Norio Matsushima

Crystal Structure of the TLR4-MD-2 Complex with Bound Endotoxin Antagonist Eritoran

TLR4 and MD-2 form a heterodimer that recognizes LPS (lipopolysaccharide) from Gram-negative bacteria. Eritoran is an analog of LPS that antagonizes its activity by binding to the TLR4-MD-2 complex. We determined the structure of the full-length ectodomain of the mouse TLR4 and MD-2 complex. We also produced a series of hybrids of human TLR4 and hagfish VLR and determined their structures with and without bound MD-2 and Eritoran. TLR4 is an atypical member of the LRR family and is composed of N-terminal, central, and C-terminal domains. The beta sheet of the central domain shows unusually small radii and large twist angles. MD-2 binds to the concave surface of the N-terminal and central domains. The interaction with Eritoran is mediated by a hydrophobic internal pocket in MD-2. Based on structural analysis and mutagenesis experiments on MD-2 and TLR4, we propose a model of TLR4-MD-2 dimerization induced by LPS.

Structural principles of leucine-rich repeat (LRR) proteins

LRR‐containing proteins are present in over 2000 proteins from viruses to eukaryotes. Most LRRs are 20–30 amino acids long, and the repeat number ranges from 2 to 42. The known structures of 14 LRR proteins, each containing 4–17 repeats, have revealed that the LRR domains fold into a horseshoe (or arc) shape with a parallel β‐sheet on the concave face and with various secondary structures, including α‐helix, 310‐helix, and pII helix on the convex face. We developed simple methods to charactere quantitatively the arc shape of LRR and then applied them to all known LRR proteins. A quantity of 2Rsin(φ/2), in which R and φ are the radii of the LRR arc and the rotation angle about the central axis per repeating unit, respectively, is highly conserved in all the LRR proteins regardless of a large variety of repeat number and the radius of the LRR arc. The radii of the LRR arc with β‐α structural units are smaller than those with β‐310 or β‐pII units. The concave face of the LRR β‐sheet forms a surface analogous to a part of a Möbius strip. Proteins 2004;54:000–000.

Orientation of bone mineral and its role in the anisotropic mechanical properties of bone—Transverse anisotropy

An orientation of hydroxyapatite (HAP) crystals in bovine femur mineral was investigated by means of X-ray pole figure analysis (XPFA). It was found that the c-axis of HAP generally orients parallel to the longitudinal axis of bone (bone axis) and a significant amount of c-axis was oriented in other directions, in particular, perpendicular to the bone axis. Comparing these results with those of the small angle X-ray scattering (SAXS) investigation by Matsushima et al. (Jap. J. appl. Phys. 21, 186-189, 1982) at least two types of morphology of bone mineral were found; rod like bone mineral having the c-axis of HAP crystal parallel to the longitudinal axis of the rod and that having the c-axis not parallel, in a particular case, perpendicular to its longitudinal axis. Transverse anisotropy in mechanical properties of bone was reproduced by the estimation of Youngs moduli by using the structural results mainly from XPFA. It is concluded that the anisotropy in mechanical properties of bone is well explained by taking account of the non-longitudinal (off-bone) axial distribution of orientation of bone mineral.

Time-resolved X-ray diffraction from tendon collagen during creep using synchrotron radiation

In order to understand the molecular mechanism of relaxation phenomena in collagenous tissue, time-resolved, small-angle X-ray diffraction measurements were performed on bovine Achilles tendon collagen under creep. A tension-induced increase in the 67 nm period (D-period) was observed, and the strain in the D-period, epsilon D, was found to be almost proportional to the external force per unit cross-sectional area (average stress) of the specimen. With an increase in epsilon D, a change in the ratio of intensities of the third-order reflection peak of the D-period to that of the second-order peak was also observed. The increase in epsilon D was decomposed into three elementary processes of D-period deformation, which are presented on the basis of the Hodge-Petruska model: (1) molecular elongation, (2) increase in gap region, and (3) relative slippage of lateral adjoining molecules. Up to 8 MPa of average stress, the contribution to epsilon D originated mostly from only mode (1). At more than 10 MPa of average stress, modes (2) and (3) also contributed to fibril elongation. For epsilon D by molecular elongation (mode (1)), the time dependence of the D-period change in the immediate response region is a sharply shaped step function, while the contribution to epsilon D by molecular rearranging modes gives a slight creep nature at the immediate response region in the time dependence of epsilon D. Because this creep nature is observed at the immediate response, it is related qualitatively to the KWW function in a stress-relaxation modulus of collagenous tissue observed in an immediate response region (Sasaki et al. (1993). Journal of Biomechanics 26, 1369-1376). The elementary process of KWW-type relaxation is concluded to be related to the tension-induced molecular rearrangement within a D-period.

