Kentaro Ihara

CRYSTAL STRUCTURE OF PHO4 BHLH DOMAIN-DNA COMPLEX : FLANKING BASE RECOGNITION

The crystal structure of a DNA‐binding domain of PHO4 complexed with DNA at 2.8 Å resolution revealed that the domain folds into a basic–helix–loop–helix (bHLH) motif with a long but compact loop that contains a short α‐helical segment. This helical structure positions a tryptophan residue into an aromatic cluster so as to make the loop compact. PHO4 binds to DNA as a homodimer with direct reading of both the core E‐box sequence CACGTG and its 3′‐flanking bases. The 3′‐flanking bases GG are recognized by Arg2 and His5. The residues involved in the E‐box recognition are His5, Glu9 and Arg13, as already reported for bHLH/Zip proteins MAX and USF, and are different from those recognized by bHLH proteins MyoD and E47, although PHO4 is a bHLH protein.

The structural basis of Rho effector recognition revealed by the crystal structure of human RhoA complexed with the effector domain of PKN/PRK1.

The small G protein Rho has emerged as a key regulator of cellular events involving cytoskeletal reorganization. Here we report the 2.2 A crystal structure of RhoA bound to an effector domain of protein kinase PKN/PRK1. The structure reveals the antiparallel coiled-coil finger (ACC finger) fold of the effector domain that binds to the Rho specificity-determining regions containing switch I, beta strands B2 and B3, and the C-terminal alpha helix A5, predominantly by specific hydrogen bonds. The ACC finger fold is distinct from those for other small G proteins and provides evidence for the diverse ways of effector recognition. Sequence analysis based on the structure suggests that the ACC finger fold is widespread in Rho effector proteins.

Grease matrix as a versatile carrier of proteins for serial crystallography

Serial femtosecond X-ray crystallography (SFX) has revolutionized atomic-resolution structural investigation by expanding applicability to micrometer-sized protein crystals, even at room temperature, and by enabling dynamics studies. However, reliable crystal-carrying media for SFX are lacking. Here we introduce a grease-matrix carrier for protein microcrystals and obtain the structures of lysozyme, glucose isomerase, thaumatin and fatty acid–binding protein type 3 under ambient conditions at a resolution of or finer than 2 Å.

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.

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.

Structure of the small GTPase Rab27b shows an unexpected swapped dimer

Members of the Rab family of small GTPases regulate membrane traffic within the cell by recruiting their specific effectors in a nucleotide-dependent manner. The Rab27 subfamily consists of Rab27a and Rab27b, which share 70% sequence identity. By interacting with a large set of effector proteins such as melanophilin and granuphilin, both Rab27a and Rab27b regulate the exocytosis of secretory lysosomes. Here, the crystal structures of mouse Rab27b in complex with GDP have been determined in three distinct crystal lattices. Surprisingly, Rab27b-GDP exists in an open conformation with protruding switch and interswitch regions, which are stabilized through dimerization by means of domain-swapping in the crystals. In contrast, small-angle X-ray scattering measurements showed an extended monomer form of Rab27b in solution. The observed dimer formation of Rab27b-GDP in the crystals would restrain the highly flexible switch regions. Possible biological implications of this atypical structure of Rab27b and its plausible influence in effector interaction are discussed.

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 Mg2+ and decreasing the stability of GDP binding. The aspartate finger of VPS9a recognizes GDP β-phosphate directly and pulls the P-loop lysine of ARA7 away from GDP β-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.

Crystal structure of the protein histidine phosphatase SixA in the multistep His-Asp phosphorelay

The multiple histidine‐aspartate phosphorelay system plays a crucial role in cellular adaptation to environments in microorganisms and plants. Like kinase‐phosphatase systems in higher eukaryotes, the multiple steps provide additional regulatory checkpoints with phosphatases. The Escherichia coli phosphatase SixA exhibits protein phosphatase activity against the histidine‐containing phosphotransfer (HPt) domain located in the C‐terminus of the histidine kinase ArcB engaged in anaerobic responses. We have determined the crystal structures of the free and tungstate‐bound forms of SixA at 2.06 Å and 1.90 Å resolution, respectively. The results provide the first three‐dimensional view of a bacterial protein histidine phosphatase, revealing a compact α/β architecture related to a family of phosphatases containing the arginine‐histidine‐glycine (RHG) motif at their active sites. Compared with these RHG phosphatases, SixA lacks an extra α‐helical subdomain as a lid over the active site, thereby forming a relatively shallow groove important for the accommodation of the HPt domain of ArcB. The tungstate ion, which mimics the substrate phosphate group, is located at the centre of the active site where the active residue, His8, points to the tungsten atom in the mode of in‐line nucleophilic attack.

Molecular Basis for Autoregulatory Interaction Between GAE Domain and Hinge Region of GGA1

Golgi‐localizing, γ‐adaptin ear domain homology, ADP ribosylation factor‐binding (GGA) proteins and the adaptor protein (AP) complex, AP‐1, are involved in membrane traffic between the trans Golgi network and the endosomes. The γ‐adaptin ear (GAE) domain of GGAs and the γ1 ear domain of AP‐1 interact with an acidic phenylalanine motif found in accessory proteins. The GAE domain of GGA1 (GGA1‐GAE) interacts with a WNSF‐containing peptide derived from its own hinge region, although the peptide sequence deviates from the standard acidic phenylalanine motif. We report here the structure of GGA1‐GAE in complex with the GGA1 hinge peptide, which revealed that the two aromatic side chains of the WNSF sequence fit into a hydrophobic groove formed by aliphatic portions of the side chains of conserved arginine and lysine residues of GGA1‐GAE, in a similar manner to the interaction between GGA‐GAEs and acidic phenylalanine sequences from the accessory proteins. Fluorescence quenching experiments indicate that the GGA1 hinge region binds to GGA1‐GAE and competes with accessory proteins for binding. Taken together with the previous observation that γ1 ear binds to the GGA1 hinge region, the interaction between the hinge region and the GAE domain underlies the autoregulation of GGA function in clathrin‐mediated trafficking through competing with the accessory proteins and the AP‐1 complex.

Structures of an ATP-independent Lon-like protease and its complexes with covalent inhibitors

The Lon proteases are a unique family of chambered proteases with a built-in AAA+ (ATPases associated with diverse cellular activities) module. Here, crystal structures of a unique member of the Lon family with no intrinsic ATPase activity in the proteolytically active form are reported both alone and in complexes with three covalent inhibitors: two peptidomimetics and one derived from a natural product. This work reveals the unique architectural features of an ATP-independent Lon that selectively degrades unfolded protein substrates. Importantly, these results provide mechanistic insights into the recognition of inhibitors and polypeptide substrates within the conserved proteolytic chamber, which may aid the development of specific Lon-protease inhibitors.