Microglia enable cross-modal plasticity by removing inhibitory synapses.

Cross-modal plasticity is the repurposing of brain regions associated with deprived sensory inputs to improve the capacity of other sensory modalities. The functional mechanisms of cross-modal plasticity can indicate how the brain recovers from various forms of injury and how different sensory modalities are integrated. Here, we demonstrate that rewiring of the microglia-mediated local circuit synapse is crucial for cross-modal plasticity induced by visual deprivation (monocular deprivation [MD]). MD relieves the usual inhibition of functional connectivity between the somatosensory cortex and secondary lateral visual cortex (V2L). This results in enhanced excitatory responses in V2L neurons during whisker stimulation and a greater capacity for vibrissae sensory discrimination. The enhanced cross-modal response is mediated by selective removal of inhibitory synapse terminals on pyramidal neurons by the microglia in the V2L via matrix metalloproteinase 9 signaling. Our results provide insights into how cortical circuits integrate different inputs to functionally compensate for neuronal damage.

Targeting Ras-binding domain of ELMO1 by computational nanobody design.

The control of cell movement through manipulation of cytoskeletal structure has therapeutic prospects notably in the development of novel anti-metastatic drugs. In this study, we determine the structure of Ras-binding domain (RBD) of ELMO1, a protein involved in cytoskeletal regulation, both alone and in complex with the activator RhoG and verify its targetability through computational nanobody design. Using our dock-and-design approach optimized with native-like initial pose selection, we obtain Nb01, a detectable binder from scratch in the first-round design. An affinity maturation step guided by structure-activity relationship at the interface generates 23 Nb01 sequence variants and 17 of them show enhanced binding to ELMO1-RBD and are modeled to form major spatial overlaps with RhoG. The best binder, Nb29, inhibited ELMO1-RBD/RhoG interaction. Molecular dynamics simulation of the flexibility of CDR2 and CDR3 of Nb29 reveal the design of stabilizing mutations at the CDR-framework junctions potentially confers the affinity enhancement.

Structural basis for the dual GTPase specificity of the DOCK10 guanine nucleotide exchange factor.

Dedicator of cytokinesis 10 (DOCK10), an evolutionarily conserved guanine nucleotide exchange factor (GEF) for Rho GTPases, has the unique specificity within the DOCK-D subfamily to activate both Cdc42 and Rac, but the structural bases for these activities remained unknown. Here we present the crystal structures of the catalytic DHR2 domain of mouse DOCK10, complexed with either Cdc42 or Rac1. The structures revealed that DOCK10DHR2 binds to Cdc42 or Rac1 by slightly changing the arrangement of its two catalytic lobes. DOCK10 also has a flexible binding pocket for the 56th GTPase residue, allowing a novel interaction with Trp56Rac1. The conserved residues in switch 1 of Cdc42 and Rac1 showed common interactions with the unique Lys-His sequence in the ß5/ß6 loop of DOCK10DHR2. However, the interaction of switch 1 in Rac1 was less stable than that of switch 1 in Cdc42, due to amino acid differences at positions 27 and 30. Structure-based mutagenesis identified the DOCK10 residues that determine the Cdc42/Rac1 dual specificity.

Crystal Structure of an Archaeal Tyrosyl-tRNA Synthetase Bound to Photocaged L-Tyrosine and Its Potential Application to Time-Resolved X-ray Crystallography.

Genetically encoded caged amino acids can be used to control the dynamics of protein activities and cellular localization in response to external cues. In the present study, we revealed the structural basis for the recognition of O-(2-nitrobenzyl)-L-tyrosine (oNBTyr) by its specific variant of Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (oNBTyrRS), and then demonstrated its potential availability for time-resolved X-ray crystallography. The substrate-bound crystal structure of oNBTyrRS at a 2.79 Å resolution indicated that the replacement of tyrosine and leucine at positions 32 and 65 by glycine (Tyr32Gly and Leu65Gly, respectively) and Asp158Ser created sufficient space for entry of the bulky substitute into the amino acid binding pocket, while Glu in place of Leu162 formed a hydrogen bond with the nitro moiety of oNBTyr. We also produced an oNBTyr-containing lysozyme through a cell-free protein synthesis system derived from the Escherichia coli B95. ΔA strain with the UAG codon reassigned to the nonnatural amino acid. Another crystallographic study of the caged protein showed that the site-specifically incorporated oNBTyr was degraded to tyrosine by light irradiation of the crystals. Thus, cell-free protein synthesis of caged proteins with oNBTyr could facilitate time-resolved structural analysis of proteins, including medically important membrane proteins.

