Atsushi Nakagawa

Host innate immune receptors and beyond: making sense of microbial infections.

The complexity of the immune system mirrors its manifold mechanisms of host-microbe interactions. A relatively simplified view was posited after the identification of host innate immune receptors that their distinct mechanisms of sensing microbial signatures create unique molecular switches to trigger the immune system. Recently, more sophisticated and cooperative strategies for these receptors have been revealed during receptor-ligand interactions, trafficking, and intra- and intercellular signaling, in order to deal with a diverse range of microbes. Continued mapping of the complex networks of host-microbe interactions may improve our understanding of self/non-self discrimination in immunity and its intervention.

14-3-3 proteins act as intracellular receptors for rice Hd3a florigen

‘Florigen’ was proposed 75 years ago to be synthesized in the leaf and transported to the shoot apex, where it induces flowering. Only recently have genetic and biochemical studies established that florigen is encoded by FLOWERING LOCUS T (FT), a gene that is universally conserved in higher plants. Nonetheless, the exact function of florigen during floral induction remains poorly understood and receptors for florigen have not been identified. Here we show that the rice FT homologue Hd3a interacts with 14-3-3 proteins in the apical cells of shoots, yielding a complex that translocates to the nucleus and binds to the Oryza sativa (Os)FD1 transcription factor, a rice homologue of Arabidopsis thaliana FD. The resultant ternary ‘florigen activation complex’ (FAC) induces transcription of OsMADS15, a homologue of A. thaliana APETALA1 (AP1), which leads to flowering. We have determined the 2.4u2009Å crystal structure of rice FAC, which provides a mechanistic basis for florigen function in flowering. Our results indicate that 14-3-3 proteins act as intracellular receptors for florigen in shoot apical cells, and offer new approaches to manipulate flowering in various crops and trees.

Crystal structure of the membrane fusion protein, MexA, of the multidrug transporter in Pseudomonas aeruginosa

The MexAB-OprM efflux pump of Pseudomonas aeruginosa is central to multidrug resistance of this organism, which infects immunocompromised hospital patients. The MexA, MexB, and OprM subunits were assumed to function as the membrane fusion protein, the body of the transporter, and the outer membrane channel protein, respectively. For better understanding of this important xenobiotic transporter, we show the x-ray crystallographic structure of MexA at a resolution of 2.40 Å. The global MexA structure showed unforeseen new features with a spiral assembly of six and seven protomers that were joined together at one end by a pseudo 2-fold image. The protomer showed a new protein structure with a tandem arrangement consisting of at least three domains and presumably one more. The rod domain had a long hairpin of twisted coiled-coil that extended to one end. The second domain adjacent to the rod α-helical domain was globular and constructed by a cluster of eight short β-sheets. The third domain located distal to the α-helical rod was globular and composed of seven short β-sheets and one short α-helix. The 13-mer was shaped like a woven rattan cylinder with a large internal tubular space and widely opened flared ends. The 6-mer and 7-mer had a funnel-like structure consisting of a tubular rod at one side and a widely opened flared funnel top at the other side. Based on these results, we constructed a model of the MexAB-OprM pump assembly. The three pairs of MexA dimers interacted with the periplasmic α-barrel domain of OprM via the α-helical hairpin, the second domain interacted with both MexB and OprM at their contact site, and the third and disordered domains probably interacted with the distal domain of MexB. In this fashion, the MexA subunit connected MexB and OprM, indicating that MexA is the membrane bridge protein.

