Issue Highlights

[Mini review]

•Structural insights on ATP hydrolysis-driven mechanical work of AAA+ hexamers

Changwon Kim 1,2,4 , Sang-Hyun Rah 1,2,3,4 and Tae-Young Yoon 1,2,4

1 Center for Nanomedicine, Institute for Basic Science (IBS), Yonsei University, Seoul 03722, Korea, 2 Yonsei-IBS Institute, Yonsei University, Seoul 03722, Korea, 3 Department of Physics, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, South Korea, 4 present address : School of Biological Sciences, Seoul National University, Seoul 08826, Korea

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  • [Mini review] Structural insights on ATP hydrolysis-driven mechanical work of AAA+ hexamers

    N-ethylmaleimide-sensitive factor (NSF) and ClpX are homo-hexameric proteins of the AAA+ (ATPases Associated with diverse cellular Activities) family. Using ATP, NSF recycles SNARE complexes following membrane fusion, while ClpX unfolds and translocates proteins through its pore. However, their molecular mechanisms were unclear until recently. NSF efficiently disassembles a SNARE complex using ATP that were bound before SNARE binding, by changing from a ‘split washer’ to a ‘flat washer’ conformation. ClpX utilizes numerous ATP binding and hydrolyses for translocation. Structural studies of ClpX show that two of the six ATP sites are unloadable. Hence, in ClpX, it is believed ATP hydrolyses occur in pairs and in symmetric motifs to work. Overall, NSF follows a spring loaded model, while ClpX follows a power-stroke model – showing that even for proteins that belong to the same family and that have similar structures, functions and models of action can be very different.

  • [Technical Report] Structure determination of the C-terminal fragment of yeast Ski7 using twinned crystal data

    The structure determination using twinned crystals is challenging although several algorithms have been developed for detwinning the X-ray data. Our crystal of the C-terminal domain 2 and 3 of Ski7 (Ski7-D2/3), a key part of non-stop mRNA decay has a perfect twin with the twin operator [h, -h-k, -l]. Many different efforts for phasing with multiple anomalous dispersion techniques using selenomethionine substituted wild-type and mutant proteins were not successful and the phases were obtained through the molecular replacement method using recently reported structure of C-terminal GTPase domain of Ski7 from Saccharomyces cerevisiae. The overall structure of Ski7-D2/3 is very similar to that of the corresponding domain of ribosome-associated GTPases including eIF5B, eEF1α, and eRF3. Domains 2 and 3 form a β-barrel structure containing several structurally deviated long connecting loops. Although the linker between domain 2 and 3 is very flexible, the relative orientation between them is virtually the same among all structures, showing that the Ski7-D2/3 does not show major conformational movement upon contacting with G domain.

  • [Resource] Small-angle X-ray Scattering Beamline BL4C SAXS at Pohang Light Source II

    BL4C SAXS at the Pohang Light Source II is a small-angle X-ray scattering beamline based on an in-vacuum undulator insertion device, Si(111) DCM, and toroidal focusing mirror. The beamline normally provides high-flux synchrotron radiation X-ray sources with energies from 10.3 to 20.6 keV and a 100 µm (vertical) × 300 µm (horizontal) full width at half-maximum focal spot. The analysis of the SAXS data would be facilitated by means of useful ancillary equipment. The design of the beamline, the key components, and its role are described.

  • [Resource] The Protein Crystallography Beamlines at the Pohang Light Source II

    Pohang Light Source II (PLS-II) is a 3.0 GeV national synchrotron radiation facility in Korea. Three protein crystallography beamlines at PLS-II are available for national and international users in the structural biology community. All three beamlines (BL-5C, BL-7A and BL-11C) are illuminated with an in-vacuum undulator source, which provides a tunable high brilliant X-ray. BL-5C and BL-7A are designed for routine protein crystallography with a default beam size of 100 µm, while BL-11C is designed for micro-crystallography with a default beam size of 10 μm. This report summarizes the primary parameters of the three beamlines, and describes user productivity and recent experimental results.

