antibacterial protein sapecin has been determined by 1H nuclear magnetic resonance (NMR) and dynamical simulated annealing calculations. It has been shown that the polypeptide fold consists of one flexible loop (residues 4–12), one helix (residues 15–23), and two extended strands (residues 24–31 and 34–40). It was found that the tertiary structure of sapecin is completely different from that of rabbit neutrophil defensin NP‐5, which is homologous to sapecin in the amino acid sequences and also has the antibacterial activity. The three‐dimensional structure determination has revealed that a basic‐residue rich region and the hydrophobic surface face each other on the surface of sapecin.

Determination of the interface of a large protein complex by transferred cross-saturation measurements.

In an earlier paper, it was shown that the cross-saturation method enables us to identify the contact residues of large protein complexes in a more rigorous manner than is possible using chemical shift perturbation and hydrogen-deuterium exchange experiments. However, there are limitations within the determination of the contact residues by the cross-saturation method, in that the method is difficult to apply to protein complexes with a molecular mass over 150 kDa and/or with weak binding, since the resonances originating from the complexes should be observed directly in the method. In the present work, to overcome these limitations, we carried out the cross-saturation measurements under conditions of a fast exchange between free and bound states on the NMR time-scale, and determined the contact residues of the complex of the B domain of protein A and intact IgG, which has a molecular mass of 164 kDa and shows weak binding.

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.

Efficacy of the β₂-adrenergic receptor is determined by conformational equilibrium in the transmembrane region.

Many drugs that target G-protein-coupled receptors (GPCRs) induce or inhibit their signal transduction with different strengths, which affect their therapeutic properties. However, the mechanism underlying the differences in the signalling levels is still not clear, although several structures of GPCRs complexed with ligands determined by X-ray crystallography are available. Here we utilized NMR to monitor the signals from the methionine residue at position 82 in neutral antagonist- and partial agonist-bound states of β2-adrenergic receptor (β2AR), which are correlated with the conformational changes of the transmembrane regions upon activation. We show that this residue exists in a conformational equilibrium between the inverse agonist-bound states and the full agonist-bound state, and the population of the latter reflects the signal transduction level in each ligand-bound state. These findings provide insights into the multi-level signalling of β2AR and other GPCRs, including the basal activity, and the mechanism of signal transduction mediated by GPCRs.

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.

Pairing of oligosaccharides in the Fc region of immunoglobulin G

The Fc portion of immunoglobulin G (IgG) expresses paired oligosaccharides with microheterogeneities, which are associated with efficiencies of effector functions and with pathological states. A comparison of electrospray ionization mass spectrometry data obtained using a variety of Fc fragments derived from human and mouse IgG that do and do not retain the inter‐chain disulfide bridge(s) revealed that (1) the Fc portion can be asymmetric as well as symmetric with respect to glycosylation and (2) the ratios of the individual glycoforms are different from what is expected from the random pairing.

Identification and Characterization of the Slowly Exchanging pH-dependent Conformational Rearrangement in KcsA

Gating of ion channels is strictly regulated by physiological conditions as well as intra/extracellular ligands. To understand the underlying structures mediating ion channel gating, we investigated the pH-dependent gating of the K+ channel KcsA under near-physiological conditions, using solution-state NMR. In a series of 1H15N-TROSY HSQC (transverse relaxation optimized spectroscopy-heteronuclear single quantum coherence) spectra measured at various pH values, significant chemical shift changes were detected between pH 3.9 and 5.2, reflecting a conformational rearrangement associated with the gating. The pH-dependent chemical shift changes were mainly observed for the resonances from the residues near the intracellular helix bundle, which has been considered to form the primary gate in the K+ channel, as well as the intracellular extension of the inner helix. The substitution of His-25 by Ala abolished this pH-dependent conformational rearrangement, indicating that the residue serves as a “pH-sensor” for the channel. Although the electrophysiological open probability of KcsA is less than 10%, the conformations of the intracellular helix bundle between the acidic and neutral conditions seem to be remarkably different. This supports the recently proposed “dual gating” properties of the K+ channel, in which the activation-coupled inactivation at the selectivity filter determines the channel open probability of the channel. Indeed, a pH-dependent chemical shift change was also observed for the signal from the Trp-67 indole, which is involved in a hydrogen bond network related to the activation-coupled inactivation. The slow kinetic parameter obtained for the intracellular bundle seems to fit better into the time scale for burst duration than very fast fluctuations within a burst period, indicating the existence of another gating element with faster kinetic properties.

Observation of NMR Signals from Proteins Introduced into Living Mammalian Cells by Reversible Membrane Permeabilization Using a Pore-Forming Toxin, Streptolysin O

We have developed a new in-cell NMR method that is applicable to any type of cell and does not require target protein modification or specialized equipment. The stable-isotope-labeled target protein, thymosin beta4 (Tbeta4), was delivered to 293F cells, which were permeabilized by a pore-forming toxin, streptolysin O, and resealed by Ca(2+) after Tbeta4 uptake. As a result, we successfully observed (1)H-(15)N HSQC signals originating from the Tbeta4, including those from the N-terminal acetylation, which had occurred inside the cell as a post-translational modification.