Crystal structure of the drug discharge outer membrane protein, OprM, of Pseudomonas aeruginosa: dual modes of membrane anchoring and occluded cavity end

The OprM lipoprotein of Pseudomonas aeruginosa is a member of the MexAB-OprM xenobiotic-antibiotic transporter subunits that is assumed to serve as the drug discharge duct across the outer membrane. The channel structure must differ from that of the porin-type open pore because the protein facilitates the exit of antibiotics but not the entry. For better understanding of the structure-function linkage of this important pump subunit, we studied the x-ray crystallographic structure of OprM at the 2.56-Å resolution. The overall structure exhibited trimeric assembly of the OprM monomer that consisted mainly of two domains: the membrane-anchoring β-barrel and the cavity-forming α-barrel. OprM anchors the outer membrane by two modes of membrane insertions. One is via the covalently attached NH2-terminal fatty acids and the other is the β-barrel structure consensus on the outer membrane-spanning proteins. The β-barrel had a pore opening with a diameter of about 6–8 Å, which is not large enough to accommodate the exit of any antibiotics. The periplasmic α-barrel was about 100 Å long formed mainly by a bundle of α-helices that formed a solvent-filled cavity of about 25,000 Å3. The proximal end of the cavity was tightly sealed, thereby not permitting the entry of any molecule. The result of this structure was that the resting state of OprM had a small outer membrane pore and a tightly closed periplasmic end, which sounds plausible because the protein should not allow free access of antibiotics. However, these observations raised another unsolved problem about the mechanism of opening of the OprM cavity ends. The crystal structure offers possible mechanisms of pore opening and pump assembly.

A high-resolution structure of the pre-microRNA nuclear export machinery

Pre-MicroRNA Export Machinery Micro (mi) RNAs play a role in the regulation of many biological processes. Long transcripts are initially processed in the nucleus to yield pre-miRNAs that are translocated through the nuclear pore complex and further processed to mature miRNAs in the cytoplasm. Okada et al. (p. 1275; see the Perspective by Stewart) describe the crystal structure of pre-miRNA complexed with the exportin Exp5 and the small nuclear GTPase RanGTP. The structure shows that Exp5 and RanGTP protect the miRNA from degradation by nucleases, as well as facilitate transport to the cytoplasm. RNA recognition is mainly through ionic interactions that are sequence independent, and model-building suggests that this nuclear export machinery could accommodate other small-structured RNAs. Exportin-5:RanGTP surrounds microRNAs to protect them from degradation as it exports them from the nucleus. Nuclear export of microRNAs (miRNAs) by exportin-5 (Exp-5) is an essential step in miRNA biogenesis. Here, we present the 2.9 angstrom structure of the pre-miRNA nuclear export machinery formed by pre-miRNA complexed with Exp-5 and a guanine triphosphate (GTP)–bound form of the small nuclear guanine triphosphatase (GTPase) Ran (RanGTP). The x-ray structure shows that Exp-5:RanGTP recognizes the 2-nucleotide 3′ overhang structure and the double-stranded stem of the pre-miRNA. Exp-5:RanGTP shields the pre-miRNA stem from degradation in a baseball mitt–like structure where it is held by broadly distributed weak interactions, whereas a tunnel-like structure of Exp-5 interacts strongly with the 2-nucleotide 3′ overhang through hydrogen bonds and ionic interactions. RNA recognition by Exp-5:RanGTP does not depend on RNA sequence, implying that Exp-5:RanGTP can recognize a variety of pre-miRNAs.

Crystal structure of the macrophage migration inhibitory factor from rat liver

The tertiary structure of the macrophage migration inhibitory factor (MIF) from rat liver (12,300 Mr) is presented at 2.2 Å resolution. Each monomer consists of two β/α/β motifs aligned in quasi two-fold symmetry, comprising a domain consisting of a four-stranded mixed β-sheet and two antiparallel α-helices. The protein exists as a trimer in the crystal. An extra β-strand that is almost perpendicular to the other β-strands joins to the β-sheet of the neighbouring monomer in the trimer. Unexpected similarities were detected between MIF and two kinds of isomerase.