Expression, purification and characterization of isoforms of peripheral stalk subunits of human V-ATPase.

The vacuolar-type H+-ATPase (V-ATPase) is a multi-subunit proton pump that is involved in both intra- and extracellular acidification processes throughout human body. Subunits constituting the peripheral stalk of the V-ATPase are known to have several isoforms responsible for tissue/cell specific different physiological roles. To study the different interaction of these isoforms, we expressed and purified the isoforms of human V-ATPase peripheral stalk subunits using Escherichia coli cell-free protein synthesis system: E1, E2, G1, G2, G3, C1, C2, H and N-terminal soluble part of a1 and a2 isoforms. The purification conditions were different depending on the isoforms, maybe reflecting the isoform specific biochemical characteristics. The purified proteins are expected to facilitate further experiments to study about the cell specific interaction and regulation and thus provide insight into physiological meaning of the existence of several isoforms of each subunit in V-ATPase.

Structure of an N-terminally truncated selenophosphate synthetase from Aquifex aeolicus.

Selenophosphate synthetase (SPS) catalyzes the activation of selenide with ATP to synthesize selenophosphate, the reactive selenium donor for biosyntheses of both the 21st amino acid selenocysteine and 2-selenouridine nucleotides in tRNA anticodons. The crystal structure of an N-terminally (25 residues) truncated fragment of SPS (SPS-DeltaN) from Aquifex aeolicus has been determined at 2.0 A resolution. The structure revealed SPS to be a two-domain alpha/beta protein, with domain folds that are homologous to those of PurM-superfamily proteins. In the crystal, six monomers of SPS-DeltaN form a hexamer of 204 kDa, whereas the molecular weight estimated by ultracentrifugation was approximately 63 kDa, which is comparable to the calculated weight of the dimer (68 kDa).

Alteration of enzymatic properties of cell-surface antigen CD38 by agonistic anti-CD38 antibodies that prolong B cell survival and induce activation.

Leukocyte cell-surface antigen CD38 is a single-transmembrane protein. CD38 ligation by anti-CD38 antibodies triggers the growth or apoptosis of immune cells. Although the extracellular domain of CD38 has multifunctional catalytic activities including NAD(+) glycohydrolase and cyclase, the CD38-mediated cell survival or death appears to be independent of its catalytic activity. It is proposed that a conformational change of CD38 triggers the signalling. The conformational change of CD38 could influence its catalytic activity. However, the agonistic anti-CD38 antibody that alters the catalytic activity of CD38 has not been reported so far. In the present study, we demonstrated that two agonistic anti-mouse CD38 mAbs (CS/2 and clone 90) change the catalytic activities of CD38. CS/2 was clearly more potent than clone 90 in prolonging B cell survival and activation. CS/2 inhibited the NAD(+) glycohydrolase activity of both the isolated extracellular domain of CD38 (FLAG-CD38) and cell-surface CD38. Kinetic analysis suggested a non-competitive inhibition. On the other hand, clone 90 stimulated the NAD(+) glycohydrolase activity of FLAG-CD38 and had little effect on the NAD(+) glycohydrolase activity of cell-surface CD38. CS/2 and clone 90 had no effect on the cyclase activity of FLAG-CD38 and inhibited the cyclase activity of cell-surface CD38. Accordingly, these agonistic antibodies probably induce the conformational changes of CD38 that are evident in the distinct alterations of the catalytic site. The antibodies will be useful tools to analyze the conformational change of CD38 in the process of triggering B cell survival and the activation signal.

Complex assembly mechanism and an RNA-binding mode of the human p14-SF3b155 spliceosomal protein complex identified by NMR solution structure and functional analyses.

