Jin Ho Moon

Structural studies of human brain-type creatine kinase complexed with the ADP-Mg2+-NO3- -creatine transition-state analogue complex

Creatine kinase is a member of the phosphagen kinase family, which catalyzes the reversible phosphoryl transfer reaction that occurs between ATP and creatine to produce ADP and phosphocreatine. Here, three structural aspects of human‐brain‐type‐creatine‐kinase (hBB‐CK) were identified by X‐ray crystallography: the ligand‐free‐form at 2.2 Å; the ADP–Mg2+, nitrate, and creatine complex (transition‐state‐analogue complex; TSAC); and the ADP–Mg2+‐complex at 2.0 Å. The structures of ligand‐bound hBB‐CK revealed two different monomeric states in a single homodimer. One monomer is a closed form, either bound to TSAC or the ADP–Mg2+‐complex, and the second monomer is an unliganded open form. These structural studies provide a detailed mechanism indicating that the binding of ADP–Mg2+ alone may trigger conformational changes in hBB‐CK that were not observed with muscle‐type‐CK.

Crystal structure of the response regulator spr1814 from Streptococcus pneumoniae reveals unique interdomain contacts among NarL family proteins.

Spr1814 belongs to the NarL/FixJ subfamily of signal transduction response regulators (RR), and has been predicted to regulate the neighboring ABC transporter, which translocates antibiotic molecules in Streptococcus pneumoniae. Here, we report the crystal structure of full-length unphosphorylated spr1814 at 1.7Å resolution. The asymmetric unit contains two spr1814 molecules, which display very different conformations. Through comparisons with other RRs structures, we concluded that one molecule adopts a general inactive conformation, whereas the other molecule adopts an intermediate conformation. The superposition of each molecule showed that rotational change of the effector domain occurred in intermediate conformational state, implying that domain rearrangement could occur upon phosphorylation.

Crystal structure of receiver domain of putative NarL family response regulator spr1814 from Streptococcus pneumoniae in the absence and presence of the phosphoryl analog beryllofluoride

Spr1814 of Streptococcus pneumoniae is a putative response regulator (RR) that has four-helix helix-turn-helix DNA-binding domain and belongs to the NarL family. The prototypical RR contains two domains, an N-terminal receiver domain linked to a variable effector domain. The receiver domain functions as a phosphorylation-activated switch and contains the typical doubly wound five-stranded α/β fold. Here, we report the crystal structure of the receiver domain of spr1814 (spr1814(R)) determined in the absence and presence of beryllofluoride as a phosphoryl analog. Based on the overall structure, spr1814(R) was shown to contain the typical fold similar with other structures of the receiver domain; however, an additional linker region connecting the receiver and DNA-binding domain was inserted into the dimer interface of spr1814(R), resulting in the formation of unique dimer interface. Upon phosphorylation, the conformational change of the linker region was observed and this suggests that domain rearrangement between the receiver domain and effector domain could occur in full-length spr1814.

Structural and Kinetic Analysis of Free Methionine-R-sulfoxide Reductase from Staphylococcus aureus CONFORMATIONAL CHANGES DURING CATALYSIS AND IMPLICATIONS FOR THE CATALYTIC AND INHIBITORY MECHANISMS

Free methionine-R-sulfoxide reductase (fRMsr) reduces free methionine R-sulfoxide back to methionine, but its catalytic mechanism is poorly understood. Here, we have determined the crystal structures of the reduced, substrate-bound, and oxidized forms of fRMsr from Staphylococcus aureus. Our structural and biochemical analyses suggest the catalytic mechanism of fRMsr in which Cys102 functions as the catalytic residue and Cys68 as the resolving Cys that forms a disulfide bond with Cys102. Cys78, previously thought to be a catalytic Cys, is a non-essential residue for catalytic function. Additionally, our structures provide insights into the enzyme-substrate interaction and the role of active site residues in substrate binding. Structural comparison reveals that conformational changes occur in the active site during catalysis, particularly in the loop of residues 97–106 containing the catalytic Cys102. We have also crystallized a complex between fRMsr and isopropyl alcohol, which acts as a competitive inhibitor for the enzyme. This isopropyl alcohol-bound structure helps us to understand the inhibitory mechanism of fRMsr. Our structural and enzymatic analyses suggest that a branched methyl group in alcohol seems important for competitive inhibition of the fRMsr due to its ability to bind to the active site.

