Structural basis of RNA polymerase II transcription on the chromatosome containing linker histone H1.

In chromatin, linker histone H1 binds to nucleosomes, forming chromatosomes, and changes the transcription status. However, the mechanism by which RNA polymerase II (RNAPII) transcribes the DNA in the chromatosome has remained enigmatic. Here we report the cryo-electron microscopy (cryo-EM) structures of transcribing RNAPII-chromatosome complexes (forms I and II), in which RNAPII is paused at the entry linker DNA region of the chromatosome due to H1 binding. In the form I complex, the H1 bound to the nucleosome restricts the linker DNA orientation, and the exit linker DNA is captured by the RNAPII DNA binding cleft. In the form II complex, the RNAPII progresses a few bases ahead by releasing the exit linker DNA from the RNAPII cleft, and directly clashes with the H1 bound to the nucleosome. The transcription elongation factor Spt4/5 masks the RNAPII DNA binding region, and drastically reduces the H1-mediated RNAPII pausing.

Structural Basis for the Inhibition of Cyclin G-Associated Kinase by Gefitinib.

Gefitinib is the molecular target drug for advanced non-small-cell lung cancer. The primary target of gefitinib is the positive mutation of epidermal growth factor receptor, but it also inhibits cyclin G-associated kinase (GAK). To reveal the molecular bases of GAK and gefitinib binding, structure analyses were conducted and determined two forms of the gefitinib-bound nanobody⋅GAK kinase domain complex structures. The first form, GAK_1, has one gefitinib at the ATP binding pocket, whereas the second form, GAK_2, binds one each in the ATP binding site and a novel binding site adjacent to the activation segment C-terminal helix, a unique element of the Numb-associated kinase family. In the novel binding site, gefitinib binds in the hydrophobic groove around the activation segment, disrupting the conserved hydrogen bonds for the catalytic activity. These structures suggest possibilities for the development of selective GAK inhibitors for viral infections, such as the hepatitis C virus.

Charge-state-distribution analysis of Bach2 intrinsically disordered heme binding region.

Bach2 is a transcriptional repressor that plays an important role in the differentiation of T-cells and B-cells. Bach2 is functionally regulated by heme binding, and possesses five Cys-Pro Cys-Pro (CP)-motifs as the heme binding site. To reveal the molecular mechanism of heme binding by Bach2, the intrinsically disordered heme binding region (a.a. 331-520; Bach2331-520) and its CP-motif mutant were prepared and characterized with and without heme, by UV-Vis spectroscopy and thermal profiles. In addition, the charge-state-distributions (CSDs) were assessed by electrospray ionization mass spectrometry. The UV-Vis spectroscopy revealed a lack of five-coordinated heme binding in the CP-motif mutant of Bach2331-520 The thermal profile and CSDs of Bach2331-520 indicated that heme binding induces the destabilization of Bach2331-520 The thermal profile revealed that the wild type Bach2331-520 was destabilized more than the CP-motif mutant. The shift in the CSDs by heme binding suggested that heme binding causes Bach2331-520 to adopt a more compact conformation. In addition, heme binding to the CP-motif could reduce the flexibility of Bach2331-520 Consequently, the five-coordinated heme binding destabilizes Bach2331-520, by reducing the flexibility of the polypeptide chain.

Direct metal recognition by guanine nucleotide-exchange factor in the initial step of the exchange reaction.

Rab small GTPases regulate vesicle transport in eukaryotes by interacting with various effectors. Guanine nucleotide-exchange factor (GEF) catalyzes the transition from inactive GDP-bound Rab to active GTP-bound Rab. The existence of several GDP-bound intermediates containing the Arabidopsis thaliana Rab5 homologue ARA7 and the GEF VPS9a prior to the formation of a nucleotide-free binary complex has been proposed [Uejima et al. (2010), J. Biol. Chem. 285, 36689-36697]. During this process, VPS9a directly interacts with the ß-phosphate of GDP and the P-loop lysine of ARA7 via a catalytically important aspartate finger, which promotes the release of GDP from ARA7. However, it is unclear how VPS9a removes Mg2+ from ARA7 before forming the GDP-bound ternary complex. Here, the structure of the ARA7-GDP-Ca2+-VPS9a complex is reported, in which the aspartate finger directly coordinates the divalent metal ion. Ca2+ is bound to the canonical Mg2+-binding site, coordinated by the ß-phosphate of GDP and the P-loop serine of ARA7. Unexpectedly, Ca2+ is further coordinated by the aspartate finger and the main chain of VPS9a. This structure may represent the earliest intermediate step in the GEF-catalyzed nucleotide-exchange reaction of ARA7 before the metal-free GDP-bound intermediates are created.

