Miki Senda

Relationship between the structure of SET/TAF-Iβ/INHAT and its histone chaperone activity

Histone chaperones assemble and disassemble nucleosomes in an ATP-independent manner and thus regulate the most fundamental step in the alteration of chromatin structure. The molecular mechanisms underlying histone chaperone activity remain unclear. To gain insights into these mechanisms, we solved the crystal structure of the functional domain of SET/TAF-Iβ/INHAT at a resolution of 2.3 Å. We found that SET/TAF-Iβ/INHAT formed a dimer that assumed a “headphone”-like structure. Each subunit of the SET/TAF-Iβ/INHAT dimer consisted of an N terminus, a backbone helix, and an “earmuff” domain. It resembles the structure of the related protein NAP-1. Comparison of the crystal structures of SET/TAF-Iβ/INHAT and NAP-1 revealed that the two proteins were folded similarly except for an inserted helix. However, their backbone helices were shaped differently, and the relative dispositions of the backbone helix and the earmuff domain between the two proteins differed by ≈40°. Our biochemical analyses of mutants revealed that the region of SET/TAF-Iβ/INHAT that is engaged in histone chaperone activity is the bottom surface of the earmuff domain, because this surface bound both core histones and double-stranded DNA. This overlap or closeness of the activity surface and the binding surfaces suggests that the specific association among SET/TAF-Iβ/INHAT, core histones, and double-stranded DNA is requisite for histone chaperone activity. These findings provide insights into the possible mechanisms by which histone chaperones assemble and disassemble nucleosome structures.

Crystal structure of a zinc-dependent D-serine dehydratase from chicken kidney

d-Serine is a physiological co-agonist of the N-methyl-d-aspartate receptor. It regulates excitatory neurotransmission, which is important for higher brain functions in vertebrates. In mammalian brains, d-amino acid oxidase degrades d-serine. However, we have found recently that in chicken brains the oxidase is not expressed and instead a d-serine dehydratase degrades d-serine. The primary structure of the enzyme shows significant similarities to those of metal-activated d-threonine aldolases, which are fold-type III pyridoxal 5′-phosphate (PLP)-dependent enzymes, suggesting that it is a novel class of d-serine dehydratase. In the present study, we characterized the chicken enzyme biochemically and also by x-ray crystallography. The enzyme activity on d-serine decreased 20-fold by EDTA treatment and recovered nearly completely by the addition of Zn2+. None of the reaction products that would be expected from side reactions of the PLP-d-serine Schiff base were detected during the >6000 catalytic cycles of dehydration, indicating high reaction specificity. We have determined the first crystal structure of the d-serine dehydratase at 1.9 Å resolution. In the active site pocket, a zinc ion that coordinates His347 and Cys349 is located near the PLP-Lys45 Schiff base. A theoretical model of the enzyme-d-serine complex suggested that the hydroxyl group of d-serine directly coordinates the zinc ion, and that the ϵ-NH2 group of Lys45 is a short distance from the substrate Cα atom. The α-proton abstraction from d-serine by Lys45 and the elimination of the hydroxyl group seem to occur with the assistance of the zinc ion, resulting in the strict reaction specificity.

Direct observation of protein microcrystals in crystallization buffer by atmospheric scanning electron microscopy.

X-ray crystallography requires high quality crystals above a given size. This requirement not only limits the proteins to be analyzed, but also reduces the speed of the structure determination. Indeed, the tertiary structures of many physiologically important proteins remain elusive because of the so-called “crystallization bottleneck”. Once microcrystals have been obtained, crystallization conditions can be optimized to produce bigger and better crystals. However, the identification of microcrystals can be difficult due to the resolution limit of optical microscopy. Electron microscopy has sometimes been utilized instead, with the disadvantage that the microcrystals usually must be observed in vacuum, which precludes the usage for crystal screening. The atmospheric scanning electron microscope (ASEM) allows samples to be observed in solution. Here, we report the use of this instrument in combination with a special thin-membrane dish with a crystallization well. It was possible to observe protein crystals of lysozyme, lipase B and a histone chaperone TAF-Iβ in crystallization buffers, without the use of staining procedures. The smallest crystals observed with ASEM were a few μm in width, and ASEM can be used with non-transparent solutions. Furthermore, the growth of salt crystals could be monitored in the ASEM, and the difference in contrast between salt and protein crystals made it easy to distinguish between these two types of microcrystals. These results indicate that the ASEM could be an important new tool for the screening of protein microcrystals.

