Structure and dynamics study of translation initiation factor 1 from Staphylococcus aureus suggests its RNA binding mode.

Translation initiation, the rate-limiting step in the protein synthesis, is tightly regulated. As one of the translation initiation factors, translation initiation factor 1 (IF1) plays crucial roles not only in translation but also in many cellular processes that are important for genomic stability, such as the activity of RNA chaperones. Here, we characterize the RNA interactions and dynamics of IF1 from Staphylococcus aureus Mu50 (IF1Sa) by NMR spectroscopy. First, the NMR-derived solution structure of IF1Sa revealed that IF1Sa adopts an oligonucleotide/oligosaccharide binding (OB)-fold. Structural comparisons showed large deviations in the α-helix and the following loop, which are potential RNA-binding regions of the OB-fold, as well as differences in the electrostatic potential surface among bacterial IF1s. Second, the 15N NMR relaxation data for IF1Sa indicated the flexible nature of the α-helix and the following loop region of IF1Sa. Third, RNA-binding properties were studied using FP assays and NMR titrations. FP binding assays revealed that IF1Sa binds to RNAs with moderate affinity. In combination with the structural analysis, the NMR titration results revealed the RNA binding sites. Taken together, these results show that IF1Sa binds RNAs with moderate binding affinity via the residues that occupy the large surface area of its ß-barrel. These findings suggest that IF1Sa is likely to bind RNA in various conformations rather than only at a specific site and indicate that the flexible RNA binding mode of IF1Sa is necessary for its interaction with various RNA substrates.

Alba from Thermoplasma volcanium belongs to α-NAT's: An insight into the structural aspects of Tv Alba and its acetylation by Tv Ard1.

The Alba superfamily proteins have been regarded as a conserved group of proteins in archaea and eukarya, which have shown to be important in nucleic acid binding, chromatic organization and gene regulation. These proteins often belong to the N-acetyltransferase (NAT) category (N(α)-acetyltransferases or N(ε)-acetyltransferases) and undergo post-translational modifications. Here, we report the crystal structure of Alba from Thermoplasma volcanium (Tv Alba) at 2.4 Å resolution. The acetylation of Tv Alba was monitored and the N-terminal of Tv Alba has been shown to interact with acetyl coenzyme A (Ac-CoA). The chemical shift perturbation experiments of Tv Alba were performed in the presence of Ac-CoA and/or Tv Ard1, another T. volcanium protein that treats Tv Alba as a substrate. To examine the DNA binding capabilities of Tv Alba alone and in the presence of Ac-CoA and/or Tv Ard1, EMSA experiments were carried out. It is shown that although Tv Alba binds to Ac-CoA, the acetylation of Tv Alba is not related with its binding to dsDNA, and the involvement of the N-terminus in Ac-CoA binding demonstrates that Tv Alba belongs to the N(α)-acetyltransferase family.

ß-Arm flexibility of HU from Staphylococcus aureus dictates the DNA-binding and recognition mechanism.

HU, one of the major nucleoid-associated proteins, interacts with the minor groove of DNA in a nonspecific manner to induce DNA bending or to stabilize bent DNA. In this study, crystal structures are reported for both free HU from Staphylococcus aureus Mu50 (SHU) and SHU bound to 21-mer dsDNA. The structures, in combination with electrophoretic mobility shift assays (EMSAs), isothermal titration calorimetry (ITC) measurements and molecular-dynamics (MD) simulations, elucidate the overall and residue-specific changes in SHU upon recognizing and binding to DNA. Firstly, structural comparison showed the flexible nature of the ß-sheets of the DNA-binding domain and that the ß-arms bend inwards upon complex formation, whereas the other portions are nearly unaltered. Secondly, it was found that the disruption and formation of salt bridges accompanies DNA binding. Thirdly, residue-specific free-energy analyses using the MM-PBSA method with MD simulation data suggested that the successive basic residues in the ß-arms play a central role in recognizing and binding to DNA, which was confirmed by the EMSA and ITC analyses. Moreover, residue Arg55 resides in the hinge region of the flexible ß-arms, exhibiting a remarkable role in their flexible nature. Fourthly, EMSAs with various DNAs revealed that SHU prefers deformable DNA. Taken together, these data suggest residue-specific roles in local shape and base readouts, which are primarily mediated by the flexible ß-arms consisting of residues 50-80.

Tetraspan TM4SF5-dependent direct activation of FAK and metastatic potential of hepatocarcinoma cells.