Design and performance of an imaging plate system for X-ray diffraction study

A new readout system for a BaFBr: Eu2+ photostimulable phosphor screen (imaging plate) was constructed by modifying a drum scanner, with a design optimized for X-ray diffraction and scattering applications. An effort was made to achieve a high detective quantum efficiency below 20 keV, a small pixel size (25 μm × 25 μm), a low quantization noise (0.22%) using 12-bit A/D converters, and the capability to cover an inherent dynamic range (1:105) of the photostimulated luminescence by using two photomultiplier tubes. This system is being used in several synchrotron radiation experiments: Laue diffraction of protein crystals, small angle diffraction from a single muscle fiber, powder diffraction from crystals in a diamond anvil cell, and time-resolved small-angle X-ray scattering from a synthetic polymer during stretching.

Structure of the carboxyl-terminal Src kinase, Csk.

The carboxyl-terminal Src kinase (Csk) is an indispensable negative regulator for the Src family tyrosine kinases (SFKs) that play pivotal roles in various cell signalings. To understand the molecular basis of the Csk-mediated regulation of SFKs, we elucidated the crystal structure of full-length Csk. The Csk crystal consists of six molecules classified as active or inactive states according to the coordinations of catalytic residues. Csk assembles the SH2 and SH3 domains differently from inactive SFKs, and their binding pockets are oriented outward enabling the intermolecular interaction. In active molecules, the SH2-kinase and SH2-SH3 linkers are tightly stuck to the N-lobe of the kinase domain to stabilize the active conformation, and there is a direct linkage between the SH2 and the kinase domains. In inactive molecules, the SH2 domains are rotated destroying the linkage to the kinase domain. Cross-correlation matrices for the active molecules reveal that the SH2 domain and the N-lobe of the kinase domain move as a unit. These observations suggest that Csk can be regulated through coupling of the SH2 and kinase domains and that Csk provides a novel built-in activation mechanism for cytoplasmic tyrosine kinases.

Immunogenicity of whole-parasite vaccines against Plasmodium falciparum involves malarial hemozoin and host TLR9.

Although whole-parasite vaccine strategies for malaria infection have regained attention, their immunological mechanisms of action remain unclear. We find that immunization of mice with a crude blood stage extract of the malaria parasite Plasmodium falciparum elicits parasite antigen-specific immune responses via Toll-like receptor (TLR) 9 and that the malarial heme-detoxification byproduct, hemozoin (HZ), but not malarial DNA, produces a potent adjuvant effect. Malarial and synthetic (s)HZ bound TLR9 directly to induce conformational changes in the receptor. The adjuvant effect of sHZ depended on its method of synthesis and particle size. Although natural HZ acts as a TLR9 ligand, the adjuvant effects of synthetic HZ are independent of TLR9 or the NLRP3-inflammasome but are dependent on MyD88. The adjuvant function of sHZ was further validated in a canine antiallergen vaccine model. Thus, HZ can influence adaptive immune responses to malaria infection and may have therapeutic value in vaccine adjuvant development.

Structural insight into maintenance methylation by mouse DNA methyltransferase 1 (Dnmt1)

Methylation of cytosine in DNA plays a crucial role in development through inheritable gene silencing. The DNA methyltransferase Dnmt1 is responsible for the propagation of methylation patterns to the next generation via its preferential methylation of hemimethylated CpG sites in the genome; however, how Dnmt1 maintains methylation patterns is not fully understood. Here we report the crystal structure of the large fragment (291–1620) of mouse Dnmt1 and its complexes with cofactor S-adenosyl-L-methionine and its product S-adenosyl-L-homocystein. Notably, in the absence of DNA, the N-terminal domain responsible for targeting Dnmt1 to replication foci is inserted into the DNA-binding pocket, indicating that this domain must be removed for methylation to occur. Upon binding of S-adenosyl-L-methionine, the catalytic cysteine residue undergoes a conformation transition to a catalytically competent position. For the recognition of hemimethylated DNA, Dnmt1 is expected to utilize a target recognition domain that overhangs the putative DNA-binding pocket. Taking into considerations the recent report of a shorter fragment structure of Dnmt1 that the CXXC motif positions itself in the catalytic pocket and prevents aberrant de novo methylation, we propose that maintenance methylation is a multistep process accompanied by structural changes.