Super-motifs and evolution of tandem leucine-rich repeats within the small proteoglycans--biglycan, decorin, lumican, fibromodulin, PRELP, keratocan, osteoadherin, epiphycan, and osteoglycin.

Leucine‐rich repeats (LRRs) with 20–30 amino acids in unit length are present in many proteins from prokaryotes to eukaryotes. The LRR‐containing proteins include a family of nine small proteoglycans, forming three distinct subfamilies: class I contains biglycan/PG‐I and decorin/PG‐II; class II: lumican, fibromodulin, PRELP, keratocan, and osteoadherin; and class III: epiphycan/PG‐Lb and osteoglycin or osteoinductive factor. Comparative sequence analysis of the 34 available protein sequences reveals that these proteoglycans have two types of LRRs, which we call S and T. The type S LRR is 21 residues long and has the consensus sequence of xxaPzxLPxxLxxLxLxxNxI. The type T LRR has 26 residues; its consensus sequence is zzxxaxxxxFxxaxxLxxLxLxxNxL. In both “x” indicates variable residue; “z” is frequently a gap; “a” is Val, Leu, or Ile; and I is Ile or Leu. These type S and T LRRs are ordered into two super‐motifs—STT with about 73 residues in classes I and II and ST with about 47 residues in class III. The 12 LRRs in the small proteoglycans of I and II are best represented as (STT)4; the seven LRRs of class III as (ST)T(ST)2. Our analyses indicate that classes I/II and III evolved along different paths after the establishment of the precursor ST, and classes I and II also diverged after the establishment of the precursor (STT)4. Proteins 2000;38:210–225.

Structural analysis of leucine-rich-repeat variants in proteins associated with human diseases

Abstract.A number of human diseases have been shown to be associated with mutation in the genes encoding leucine-rich-repeat (LRR)-containing proteins. They include 16 different LRR proteins. Mutations of these proteins are associated with 19 human diseases. The mutations occur frequently within the LRR domains as well as their neighboring domains, including cysteine clusters. Here, based on the sequence analysis of the LRR domains and the known structure of LRR proteins, we describe some features of different sequence variants and discuss their adverse effects. The mutations in the cysteine clusters, which preclude the formation of sulfide bridges or lead to a wrong paring of cysteines in extracellular proteins or extracellular domains, occur with high frequency. In contrast, missense mutations at some specific positions in LRRs are very rare or are not observed at all.

Molecular evolution of vertebrate Toll-like receptors: Evolutionary rate difference between their leucine-rich repeats and their TIR domains

Toll-like receptors (TLRs) that initiate an innate immune response contain an extracellular leucine rich repeat (LRR) domain and an intracellular Toll IL-receptor (TIR) domain. There are fifteen different TLRs in vertebrates. The LRR domains, which adopt a solenoid structure, usually have higher rates of evolution than do the TIR globular domains. It is important to understand the molecular evolution and functional roles of TLRs from this standpoint. Both pairwise genetic distances and Ka/Kss (the ratios between non synonymous and synonymous substitution rates) were compared between the LRR domain and the TIR domain of 366 vertebrate TLRs from 96 species (from fish to primates). In fourteen members (TLRs 1, 2, 3, 4, 5, 6, 7, 8, 9, 11/12, 13, 14, 21, and 22/23) the LRR domains evolved significantly more rapidly than did the corresponding TIR domains. The evolutionary rates of the LRR domains are significantly different among these members; LRR domains from TLR3 and TLR7 from primates to fishes have the lowest rate of evolution. In contrast, the fifteenth member, TLR10, shows no significant differences; its TIR domain is not highly conserved. The present results suggest that TLR10 may have a different function in signaling from those other members and that a higher conservation of TLR3 and TLR7 may reflect a more ancient mechanism and/or structure in the innate immune response system. Gene conversions are suggested to have occurred in platypus TLR6 and TLR10. This study provides new insight about structural and functional diversification of vertebrate TLRs.