  • [Crystallization] Purification, crystallization and X-ray crystallographic analysis of cystathionine gamma-synthase from Corynebacterium glutamicum

    Cystathionine gamma synthase from Corynebacterium glutamicum (CgMetB) is a key enzyme for the production of L-methionine and it condenses O-acetyl-L-homoserine (OAHS) and cysteine to produce cystathionine. MetB is also an attractive target for the development of antimicrobial compounds because it catalyzes the first reaction of the L-methionine biosynthetic pathway. The CgMetB was overexpressed and purified to homogeneity by affinity and size-exclusion chromatography. The CgMetB protein was crystallized using hanging-drop vapor-diffusion method in the presence of 13% polyethylene glycol 3350 and 0.1 M Magnesium formate dihydrate at 295 K. X-ray diffraction data were collected to a maximum resolution of 1.5 Å. The crystal belonged to space group F222, with unit cell parameters a = 58.57 Å, b = 149.85 Å, c = 161.86, α = β = γ = 90.0°. With one molecules per asymmetric unit, the crystal volume per unit protein mass was 2.13 Å 3 Da -1 , which correspond to a solvent content of approximately 42.27%.

  • [Crystallization] Crystallization and preliminary crystallographic analysis of IlvC, a ketol-acid reductoisomerase, from Streptococcus pneumoniae

    IlvC, a ketol-acid reductoisomerase, plays a critical role in alkyl migration and catalyzes the second step in the biosynthesis of branched amino acids such as leucine, valine and isoleucine. As an initial step to investigate whether IlvC is involved in pneumococcal growth and virulence from the structural background, ilvC from Streptococcus pneumoniae D39 (SpIlvC) was cloned and overexpressed in Escherichia coli. Crystals of SpIlvC were obtained by hanging-drop vapour diffusion in 0.1 M HEPES pH 7.5, 0.1 M NaCl, 1.5 M ammonium sulfate and diffracted to 1.69 Å resolution. The SpIlvC crystal belonged to space group P2 1 2 1 2 1 with unit cell parameters a = 69.1°, b = 104.3°, c = 110.9° and contained two molecules in the asymmetric unit.

  • [Crystallization] Purification, crystallization, and preliminary X-ray crystallographic analysis of VV2_1132, a LysR-type transcriptional regulator from Vibrio vulnificus

    LysR-type transcriptional regulators (LTTRs) belong to the largest family of transcriptional regulators in prokaryotes. However, the crystal structures of only a few full-length LTTRs have been determined. LTTR HypT (also known as YjiE or QseD) from Escherichia coli specifically senses hypochlorite to protect oxidation damage by hypochlorite generated by the host immune system. In the genome of the highly virulent bacteria, Vibrio vulnificus, VV2_1132 showed the highest sequence similarity to E. coli HypT. In this study, we overexpressed full-length VV2_1132 in an E. coli expression system and crystallized the protein. The crystal diffracted X-rays to 2.2 Å resolution and belonged to the orthogonal space group P2 1 2 1 2, with unit cell parameters a = 57.8, b = 113.5, and c = 220.7 Å. Cell content analysis predicted that the asymmetric unit may contain four molecules of VV2_1132. For the case of four molecules in the asymmetric unit, the Matthews coefficient was 2.70 Å 3 /Da with a solvent content of 54.5%. We are currently identifying the crystal structure of VV2_1132 using anomalous signals from selenomethione-substituted crystals. This crystal structure will help elucidate the function and action mechanism of VV2_1132, which may be involved in the pathogenesis of V. vulnificus.

  • [Crystallization] Expression, crystallization, and preliminary X-ray crystallographic analysis of peptide deformylase from Acinetobacter baumanii

    The emergences of multi-drug resistant bacteria such as Acinetobacter baumanni have emphasized the necessity of new antibiotics. Peptidyl deformylase (PDF) catalyzes the removal of the formyl group from the N-terminal formylated methionine residue present in all nascent polypeptides in bacteria. In this study, the PDF gene from Acinetobacter baumannii K0420859 was cloned and its protein was overexpressed in E. coli, purified, and crystallized. The purified protein was crystallized using the hanging-drop vapour-diffusion method and the crystal diffracted to 2.4 Å resolution. The crystal belonged to the trigonal space group P3 2 with unit cell parameters of a = b= 39.4 Å and c = 187.9 Å. Two protomers were presented in the asymmetric unit with a corresponding V M of 2.10 Å 3 Da -1 and a solvent content of 41.5%.