The spliceosomal protein p14, a component of the SF3b complex in the U2 small nuclear ribonucleoprotein (snRNP), is essential for the U2 snRNP to recognize the branch site adenosine. The elucidation of the dynamic process of the splicing machinery rearrangement awaited the solution structural information. We identified a suitable complex of human p14 and the SF3b155 fragment for the determination of its solution structure by NMR. In addition to the overall structure of the complex, which was recently reported in a crystallographic study (typical RNA recognition motif fold beta1-alpha1-beta2-beta3-alpha2-beta4 of p14, and alphaA-betaA fold of the SF3b155 fragment), we identified three important features revealed by the NMR solution structure. First, the C-terminal extension and the nuclear localization signal of p14 (alpha3 and alpha4 in the crystal structure, respectively) were dispensable for the complex formation. Second, the proline-rich segment of SF3b155, following betaA, closely approaches p14. Third, interestingly, the beta1-alpha1 loop and the alpha2-beta4 beta-hairpin form a positively charged groove. Extensive mutagenesis analyses revealed the functional relevance of the residues involved in the protein-protein interactions: two aromatic residues of SF3b155 (Phe408 and Tyr412) play crucial roles in the complex formation, and two hydrophobic residues (Val414 and Leu415) in SF3b 155 serve as an anchor for the complex formation, by cooperating with the aromatic residues. These findings clearly led to the conclusion that SFb155 binds to p14 with three contact points, involving Phe408, Tyr412, and Val414/Leu415. Furthermore, to dissect the interactions between p14 and the branch site RNA, we performed chemical-shift-perturbation experiments, not only for the main-chain but also for the side-chain resonances, for several p14-SF3b155 complex constructs upon binding to RNA. These analyses identified a positively charged groove and the C-terminal extension of p14 as RNA-binding sites. Strikingly, an aromatic residue in the beta1-alpha1 loop, Tyr28, and a positively charged residue in the alpha2-beta4 beta-hairpin, Agr85, are critical for the RNA-binding activity of the positively charged groove. The Tyr28Ala and Arg85Ala point mutants and a deletion mutant of the C-terminal extension clearly revealed that their RNA binding activities were independent of each other. Collectively, this study provides details for the protein-recognition mode of p14 and insight into the branch site recognition.

Establishment of stable hFis1 knockdown cells with an siRNA expression vector.

Yeast Fis1p participates in mitochondrial fission, together with Dnm1p and Mdv1p. Recently, human Fis1 (hFis1) was reported to be involved in mitochondrial fission, together with Drp1. We established stable transformants with an hFis1 siRNA expression vector. In the stable hFis1 knockdown cells, hFis1 expression was suppressed to approximately 10%, and mitochondrial fission, induced by cisplatin treatment, was delayed. In addition, mouse Fis1 (mFis1) expression promoted mitochondrial fission and cell death in the hFis1 knockdown cells, suggesting that mFis1 complements the function of hFis1. These hFis1 siRNA expression vectors may be useful for studying the molecular function of mammalian Fis1.

Crystal structure of the GTP-binding protein Obg from Thermus thermophilus HB8.

Obg comprises a unique family of high-molecular mass GTPases conserved from bacteria to eukaryotes. Bacterial Obg is essential for cellular growth, sporulation, and differentiation. Here, we report the crystal structure of the full-length form of Obg from Thermus thermophilus HB8 at 2.07 A resolution, in the nucleotide-free state. It reveals a three-domain arrangement, composed of the N-terminal domain, the guanine nucleotide-binding domain (G domain), and the C-terminal domain. The N-terminal and G domains have the Obg fold and the Ras-like fold, respectively. These global folds are similar to those of the recently published structure of the C-terminal domain-truncated form of Obg from Bacillus subtilis. On the other hand, the C-terminal domain of Obg was found to have a novel fold (the OCT fold). A comparison of the T.thermophilus and B.subtilis nucleotide-free Obg structures revealed significant conformational changes in the switch-I and switch-II regions of the G domain. Notably, the N-terminal domain is rotated drastically, by almost 180 degrees, around the G domain axis. In the T.thermophilus Obg crystal, the nucleotide-binding site of the G domain interacts with the C-terminal domain of the adjacent molecule. These data suggest a possible domain rearrangement of Obg, and a potential role of the C-terminal domain in the regulation of the nucleotide-binding state.

Crystal structure of the complex of human epidermal growth factor and receptor extracellular domains.

Epidermal growth factor (EGF) regulates cell proliferation and differentiation by binding to the EGF receptor (EGFR) extracellular region, comprising domains I-IV, with the resultant dimerization of the receptor tyrosine kinase. In this study, the crystal structure of a 2:2 complex of human EGF and the EGFR extracellular region has been determined at 3.3 A resolution. EGFR domains I-III are arranged in a C shape, and EGF is docked between domains I and III. The 1:1 EGF*EGFR complex dimerizes through a direct receptor*receptor interaction, in which a protruding beta-hairpin arm of each domain II holds the body of the other. The unique "receptor-mediated dimerization" was verified by EGFR mutagenesis.