Nef of HIV‐1 interacts directly with calcium‐bound calmodulin

It was recently found that the myristoyl group of CAP‐23/NAP‐22, a neuron‐specific protein kinase C substrate, is essential for the interaction between the protein and Ca2+‐bound calmodulin (Ca2+/CaM). Based on the N‐terminal amino acid sequence alignment of CAP‐23/NAP‐22 and other myristoylated proteins, including the Nef protein from human immunodeficiency virus (HIV), we proposed a new hypothesis that the protein myristoylation plays important roles in protein–calmodulin interactions. To investigate the possibility of direct interaction between Nef and calmodulin, we performed structural studies of Ca2+/CaM in the presence of a myristoylated peptide corresponding to the N‐terminal region of Nef. The dissociation constant between Ca2+/CaM and the myristoylated Nef peptide was determined to be 13.7 nM by fluorescence spectroscopy analyses. The NMR experiments indicated that the chemical shifts of some residues on and around the hydrophobic clefts of Ca2+/CaM changed markedly in the Ca2+/CaM‐Nef peptide complex with the molar ratio of 1:2. Correspondingly, the radius of gyration determined by the small angle X‐ray scattering measurements is 2–3 Å smaller that of Ca2+/CaM alone. These results demonstrate clearly that Nef interacts directly with Ca2+/CaM.

310‐helices in proteins are parahelices

The 310‐helix is characterized by having at least two consecutive hydrogen bonds between the main‐chain carbonyl oxygen of residue i and the main‐chain amide hydrogen of residue i + 3. The helical parameters — pitch, residues per turn, radius, and root mean square deviation (rmsd) from the best‐fit helix — were determined by using the HELFIT program. All 310‐helices were classified as regular or irregular based on rmsd/(N − 1)1/2 where N is the helix length. For both there are systematic, position‐specific shifts in the backbone dihedral angles. The average ϕ, ψ shift systematically from ∼ −58°, ∼ −32° to ∼ −90°, ∼ −4° for helices 5, 6, and 7 residues long. The same general pattern is seen for helices, N = 8 and 9; however, in N = 9, the trend is repeated with residues 6, 7, and 8 approximately repeating the ϕ, ψ of residues 2, 3, and 4. The residues per turn and radius of regular 310‐helices decrease with increasing length of helix, while the helix pitch and rise per residue increase. That is, regular 310‐helices become thinner and longer as N increases from 5 to 8. The fraction of regular 310‐helices decreases linearly with helix length. All longer helices, N ≥ 9 are irregular. Energy minimizations show that regular helices become less stable with increasing helix length. These findings indicate that the definition of 310‐helices in terms of average, uniform dihedral angles is not appropriate and that it is inherently unstable for a polypeptide to form an extended, regular 310‐helix. The 310‐helices observed in proteins are better referred to parahelices. Proteins 2006.

Recombinant Soluble Forms of Extracellular TLR4 Domain and MD-2 Inhibit Lipopolysaccharide Binding on Cell Surface and Dampen Lipopolysaccharide-Induced Pulmonary Inflammation in Mice

In this study, we sought the possibility of a new therapeutic strategy for dampening endotoxin-induced inflammation using soluble form of extracellular rTLR4 domain (sTLR4) and soluble form of rMD-2 (sMD-2). Addition of sTLR4 plus sMD-2 was significantly effective in inhibiting LPS-elicited IL-8 release from U937 cells and NF-κB activation in the cells transfected with TLR4 and MD-2 when compared with a single treatment with sTLR4 or sMD-2. Thus, we investigated the role of the extracellular TLR4 domain in interaction of lipid A with MD-2. Biotinylated sTLR4 failed to coprecipitate [3H]lipid A when it was sedimented with streptavidin-agarose, demonstrating that the extracellular TLR4 domain does not directly bind lipid A by itself. The amounts of lipid A coprecipitated with sMD-2 significantly increased when coincubated with sTLR4, and sTLR4 increased the affinity of lipid A for the binding to sMD-2. Soluble CD14 is required for the sTLR4-stimulated increase of lipid A binding to sMD-2. We also found that addition of sTLR4 plus sMD-2 inhibited the binding of Alexa-conjugated LPS to the cells expressing TLR4 and MD-2. Murine lungs that had received sTLR4 plus sMD-2 with LPS did not show any findings indicative of interstitial edema, neutrophil flux, and hemorrhage. Coinstillation of sTLR4 plus sMD-2, but not sTLR4 or sMD-2 alone, significantly decreased neutrophil infiltration and TNF-α levels in bronchoalveolar lavage fluids from LPS-treated mice. This study provides novel usage of sTLR4 and sMD-2 as an antagonist against endotoxin-induced pulmonary inflammation.