Kanako Sugiyama

Structural insight into the essential PB1-PB2 subunit contact of the influenza virus RNA polymerase

Influenza virus RNA‐dependent RNA polymerase is a multi‐functional heterotrimer, which uses a ‘cap‐snatching’ mechanism to produce viral mRNA. Host cell mRNA is cleaved to yield a cap‐bearing oligonucleotide, which can be extended using viral genomic RNA as a template. The cap‐binding and endonuclease activities are only activated once viral genomic RNA is bound. This requires signalling from the RNA‐binding PB1 subunit to the cap‐binding PB2 subunit, and the interface between these two subunits is essential for the polymerase activity. We have defined this interaction surface by protein crystallography and tested the effects of mutating contact residues on the function of the holo‐enzyme. This novel interface is surprisingly small, yet, it has a crucial function in regulating the 250 kDa polymerase complex and is completely conserved among avian and human influenza viruses.

Capturing the hemoglobin allosteric transition in a single crystal form

Allostery in many oligomeric proteins has been postulated to occur via a ligand-binding-driven conformational transition from the tense (T) to relaxed (R) state, largely on the basis of the knowledge of the structure and function of hemoglobin, the most thoroughly studied of all allosteric proteins. However, a growing body of evidence suggests that hemoglobin possesses a variety of intermediates between the two end states. As such intermediate forms coexist with the end states in dynamic equilibrium and cannot be individually characterized by conventional techniques, very little is known about their properties and functions. Here we present complete structural and functional snapshots of nine equilibrium conformers of human hemoglobin in the half-liganded and fully liganded states by using a novel combination of X-ray diffraction analysis and microspectrophotometric O2 equilibrium measurements on three isomorphous crystals, each capturing three distinct equilibrium conformers. Notably, the conformational set of this crystal form varies according to shifts in the allosteric equilibrium, reflecting the differences in hemoglobin ligation state and crystallization solution conditions. We find that nine snapshot structures cover the complete conformational space of hemoglobin, ranging from T to R2 (the second relaxed quaternary structure) through R, with various relaxed intermediate forms between R and R2. Moreover, we find a previously unidentified intermediate conformer, between T and R, with an intermediate O2 affinity, sought by many research groups over a period of decades. These findings reveal a comprehensive picture of the equilibrium conformers and transition pathway for human hemoglobin.

Crystal structure of the biotin carboxylase domain of human acetyl-CoA carboxylase 2

Crystal structure of the biotin carboxylase domain of human acetyl-CoA carboxylase 2 Yong Soon Cho,1,2y Jae Il Lee,1y Dongkyu Shin,1,3y Hyun Tae Kim, Young Hoon Cheon, Chang Il Seo, Yong Eun Kim, Young-Lan Hyun, Yoon Sup Lee, Kanako Sugiyama, Sam-Yong Park, Seonggu Ro, Joong Myung Cho, Tae Gyu Lee,* and Yong-Seok Heo* 1 R&D Center, CrystalGenomics, Inc., Seoul 138-739, Korea 2Department of Biology, Yonsei University, Seoul 120-749, Korea 3Department of Chemistry and School of Molecular Science (BK21), KAIST, Daejeon 305-701, Korea 4 Protein Design Laboratory, Yokohama City University, Yokohama 230-0045, Japan 5Department of Chemistry, Konkuk University, Seoul 143-701, Korea

Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium

Significance Optogenetics is a rapidly growing field in which light is used to control biological systems. We show that Oscillatoria acuminata photoactivated adenylate cyclase (OaPAC) protein produces the fundamental second messenger cyclic-AMP (cAMP) in response to blue light, is stable and functional in different mammalian cell types, and can be used to trigger events by raising cAMP level. OaPAC consists of a catalytic domain controlled by a photosensitive blue light using flavin (BLUF) domain. We have solved the crystal structure to show how activity is triggered by light, and guide mutagenesis experiments. Although the catalytic domain resembles known cyclases, the BLUF domains form an unusual intertwined structure. The protein activity is the same in solution as in the crystal, showing that the activation mechanism involves only small molecular movements. Cyclic-AMP is one of the most important second messengers, regulating many crucial cellular events in both prokaryotes and eukaryotes, and precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a small homodimer eminently suitable for this task, requiring only a simple flavin chromophore within a blue light using flavin (BLUF) domain. These domains, one of the most studied types of biological photoreceptor, respond to blue light and either regulate the activity of an attached enzyme domain or change its affinity for a repressor protein. BLUF domains were discovered through studies of photo-induced movements of Euglena gracilis, a unicellular flagellate, and gene expression in the purple bacterium Rhodobacter sphaeroides, but the precise details of light activation remain unknown. Here, we describe crystal structures and the light regulation mechanism of the previously undescribed OaPAC, showing a central coiled coil transmits changes from the light-sensing domains to the active sites with minimal structural rearrangement. Site-directed mutants show residues essential for signal transduction over 45 Å across the protein. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase, showing a rapid and stable response to light over many hours and activation cycles. The structures determined in this study will assist future efforts to create artificial light-regulated control modules as part of a general optogenetic toolkit.

Structures and oxygen affinities of crystalline human hemoglobin C (β6 Glu->Lys) in the R and R2 quaternary structures.

Recent crystallographic studies suggested that fully liganded human hemoglobin can adopt multiple quaternary conformations that include the two previously solved relaxed conformations, R and R2, whereas fully unliganded deoxyhemoglobin may adopt only one T (tense) quaternary conformation. An important unanswered question is whether R, R2, and other relaxed quaternary conformations represent different physiological states with different oxygen affinities. Here, we answer this question by showing the oxygen equilibrium curves of single crystals of human hemoglobin in the R and R2 state. In this study, we have used a naturally occurring mutant hemoglobin C (β6 Glu→Lys) to stabilize the R and R2 crystals. Additionally, we have refined the x-ray crystal structure of carbonmonoxyhemoglobin C, in the R and R2 state, to 1.4 and 1.8 Å resolution, respectively, to compare precisely the structures of both types of relaxed states. Despite the large quaternary structural difference between the R and R2 state, both crystals exhibit similar noncooperative oxygen equilibrium curves with a very high affinity for oxygen, comparable with the fourth oxygen equilibrium constant (K4) of human hemoglobin in solution. One small difference is that the R2 crystals have an oxygen affinity that is 2–3 times higher than that of the R crystals. These results demonstrate that the functional difference between the two typical relaxed quaternary conformations is small and physiologically less important, indicating that these relaxed conformations simply reflect a structural polymorphism of a high affinity relaxed state.

Structure-based analysis of domain function of chitin oligosaccharide deacetylase from Vibrio parahaemolyticus

The X‐ray crystal structure of chitin oligosaccharide deacetylase from Vibrio parahaemolyticus (Vp‐COD) was determined at an 1.35 Å resolution. The amino acid sequence and structure of Vp‐COD show that the enzyme comprises one polysaccharide deacetylase domain (PDD) and two carbohydrate‐binding domains (CBDs). On the basis of a chitin‐binding assay with Vp‐COD and its CBDs‐deleted mutant, it was confirmed that CBDs can adhere to chitin. The catalytic activity of the CBDs‐deleted mutant was only mildly depressed compared with that of Vp‐COD, indicating that CBDs are unlikely to affect the configuration of the active center residues in active site of PDD.

The crystal structure of the active domain of Anopheles anti-platelet protein, a powerful anti-coagulant, in complex with an antibody

