soo Chang

Crystal structure of NAD(+)-dependent DNA ligase: modular architecture and functional implications.

DNA ligases catalyze the crucial step of joining the breaks in duplex DNA during DNA replication, repair and recombination, utilizing either ATP or NAD+ as a cofactor. Despite the difference in cofactor specificity and limited overall sequence similarity, the two classes of DNA ligase share basically the same catalytic mechanism. In this study, the crystal structure of an NAD+‐dependent DNA ligase from Thermus filiformis, a 667 residue multidomain protein, has been determined by the multiwavelength anomalous diffraction (MAD) method. It reveals highly modular architecture and a unique circular arrangement of its four distinct domains. It also provides clues for protein flexibility and DNA‐binding sites. A model for the multidomain ligase action involving large conformational changes is proposed.

Crystal structure of human nucleoside diphosphate kinase A, a metastasis suppressor.

Introduction. Nucleoside diphosphate kinase (NDK) catalyzes the transfer of the g-phosphoryl group from a nucleoside triphosphate (NTP) to a nucleoside diphosphate (NDP) by using ATP as a major phosphate donor. During the catalytic reaction, the enzyme is transiently phosphorylated on a conserved histidine residue. NDP kinases play a primary role in maintaining cellular pools of all NTPs but also in the regulation of important cellular processes. In humans, the two isoforms NDK-A (NM23-H1) and NDK-B (NM23-H2) are closely related in amino acid sequence (88% identity) but display significant differences in cellular functions. NDK-A acts as a metastasis suppressor for some tumor types. NDK-B, also known as PuF, binds to the promoter of the c-myc oncogene and activates its transcription. These cellular functions of the two isoforms are independent of their NDP kinase activity, and two isoforms can form homoand heterohexamers, resulting in different ratios of the respective subunits. We present here the crystal structure of human NDK-A determined at 2.2 Å resolution. This enables a detailed structural comparison of NDK-A with NDK-B, contributing to understanding the difference in their cellular functions.

Crystal structure of a truncated version of the phage λ protein gpD

Introduction. Bacteriophage contains a linear doublestranded DNA genome enclosed within an icosahedral capsid of triangulation number T 7, and a flexible, non-contractile, tail. It has been adapted for phage display, providing a valuable alternative to filamentous phages. As a lytic phage, the phage coat is directly assembled in the cytoplasm, and does not pass through the secretion machinery and the oxidizing periplasm, thereby providing a display system for proteins not suitable for secretion. Both the head protein, gpD, and the tail protein, gpV, have been used as fusion partners in applications of phage display. To further develop the technology of phage display, the structures of the proteins utilized in this technique are of considerable interest. The high-resolution crystal structure of gpD, the capsid-stabilizing protein of bacteriophage , which sits as protrusions at trigonal sites, has been solved previously. It provides a structural basis for the understanding of how gpD could play the role of a display platform. Molecules of gpD were found to be arranged in noncrystallographic trimers with substantial inter-subunit interfaces. Their C termini are well ordered and located on one side of the trimer, relatively far from its threefold axis. By contrast, the N termini are disordered up to Ser15, which is close to the threefold axis and on the same side as the C termini. Even though these 14 N-terminal residues are disordered, they appear to have an important function, since an N-terminal truncated version of gpD (gpD N1) does not complement a phage mutant devoid of gpD. Both gpD and gpD N1 are monomeric in solution, and gpD trimerizes on the phage and upon crystallization. It was, therefore, of interest to characterize gpD N1 more closely. While gpD N1 contains all visible atoms of the gpD trimer, it was conceivable that the N-terminal stretch might play an important role in trimer formation, which might occur concomitant with binding to the phage procoat. Furthermore, Pro17 and His19 form a characteristic ring in the trimer, and this might be compromised when the first 14 residues are absent. Finally, we considered that if crystals of gpD N1 isomorphous to those of the full-length protein could be obtained, comparison of the low-resolution diffraction of both forms might conceivably elucidate the location of the N terminus of the molecule, even if disordered, by applying an approach similar to the X-ray contrast variation used to outline the protein molecules. We thus solved the structure of gpD N1 at the resolution of 1.85 Å. Despite having crystallized in a larger cell with two trimers in the asymmetric unit, the structure of this truncated version of gpD is virtually identical to the structure of the unmodified protein.

Nucleoside diphosphate kinase from the hyperthermophilic archaeon Methanococcus jannaschii: overexpression, crystallization and preliminary X-ray crystallographic analysis

Nucleoside diphosphate (NDP) kinase is a key enzyme in maintaining cellular pools of all nucleoside triphosphates. NDP kinase from the hyperthermophilic archaebacterium Methanococcus jannaschii has been overexpressed in Escherichia coli and crystallized at 297 K using polyethylene glycol 4000 as precipitant. The crystal is hexagonal, belonging to the space group P6(3), with unit-cell parameters a = b = 72.89, c = 100.87 A. The asymmetric unit contains two subunits of NDP kinase, with a corresponding crystal volume per protein mass (V(M)) of 2.38 A(3) Da(-1) and a solvent content of 48.3%. Native X-ray diffraction data to 2.30 A resolution have been collected using synchrotron X-rays.