The Lipid Kinase PI5P4Kβ Is an Intracellular GTP Sensor for Metabolism and Tumorigenesis.

While cellular GTP concentration dramatically changes in response to an organisms cellular status, whether it serves as a metabolic cue for biological signaling remains elusive due to the lack of molecular identification of GTP sensors. Here we report that PI5P4Kβ, a phosphoinositide kinase that regulates PI(5)P levels, detects GTP concentration and converts them into lipid second messenger signaling. Biochemical analyses show that PI5P4Kβ preferentially utilizes GTP, rather than ATP, for PI(5)P phosphorylation, and its activity reflects changes in direct proportion to the physiological GTP concentration. Structural and biological analyses reveal that the GTP-sensing activity of PI5P4Kβ is critical for metabolic adaptation and tumorigenesis. These results demonstrate that PI5P4Kβ is the missing GTP sensor and that GTP concentration functions as a metabolic cue via PI5P4Kβ. The critical role of the GTP-sensing activity of PI5P4Kβ in cancer signifies this lipid kinase as a cancer therapeutic target.

Molecular Mechanism of Strict Substrate Specificity of an Extradiol Dioxygenase, DesB, Derived from Sphingobium sp. SYK-6

DesB, which is derived from Sphingobium sp. SYK-6, is a type II extradiol dioxygenase that catalyzes a ring opening reaction of gallate. While typical extradiol dioxygenases show broad substrate specificity, DesB has strict substrate specificity for gallate. The substrate specificity of DesB seems to be required for the efficient growth of S. sp. SYK-6 using lignin-derived aromatic compounds. Since direct coordination of hydroxyl groups of the substrate to the non-heme iron in the active site is a critical step for the catalytic reaction of the extradiol dioxygenases, the mechanism of the substrate recognition and coordination of DesB was analyzed by biochemical and crystallographic methods. Our study demonstrated that the direct coordination between the non-heme iron and hydroxyl groups of the substrate requires a large shift of the Fe (II) ion in the active site. Mutational analysis revealed that His124 and His192 in the active site are essential to the catalytic reaction of DesB. His124, which interacts with OH (4) of the bound gallate, seems to contribute to proper positioning of the substrate in the active site. His192, which is located close to OH (3) of the gallate, is likely to serve as the catalytic base. Glu377’ interacts with OH (5) of the gallate and seems to play a critical role in the substrate specificity. Our biochemical and structural study showed the substrate recognition and catalytic mechanisms of DesB.

Crystal structure of Methanococcus jannaschii TATA box-binding protein.

As the archaeal transcription system consists of a eukaryotic‐type transcription apparatus and bacterial‐type regulatory transcription factors, analyses of the molecular interface between the transcription apparatus and regulatory transcription factors are critical to reveal the evolutionary change of the transcription system. TATA box‐binding protein (TBP), the central components of the transcription apparatus are classified into three groups: eukaryotic, archaeal‐I and archaeal‐II TBPs. Thus, comparative functional analysis of these three groups of TBP is important for the study of the evolution of the transcription system. Here, we present the first crystal structure of an archaeal‐II TBP from Methanococcus jannaschii. The highly conserved and group‐specific conserved surfaces of TBP bind to DNA and TFIIB/TFB, respectively. The phylogenetic trees of TBP and TFIIB/TFB revealed that they evolved in a coupled manner. The diversified surface of TBP is negatively charged in the archaeal‐II TBP, which is completely different from the case of eukaryotic and archaeal‐I TBPs, which are positively charged and biphasic, respectively. This difference is responsible for the diversification of the regulatory functions of TBP during evolution.