GDP-bound and nucleotide-free intermediates of the guanine nucleotide exchange in the Rab5·Vps9 system.

Many GTPases regulate intracellular transport and signaling in eukaryotes. Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing the exchange of their GDP for GTP. Here we present crystallographic and biochemical studies of a GEF reaction with four crystal structures of Arabidopsis thaliana ARA7, a plant homolog of Rab5 GTPase, in complex with its GEF, VPS9a, in the nucleotide-free and GDP-bound forms, as well as a complex with aminophosphonic acid-guanylate ester and ARA7·VPS9a(D185N) with GDP. Upon complex formation with ARA7, VPS9 wedges into the interswitch region of ARA7, inhibiting the coordination of Mg(2+) and decreasing the stability of GDP binding. The aspartate finger of VPS9a recognizes GDP ß-phosphate directly and pulls the P-loop lysine of ARA7 away from GDP ß-phosphate toward switch II to further destabilize GDP for its release during the transition from the GDP-bound to nucleotide-free intermediates in the nucleotide exchange reaction.

Specific recognition of linear ubiquitin chains by NEMO is important for NF-kappaB activation.

Activation of nuclear factor-kappaB (NF-kappaB), a key mediator of inducible transcription in immunity, requires binding of NF-kappaB essential modulator (NEMO) to ubiquitinated substrates. Here, we report that the UBAN (ubiquitin binding in ABIN and NEMO) motif of NEMO selectively binds linear (head-to-tail) ubiquitin chains. Crystal structures of the UBAN motif revealed a parallel coiled-coil dimer that formed a heterotetrameric complex with two linear diubiquitin molecules. The UBAN dimer contacted all four ubiquitin moieties, and the integrity of each binding site was required for efficient NF-kappaB activation. Binding occurred via a surface on the proximal ubiquitin moiety and the canonical Ile44 surface on the distal one, thereby providing specificity for linear chain recognition. Residues of NEMO involved in binding linear ubiquitin chains are required for NF-kappaB activation by TNF-alpha and other agonists, providing an explanation for the detrimental effect of NEMO mutations in patients suffering from X-linked ectodermal dysplasia and immunodeficiency.

Elucidation of Rab27 recruitment by its effectors: structure of Rab27a bound to Exophilin4/Slp2-a.

Rab GTPases coordinate vesicular trafficking within eukaryotic cells by collaborating with a set of effector proteins. Rab27a regulates numerous exocytotic pathways, and its dysfunction causes the Griscelli syndrome human immunodeficiency. Exophilin4/Slp2-a localizes on phosphatidylserine-enriched plasma membrane, and its N-terminal Rab27-binding domain (RBD27) specifically recognizes Rab27 on the surfaces of melanosomes and secretory granules prior to docking and fusion. To characterize the selective binding of Rab27 to 11 various effectors, we have determined the 1.8 A resolution structure of Rab27a in complex with Exophilin4 RBD27. The effector packs against the switch and interswitch elements of Rab27a, and specific affinity toward Rab27a is modulated by a shift in the orientation of the effector structural motif (S/T)(G/L)xW(F/Y)(2). The observed structural complementation between the interacting surfaces of Rab27a and Exophilin4 sheds light on the disparities among the Rab27 effectors and outlines a general mechanism for their recruitment.

Development of an automated large-scale protein-crystallization and monitoring system for high-throughput protein-structure analyses.

Protein crystallization remains one of the bottlenecks in crystallographic analysis of macromolecules. An automated large-scale protein-crystallization system named PXS has been developed consisting of the following subsystems, which proceed in parallel under unified control software: dispensing precipitants and protein solutions, sealing crystallization plates, carrying robot, incubators, observation system and image-storage server. A sitting-drop crystallization plate specialized for PXS has also been designed and developed. PXS can set up 7680 drops for vapour diffusion per hour, which includes time for replenishing supplies such as disposable tips and crystallization plates. Images of the crystallization drops are automatically recorded according to a preprogrammed schedule and can be viewed by users remotely using web-based browser software. A number of protein crystals were successfully produced and several protein structures could be determined directly from crystals grown by PXS. In other cases, X-ray quality crystals were obtained by further optimization by manual screening based on the conditions found by PXS.