ABSTRACT
A Nif3 family protein of Methanocaldococcus jannaschii, MJ0927, is highly conserved from bacteria to humans. Although several structures of bacterial Nif3 proteins are known, no structure representing archaeal Nif3 has yet been reported. The crystal structure of Methanocaldococcus jannaschii MJ0927 was determined at 2.47 Å resolution to understand the structural differences between the bacterial and archaeal Nif3 proteins. Intriguingly, MJ0927 is found to adopt an unusual assembly comprising a trimer of dimers that forms a cage-like architecture. Electrophoretic mobility-shift assays indicate that MJ0927 binds to both single-stranded and double-stranded DNA. Structural analysis of MJ0927 reveals a positively charged region that can potentially explain its DNA-binding capability. Taken together, these data suggest that MJ0927 adopts a novel quartenary architecture that could play various DNA-binding roles in Methanocaldococcus jannaschii.
Subject(s)
Bacterial Proteins/chemistry , Conserved Sequence , Methanocaldococcus/chemistry , Amino Acid Sequence , Crystallography, X-Ray , Electrophoretic Mobility Shift Assay , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Multimerization , Protein Structure, Quaternary , Sequence AlignmentABSTRACT
Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) 2-epimerase catalyzes the interconversion of UDP-GlcNAc to UDP-N-acetylmannosamine (UDP-ManNAc), which is used in the biosynthesis of cell surface polysaccharides in bacteria. Biochemical experiments have demonstrated that mutation of this enzyme causes changes in cell morphology and the thermoresistance of the cell wall. Here, we present the crystal structures of Methanocaldococcus jannaschii UDP-GlcNAc 2-epimerase in open and closed conformations. A comparison of these crystal structures shows that upon UDP and UDP-GlcNAc binding, the enzyme undergoes conformational changes involving a rigid-body movement of the C-terminal domain. We also present the crystal structure of Bacillus subtilis UDP-GlcNAc 2-epimerase in the closed conformation in the presence of UDP and UDP-GlcNAc. Although a structural overlay of these two closed-form structures reveals that the substrate-binding site is evolutionarily conserved, some areas of the allosteric site are distinct between the archaeal and bacterial UDP-GlcNAc 2-epimerases. This is the first report on the crystal structure of archaeal UDP-GlcNAc 2-epimerase, and our results clearly demonstrate the changes between the open and closed conformations of this enzyme.
Subject(s)
Archaeal Proteins , Methanocaldococcus/enzymology , Uridine Diphosphate N-Acetylglucosamine , Amino Acid Sequence , Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Carbohydrate Epimerases/chemistry , Carbohydrate Epimerases/metabolism , Catalytic Domain , Crystallography, X-Ray , Isomerism , Models, Molecular , Molecular Sequence Data , Protein Conformation , Sequence Alignment , Uridine Diphosphate N-Acetylglucosamine/chemistry , Uridine Diphosphate N-Acetylglucosamine/metabolismABSTRACT
MJ0927 is a member of the Nif3 family and is widely distributed across living organisms. Although several crystal structures of Nif3 proteins have been reported, structural information on archaeal Nif3 is still limited. To understand the structural differences between bacterial and archaeal Nif3 proteins, MJ0927 from Methanocaldococcus jannaschii was purified and crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.47â Å and belonged to the orthorhombic space group C222, with unit-cell parameters a = 81.21, b = 172.94, c = 147.42â Å. Determination of this structure may provide insights into the function of MJ0927.
Subject(s)
Archaeal Proteins/chemistry , Archaeal Proteins/isolation & purification , Methanococcaceae/chemistry , Archaeal Proteins/genetics , Cloning, Molecular , Crystallization , Crystallography, X-Ray , Protein ConformationABSTRACT
The RecQ proteins are a highly conserved group of DNA helicases which play crucial roles in the maintenance of genome stability. DrRecQ from the radioresistant bacterium Deinococcus radiodurans is a special member of the RecQ family because it contains three Helicase-and-RNase-D-C-terminal (HRDC) domains at the C-terminus. The helicase catalytic core is essential for ATPase and DNA-unwinding activities. In this work, the helicase catalytic core of DrRecQ was expressed in Escherichia coli, purified and crystallized. Crystals were obtained using the sitting-drop vapour diffusion method and X-ray diffraction data were collected to 2.9â Å resolution. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 84.75, b = 95.61, c = 183.83â Å.
Subject(s)
Deinococcus/enzymology , RecQ Helicases/chemistry , Catalytic Domain , Crystallization , Gene Expression , RecQ Helicases/genetics , RecQ Helicases/isolation & purification , X-Ray DiffractionABSTRACT
The D-alanyl lipoteichoic acids (D-alanyl LTAs) present in the cell walls of Gram-positive bacteria play crucial roles in autolysis, cation homeostasis and biofilm formation. The alanylation of LTAs requires the D-alanyl carrier protein DltC to transfer D-Ala onto a membrane-associated LTA. Here, DltC from Staphylococcus epidermidis (SeDltC) was purified and crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 1.83â Å and belonged to space group P2, with unit-cell parameters a = 66.26, b = 53.28, c = 88.05â Å, ß = 98.22°. The results give a preliminary crystallographic analysis of SeDltC and shed light on the functional role of DltC in the alanylation of LTAs.
