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.

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.

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.

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 ARA7⅐VPS9a(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 ␤-phosphate directly and pulls the P-loop lysine of ARA7 away from GDP ␤-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.Small GTPases work as a molecular switch, which is turned off by its intrinsic GTPase activity hydrolyzing GTP to GDP. To turn the switch on, the bound GDP should be removed to introduce a GTP. Normally, this exchange reaction is much slower than the rate of intrinsic GTPase activity because nucleotide binds to small GTPase tightly with the Mg 2ϩ ion. GEF 5 enhances the nucleotide exchange of its cognate GTPase by destabilizing the Mg 2ϩ ion and GDP binding to the GTPase. Recognition of the GDP-bound GTPase by GEF is a critical but transient step prior to the formation of a nucleotide-free GTPase⅐GEF binary complex (1, 2). Because the nucleotide-free complex is stable in vitro, most GTPase⅐GEF complexes have been crystallized and structurally analyzed as the nucleotidefree form in the past, with a few exceptions containing nucleotides. One of them is a structure of eFF1A⅐eEF1B␣ in complex with different di-and triphosphate nucleotides, in which ␤-and ␥-phosphate electron densities of the nucleotides were ambiguous and only GMP could have been modeled (3). Another example is the Arf1⅐Sec7 complex, in which introducing either an abortive inhibitor or a mutation on the GEF allowed for GDP binding into the Arf1⅐Sec7 complex (4, 5). The complex structure between ROP4⅐GDP, a member of the plant Rho family, and its GEF PRONE8 indicated that GDP bound to the ROP4⅐PRONE8 complex loosely (6). A complex between Cdc42 and the DHR2 GEF domain of DOCK9 GEF has been crystallized in three different nucleotide forms: nucleotide-free, GDPbound, and GTP/Mg 2ϩ -bound. These structures demonstrate that the nucleotide sensor found in the ␣10 helix of the DHR2 domain is responsible for the exclusion of GDP/Mg 2ϩ and the introduction of GTP/Mg 2ϩ (7). It should be noted that none of the nucleotides were directly recognized by the GEFs in the complex structures mentioned above. These exceptions have encouraged us to investigate the transient nucleotide recognition in the GEF-catalyzed reaction.Rab small GTPases regulate vesicular transport in eukaryotes (8, 9), including ...

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.