Background: Naturally occurring anticoagulant proteins provide models for new medications with highly desirable properties. Results: The crystal structure of the active region of mosquito protein AAPP has been solved. Conclusion: The mosquito protein AAPP uses a small turn region to block coagulation extremely effectively by binding collagen. Significance: New small molecule anti-coagulants may be developed with completely new mechanisms and none of the drawbacks of current treatments. Blood clotting is a vitally important process that must be carefully regulated to prevent blood loss on one hand and thrombosis on the other. Severe injury and hemophilia may be treated with pro-coagulants, whereas risk of obstructive clotting or embolism may be reduced with anti-coagulants. Anti-coagulants are an extremely important class of drug, one of the most widely used types of medication, but there remains a pressing need for novel treatments, however, as present drugs such as warfarin have significant drawbacks. Nature provides a number of examples of anti-coagulant proteins produced by blood-sucking animals, which may provide templates for the development of new small molecules with similar physiological effects. We have, therefore, studied an Anopheles anti-platelet protein from a malaria vector mosquito and report its crystal structure in complex with an antibody. Overall the protein is extremely sensitive to proteolysis, but the crystal structure reveals a stable domain built from two helices and a turn, which corresponds to the functional region. The antibody raised against Anopheles anti-platelet protein prevents it from binding collagen. Our work, therefore, opens new avenues to the development of both novel small molecule anti-clotting agents and anti-malarials.

Homopiperazine derivatives as a novel class of proteasome inhibitors with a unique mode of proteasome binding.

The proteasome is a proteolytic machinery that executes the degradation of polyubiquitinated proteins to maintain cellular homeostasis. Proteasome inhibition is a unique and effective way to kill cancer cells because they are sensitive to proteotoxic stress. Indeed, the proteasome inhibitor bortezomib is now indispensable for the treatment of multiple myeloma and other intractable malignancies, but is associated with patient inconvenience due to intravenous injection and emerging drug resistance. To resolve these problems, we attempted to develop orally bioavailable proteasome inhibitors with distinct mechanisms of action and identified homopiperazine derivatives (HPDs) as promising candidates. Biochemical and crystallographic studies revealed that some HPDs inhibit all three catalytic subunits (ß 1, ß 2 and ß 5) of the proteasome by direct binding, whereas bortezomib and other proteasome inhibitors mainly act on the ß5 subunit. Proteasome-inhibitory HPDs exhibited cytotoxic effects on cell lines from various hematological malignancies including myeloma. Furthermore, K-7174, one of the HPDs, was able to inhibit the growth of bortezomib-resistant myeloma cells carrying a ß5-subunit mutation. Finally, K-7174 had additive effects with bortezomib on proteasome inhibition and apoptosis induction in myeloma cells. Taken together, HPDs could be a new class of proteasome inhibitors, which compensate for the weak points of conventional ones and overcome the resistance to bortezomib.

G196 epitope tag system: a novel monoclonal antibody, G196, recognizes the small, soluble peptide DLVPR with high affinity.

The recognition specificity of monoclonal antibodies (mAbs) has made mAbs among the most frequently used tools in both basic science research and in clinical diagnosis and therapies. Precise determination of the epitope allows the development of epitope tag systems to be used with recombinant proteins for various purposes. Here we describe a new family of tag derived from the epitope recognized by a highly specific mAb G196. The minimal epitope was identified as the five amino acid sequence Asp-Leu-Val-Pro-Arg. Permutation analysis was used to characterize the binding requirements of mAb G196, and the variable regions of the mAb G196 were identified and structurally analyzed by X-ray crystallography. Isothermal titration calorimetry revealed the high affinity (Kd = 1.25 nM) of the mAb G196/G196-epitope peptide interaction, and G196-tag was used to detect several recombinant cytosolic and nuclear proteins in human and yeast cells. mAb G196 is valuable for developing a new peptide tagging system for cell biology and biochemistry research.

Crystallization and preliminary crystallographic studies of the butyrolactone autoregulator receptor protein (BarA) from Streptomyces virginiae

The Streptomyces butyrolactone autoregulator receptor protein (BarA) is a DNA-binding protein that regulates the biosynthesis of the antibiotic virginiamycin. In this study, BarA from S. virginiae was overexpressed in Escherichia coli, purified and crystallized. Crystals of purified protein have been grown that diffracted to beyond 3.0 A resolution at 100 K using synchrotron radiation. The protein crystals belonged to the hexagonal space group P6(5)22, with unit-cell parameters a = b = 128.0, c = 286.2 A. With four molecules per asymmetric unit, the crystal volume per unit protein mass (V(M)) was 3.2 A(3) Da(-1) and the solvent content was 62%.