Crystallization and preliminary X‐ray analysis of the reduced Rieske‐type [2Fe–2S] ferredoxin derived from Pseudomonas sp. strain KKS102

The reduced form of BphA3, a Rieske-type [2Fe-2S] ferredoxin component of the biphenyl dioxygenase BphA from Pseudomonas sp. strain KKS102, was crystallized by the sitting-drop vapour-diffusion method under anaerobic conditions. The crystal belongs to space group P3(1)21, with unit-cell parameters a = b = 49.6, c = 171.9 A, and diffracts to a resolution of 1.95 A. A molecular-replacement calculation using oxidized BphA3 as a search model yielded a satisfactory solution.

Crystallization and preliminary X-ray analysis of the Rieske-type [2Fe-2S] ferredoxin component of biphenyl dioxygenase from Pseudomonas sp. strain KKS102.

BphA3, a Rieske-type [2Fe-2S] ferredoxin component of a biphenyl dioxygenase (BphA) from Pseudomonas sp. strain KKS102, was crystallized by the hanging-drop vapour-diffusion method. Two crystal forms were obtained from the same conditions. The form I crystal belongs to space group P2(1)2(1)2, with unit-cell parameters a = 26.3, b = 144.3, c = 61.5 A, and diffracted to 2.45 A resolution. The form II crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 26.2, b = 121.3, c = 142.7 A, and diffracted to 2.8 A resolution. A molecular-replacement calculation using BphF as a search model yielded a satisfactory solution for both forms.

Structural Basis of the γ-Lactone-Ring Formation in Ascorbic Acid Biosynthesis by the Senescence Marker Protein-30/Gluconolactonase

The senescence marker protein-30 (SMP30), which is also called regucalcin, exhibits gluconolactonase (GNL) activity. Biochemical and biological analyses revealed that SMP30/GNL catalyzes formation of the γ-lactone-ring of l-gulonate in the ascorbic acid biosynthesis pathway. The molecular basis of the γ-lactone formation, however, remains elusive due to the lack of structural information on SMP30/GNL in complex with its substrate. Here, we report the crystal structures of mouse SMP30/GNL and its complex with xylitol, a substrate analogue, and those with 1,5-anhydro-d-glucitol and d-glucose, product analogues. Comparison of the crystal structure of mouse SMP30/GNL with other related enzymes has revealed unique characteristics of mouse SMP30/GNL. First, the substrate-binding pocket of mouse SMP30/GNL is designed to specifically recognize monosaccharide molecules. The divalent metal ion in the active site and polar residues lining the substrate-binding cavity interact with hydroxyl groups of substrate/product analogues. Second, in mouse SMP30/GNL, a lid loop covering the substrate-binding cavity seems to hamper the binding of l-gulonate in an extended (or all-trans) conformation; l-gulonate seems to bind to the active site in a folded conformation. In contrast, the substrate-binding cavities of the other related enzymes are open to the solvent and do not have a cover. This structural feature of mouse SMP30/GNL seems to facilitate the γ-lactone-ring formation.

Structural reverse genetics study of the PI5P4Kβ-nucleotide complexes reveals the presence of the GTP bioenergetic system in mammalian cells.

Reverse genetic analysis can connect a gene and its protein counterpart to a biological function(s) by knockout or knockdown of the specific gene. However, when a protein has multiple biochemical activities, the conventional genetics strategy is incapable of distinguishing which biochemical activity of the protein is critical for the particular biological function(s). Here, we propose a structural reverse genetics strategy to overcome this problem. In a structural reverse genetics study, multiple biochemical activities of a protein are segregated by mapping those activities to a structural element(s) in the atomic resolution tertiary structure. Based on the structural mapping, a mutant lacking one biochemical activity of interest can be produced with the other activities kept intact. Expression of the mutant by knockin or ectopic expression in the knockout strain along with the following analysis can connect the single biochemical activity of interest to a biological function. Using the structural reverse genetics strategy, we have dissected the newly identified GTP‐dependent activity of a lipid kinase PI5P4Kβ from its ATP‐dependent activity. The GTP‐insensitive mutant has demonstrated the existence of the GTP bioenergetic sensor system in mammalian cells and its critical role in tumorigenesis. As structural reverse genetics can identify in vivo significance of individual biochemical activity, it is a powerful approach to reveal hidden biological functions, which could be a novel pharmacological target for therapeutic intervention. Given the recent expansion of choices in structural biological methods and advances in genome editing technologies, the time is ripe for structural reverse genetics strategies.