Masayoshi Sakakura

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.

Ligand-induced structural changes of the CD44 hyaluronan-binding domain revealed by NMR.

CD44, a major cell surface receptor for hyaluronan (HA), contains a functional domain responsible for HA binding at its N terminus (residues 21-178). Accumulating evidence indicates that proteolytic cleavage of CD44 in its extracellular region (residues 21-268) leads to enhanced tumor cell migration and invasion. Hence, understanding the mechanisms underlying the CD44 proteolytic cleavage is important for understanding the mechanism of CD44-mediated tumor progression. Here we present the NMR structure of the HA-binding domain of CD44 in its HA-bound state. The structure is composed of the Link module (residues 32-124) and an extended lobe (residues 21-31 and 125-152). Interestingly, a comparison of its unbound and HA-bound structures revealed that rearrangement of the β-strands in the extended lobe (residues 143-148) and disorder of the structure in the following C-terminal region (residues 153-169) occurred upon HA binding, which is consistent with the results of trypsin proteolysis studies of the CD44 HA-binding domain. The order-to-disorder transition of the C-terminal region by HA binding may be involved in the CD44-mediated cell migration.

Hyaluronan Recognition Mode of CD44 Revealed by Cross-saturation and Chemical Shift Perturbation Experiments

CD44 is the main cell surface receptor for hyaluronic acid (HA) and contains a functional HA-binding domain (HABD) composed of a Link module with N- and C-terminal extensions. The contact residues of human CD44 HABD for HA have been determined by cross-saturation experiments and mapped on the topology of CD44 HABD, which we elucidated by NMR. The contact residues are distributed in both the consensus fold for the Link module superfamily and the additional structural elements consisting of the flanking regions. Interestingly, the contact residues exhibit small changes in chemical shift upon HA binding. In contrast, the residues with large chemical shift changes are localized in the C-terminal extension and the first α-helix and are generally inconsistent with the contact residues. These results suggest that, upon ligand binding, the C-terminal extension and the first α-helix undergo significant conformational changes, which may account for the broad ligand specificity of CD44 HABD.

Structural Investigation of the Interaction between LolA and LolB Using NMR

Lipoproteins that play critical roles in various cellular functions of Gram-negative bacteria are localized in the cells inner and outer membranes. Lol proteins (LolA, LolB, LolC, LolD, and LolE) are involved in the transportation of outer membrane-directed lipoproteins from the inner to the outer membrane. LolA is a periplasmic chaperone that transports lipoproteins, and LolB is an outer membrane receptor that accepts lipoproteins. To clarify the structural basis for the lipoprotein transfer from LolA to LolB, we examined the interaction between LolA and mLolB, a soluble mutant of LolB, using solution NMR spectroscopy. We determined the interaction mode between LolA and mLolB with conformational changes of LolA. Based upon the observations, we propose that the LolA·LolB complex forms a tunnel-like structure, where the hydrophobic insides of LolA and LolB are connected, which enables lipoproteins to transfer from LolA to LolB.

Carbohydrate Binding Mechanism of the Macrophage Galactose-type C-type Lectin 1 Revealed by Saturation Transfer Experiments

Macrophage galactose-type C-type lectins 1 and 2 (MGL1/2) are expressed on the surfaces of macrophages and immature dendritic cells. Despite the high similarity between the primary sequences of MGL1 and MGL2, they display different ligand specificities. MGL1 shows high affinity for the LewisX trisaccharide, whereas MGL2 shows affinity for N-acetylgalactosamine. To elucidate the structural basis for the ligand specificities of the MGLs, we performed NMR analyses of the MGL1-LewisX complex. To identify the LewisX binding site on MGL1, a saturation transfer experiment for the MGL1-LewisX complex where sugar-CH/CH2-selective saturation was applied was carried out. To obtain sugar moiety-specific information on the interface between MGL1 and the LewisX trisaccharide, saturation transfer experiments where each of galactose-H5-, fucose-CH3-, and N-acetylglucosamine-CH3-selective saturations was applied to the MGL1-LewisX complex were performed. Based on these results, we present a LewisX binding mode on MGL1 where the galactose moiety is bound to the primary sugar binding site, including Asp-94, Trp-96, and Asp-118, and the fucose moiety interacts with the secondary sugar binding site, including Ala-89 and Thr-111. Ala-89 and Thr-111 in MGL1 are replaced with arginine and serine in MGL2, respectively. The hydrophobic environment formed by a small side chain of Ala-89 and a methyl group of Thr-111 is a requisite for the accommodation of the fucose moiety of the LewisX trisaccharide within the sugar binding site of MGL1.

An NMR method for the determination of protein binding interfaces using TEMPOL-induced chemical shift perturbations

BACKGROUND The determination of protein-protein interfaces is of crucial importance to understand protein function and to guide the design of compounds. To identify protein-protein interface by NMR spectroscopy, 13C NMR paramagnetic shifts induced by freely diffusing 4-hydroxy-2, 2, 6, 6-tetramethyl-piperidine-1-oxyl (TEMPOL) are promising, because TEMPOL affects distinct 13C NMR chemical shifts of the solvent accessible nuclei belonging to proteins of interest, while 13C nuclei within the interior of the proteins may be distinguished by a lack of such shifts. METHOD We measured the 13C NMR paramagnetic shifts induced by TEMPOL by recording 13C-(13)C TOCSY spectra for ubiquitin in the free state and the complex state with yeast ubiquitin hydrolase1 (YUH1). RESULTS Upon complexation of ubiquitin with YUH1, 13C NMR paramagnetic shifts associated with the protein binding interface were reduced by 0.05 ppm or more. The identified interfacial atoms agreed with the prior X-ray crystallographic data. CONCLUSIONS The TEMPOL-induced 13C chemical shift perturbation is useful to determine precise protein-protein interfaces. GENERAL SIGNIFICANCE The present method is a useful method to determine protein-protein interface by NMR, because it has advantages in easy sample preparations, simple data analyses, and wide applicabilities.

Backbone resonance assignment for the outer membrane lipoprotein receptor LolB from Escherichia coli

LolB, which is anchored to the outer membrane of Gram-negative bacteria, receives outer-membrane-directed lipoproteins from LolA, and incorporates them into the outer membrane. We established backbone resonance assignments of 2H/13C/15N labeled LolB from Escherichia coli.

Backbone resonance assignments for the periplasmic chaperone LolA from Escherichia coli.

LolA is an essential periplasmic protein in Gram-negative bacteria and plays a role in transporting lipoproteins through periplasmic space from the inner to the outer membrane. We established backbone resonance assignments of 2H/13C/15N labeled LolA from Escherichia coli.