Lactate dehydrogenase from the hyperthermophilic archaeon Methanococcus jannaschii: overexpression, crystallization and preliminary X-ray analysis

L(+)-Lactate dehydrogenase (LDH) is a key enzyme in anaerobic metabolism which converts pyruvate to lactate. LDH from the hyperthermophilic archaebacterium Methanococcus jannaschii has been overexpressed in Escherichia coli and crystallized in two crystal forms at 297 K using 2-methyl-2,4-pentanediol as precipitant. Type I crystals grew rapidly and diffracted to at least 2.8 A Bragg spacing upon exposure to Cu Kalpha X-rays. X-ray diffraction data to 2.9 A have been collected from a native crystal. The type I crystal is tetragonal, belonging to the space group P4(2)2(1)2, with unit-cell parameters a = b = 99.74, c = 170.00 A. The asymmetric unit contains two LDH subunits, with a corresponding crystal volume per protein mass (V(m)) of 3.05 A(3) Da(-1) and a solvent content of 59.7%. Type II crystals, which grew more slowly, diffracted to at least 1.8 A Bragg spacing upon exposure to Cu Kalpha X-rays. X-ray diffraction data to 1.9 A have been collected from a native crystal. The type II crystal is orthorhombic, belonging to the space group P2(1)2(1)2, with unit-cell parameters a = 47.65, b = 125.10, c = 58.08 A. The asymmetric unit contains a single LDH subunit, with a corresponding crystal volume per protein mass (V(m)) of 2.50 A(3) Da(-1) and a solvent content of 50.8%. Therefore, the type II crystal is more suitable for high-resolution structure determination than the type I crystal.

A thermostable xylose isomerase from Thermus caldophilus: Biochemical characterization, crystallization and preliminary x-ray analysis

A highly thermostable xylose isomerase from Thermus caldophilus has been expressed in Escherichia coli. The purified enzyme has an optimum temperature of 363 K. It has been crystallized at room temperature using ammonium sulfate as a precipitant. The crystal belongs to the orthorhombic space group P212121, with unit-cell parameters a = 84.35, b = 123.60, c = 140.24 A. The presence of one molecule of tetrameric xylose isomerase in the asymmetric unit gives a crystal volume per protein mass (Vm) of 2.1 A3 Da-1 and a solvent content of 41% by volume. The crystals initially showed diffraction to 1.7 A Bragg spacing with synchrotron X-rays, and a set of native data extending to 2.3 A resolution has been collected.

Crystallization and preliminary X-ray crystallographic analysis of human nucleoside diphosphate kinase A

Human nucleoside diphosphate kinase A catalyzes phosphoryl transfer and acts as a suppressor of metastasis. It has been crystallized using 2-methyl-2,4-pentanediol as a precipitant at 288 K. The crystal is monoclinic, belonging to the space group P2(1), with unit-cell parameters a = 74.21, b = 78.11, c = 82.29 A, beta = 101. 33 degrees. The asymmetric unit contains a homohexamer, with a corresponding crystal volume per protein mass (V(m)) of 2.27 A(3) Da(-1) and a solvent content of 46%. Native X-ray data to 2.15 A resolution have been collected using synchrotron X-rays.

Crystallization and preliminary x-ray crystallographic analysis of NAD+-dependent DNA ligase from Thermus filiformis.

A highly thermostable DNA ligase from Thermus filiformis has been crystallized at room temperature using methoxypolyethylene glycol 5000 as a precipitant. The crystal belongs to the monoclinic space group P2(1), with unit-cell parameters a = 90.63, b = 117.80, c = 98. 65 A, beta = 115.56 degrees. Two molecules of DNA ligase are present in the asymmetric unit, giving a crystal volume per protein mass (V(m)) of 3.1 A(3) Da(-1) and a solvent content of 61%. A native data set extending to 3.0 A resolution has been collected at 100 K using synchrotron X-rays.

Crystallization and preliminary X-ray crystallographic analysis of deoxycytidylate hydroxymethylase from bacteriophage T4.

Deoxycytidylate hydroxymethylase from bacteriophage T4 is a homodimeric enzyme in which each polypeptide chain consists of 246 amino-acid residues. It has been crystallized in the presence of its substrate, deoxycytidine monophosphate, at room temperature using sodium citrate as precipitant. The crystals are monoclinic, belonging to space group C2, with unit-cell parameters a = 174.22, b = 53.12, c = 75.17 A, beta = 115.29 degrees. The asymmetric unit contains one homodimer, with a corresponding Vm of 2.65 A3 Da-1 and solvent content of 54%. Native diffraction data to 1.6 A resolution have been collected from two crystals using synchrotron radiation.

Crystallization and preliminary X-ray analysis of the Mj0684 gene product, a putative aspartate aminotransferase, from Methanococcus jannaschii.

A putative aspartate aminotransferase from the hyperthermophilic archaeon Methanococcus jannaschii encoded by the Mj0684 gene has been overexpressed in Escherichia coli and crystallized at 296 K using the sitting-drop vapour-diffusion method. The crystals belong to space group P4(1)2(1)2 (or P4(3)2(1)2), with unit-cell parameters a = b = 111.87, c = 60.86 A. They diffract to 2.2 A resolution using Cu Kalpha X-rays. The asymmetric unit contains a single subunit of the recombinant Mj0684 gene product, giving a corresponding V(M) of 2.25 A(3) Da(-1) and a solvent content of 45.3% by Volume. An X-ray diffraction data set has been collected to 2.2 A at 295 K.