Crystallization and preliminary X-ray crystallographic studies of the ice-binding protein from the Arctic [correction of Aantarctic] yeast Leucosporidium sp. AY30.

Freezing is dangerous to cellular organisms because it causes an increase in the concentration of ions and other solutes in the plasma, denatures biomolecules and ruptures cell membranes. Some cold-adapted organisms can survive at subzero temperatures by producing proteins that bind to and inhibit the growth of ice crystals. To better understand the structure and function of these proteins, the ice-binding protein from Leucosporidium sp. AY30 (LeIBP) was overexpressed, purified and crystallized. The native crystal belonged to space group P4(3)2(1)2, with unit-cell parameters a=b=98.05, c=106.13 Å. Since LeIBP lacks any cysteine or methionine residues, two leucine residues (Leu69 and Leu155) were substituted by methionine residues in order to obtain selenomethionine-substituted LeIBP for use in multiple-wavelength anomalous diffraction (MAD) phasing. The selenomethionine-substituted mutant crystallized in the same space group as the native protein.

Crystallization and preliminary X‐ray crystallographic studies of the ice‐binding protein from the Antarctic yeast Leucosporidium sp. AY30. Corrigendum

A correction to the article by Park et al. [(2011). Acta Cryst. F67, 800–802].

The structure of a shellfish specific GST class glutathione S‐transferase from antarctic bivalve Laternula elliptica reveals novel active site architecture

Glutathione‐S‐transferases have been identified in all the living species examined so far, yet little is known about their function in marine organisms. In a previous report, the recently identified GST from Antarctic bivalve Laternula elliptica (LeGST) was classified into the rho class GST, but there are several unique features of LeGST that may justify reclassification, which could represent specific shellfish GSTs. Here, we determined the crystal structure of LeGST, which is a shellfish specific class of GST. The structural analysis showed that the relatively open and wide hydrophobic H‐site of the LeGST allows this GST to accommodate various substrates. These results suggest that the H‐site of LeGST may be the result of adaptation to their environments as sedentary organisms. Proteins 2013.

Crystallization and preliminary X-ray crystallographic studies of a new class of enoyl-(acyl-carrier protein) reductase, FabV, from Vibrio fischeri

Enoyl-(acyl-carrier protein) reductase (ENR) catalyzes the last step of the fatty-acid elongation cycle of the bacterial fatty-acid biosynthesis (FAS II) pathway. Recently, a new class of ENR has been identified from Vibrio cholerae and was named FabV. In order to understand the molecular mechanism of the new class of ENR at the structural level, FabV from V. fischeri was overexpressed, purified and crystallized. Diffraction data were collected to 2.7 Å resolution from a native crystal. The crystal belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 123.53, b = 164.14, c = 97.07 Å. The presence of four molecules of FabV in the asymmetric unit gave a V(M) value of 2.81 Å(3) Da(-1), with a corresponding solvent content of 54.5%.

Crystallization and preliminary X-ray crystallographic studies of DesR, a thermosensing response regulator in a two-component signalling system from Streptococcus pneumoniae

The response regulator DesR, which activates the transcription of the des gene by binding to a regulatory region, is essential for controlling the fluidity of membrane phospholipids. DesR from Streptococcus pneumoniae was overexpressed in Escherichia coli. The protein was purified and crystallized for structural analysis. Diffraction data were collected to 1.7 A resolution using synchrotron radiation and the crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 46.91, b = 71.38, c = 117.73 A. Assuming the presence of a dimer in the asymmetric unit, this corresponds to a V(M) of 2.21 A(3) Da(-1).

Crystal structure of a PduO-type ATP:cobalamin adenosyltransferase from Burkholderia thailandensis.

Crystal structure of a PduO-type ATP:cobalamin adenosyltransferase from Burkholderia thailandensis Jin HoMoon,1y Ae Kyung Park,2y Eun Hyuk Jang, Heenam Stanley Kim, and YoungMin Chi* 1 Institute of Life Science and Natural Resources, Korea University, Seoul 136-713, Korea 2Division of Biotechnology, College of Life Sciences, Korea University, Seoul 136-713, Korea 3Department of Medicine, College of Medicine, Korea University, Seoul 136-705, Korea