Transmembrane 4 L six family member 5 (TM4SF5) plays an important role in cell migration, and focal adhesion kinase (FAK) activity is essential for homeostatic and pathological migration of adherent cells. However, it is unclear how TM4SF5 signaling mediates the activation of cellular migration machinery, and how FAK is activated during cell adhesion. Here, we showed that direct and adhesion-dependent binding of TM4SF5 to FAK causes a structural alteration that may release the inhibitory intramolecular interaction in FAK. In turn, this may activate FAK at the cell's leading edge, to promote migration/invasion and in vivo metastasis. TM4SF5-mediated FAK activation occurred during integrin-mediated cell adhesion. TM4SF5 was localized at the leading edge of the cells, together with FAK and actin-organizing molecules, indicating a signaling link between TM4SF5/FAK and actin reorganization machinery. Impaired interactions between TM4SF5 and FAK resulted in an attenuated FAK phosphorylation (the signaling link to actin organization machinery) and the metastatic potential. Our findings demonstrate that TM4SF5 directly binds to and activates FAK in an adhesion-dependent manner, to regulate cell migration and invasion, suggesting that TM4SF5 is a promising target in the treatment of metastatic cancer.

Overexpression, crystallization and preliminary X-ray crystallographic analysis of hypothetical protein SAV0479 from Staphylococcus aureus Mu50.

SAV0479, a hypothetical protein from the Mu50 strain of methicillin- and vancomycin-resistant Staphylococcus aureus, was selected for structure and function determination as part of a structural genomics project. Here, the cloning, overexpression, purification and crystallization of SAV0479 are reported. Crystals were obtained by the hanging-drop vapour-diffusion method and diffraction data were collected to a resolution of 2.8 Å. The crystals belonged to space group P3(1)21, with unit-cell parameters a = b = 81.48, c = 82.53 Å. Three monomers of SAV0479 are present in each asymmetric unit.

Backbone 1H, 15N, and 13C resonance assignments and secondary-structure of the conserved hypothetical protein HP0892 of Helicobacter pylori.

HP0892 (SwissProt/TrEMBL ID O25552) is a 90-residue conserved hypothetical protein from Helicobacter pylori strain 26695, with a calculated pI of 9.38 and a molecular mass of 10.41 kDa. It belongs to the Plasmid stabilization system protein family (PF05016) in the Pfam database. Proteins with sequence similarity to HP0892 exist in Vibrio choierae, Enterococcus faecalis, Campylobacter jejuni, Streptococcus pneumoniae, Haemophilus influenzae, Escherichia coli O157. Here we report the sequence-specific backbone resonance assignments of HP0892 using multidimentional heteronuclear NMR spectroscopy. About 97.0% (422/ 435) of the HN, N, CO, C Alpha , C Beta resonances of 90 residues of HP0892 were assigned. On the basis of the resonance assignments, three helical regions and four strand regions were identified using the CSI program. This study is a prerequisite for calculating the solution structure of HP0892, and will be useful for studying its interaction with other molecules.

Crystal structure of toxin HP0892 from Helicobacter pylori with two Zn(II) at 1.8 Å resolution.

Antibiotic resistance and microorganism virulence have been consistently exhibited by bacteria and archaea, which survive in conditions of environmental stress through toxin-antitoxin (TA) systems. The HP0892-HP0893 TA system is one of the two known TA systems belonging to Helicobacter pylori. The antitoxin, HP0893, binds and inhibits the HP0892 toxin and regulates the transcription of the TA operon. Here, we present the crystal structure of the zinc-bound HP0892 toxin at 1.8 Å resolution. Reorientation of residues at the mRNase active site was shown. The involved residues, namely E58A, H86A, and H58A/ H60A, were mutated and the binding affinity was monitored by ITC studies. Through the structural difference between the apo and the metal-bound state, and using a homology modeling tool, the involvement of the metal ion in mRNase active site could be identified. The most catalytically important residue, His86, reorients itself to exhibit RNase activity. His47, Glu58, and His60 are involved in metal binding where Glu58 acts as a general base and His47 and His60 may also act as a general acid in enzymatic activity. Glu58 and Asp64 are involved in substrate binding and specific sequence recognition. Arg83 is involved in phosphate binding and stabilization of the transition state, and Phe90 is involved in base packing and substrate orientation.

1H, 13C and 15N chemical shift assignments of Ninjurin1 Extracellular N-terminal Domain.

Cell adhesion molecules play a crucial role in fundamental biological processes via regulating cell-cell interactions. Nerve injury induced protein1 (Ninjurin1) is a novel adhesion protein that has no significant homology with other known cell adhesion molecules. Here we present the assignment of an 81 aa construct for human Ninjurin1 Extracellular N-Terminal (ENT) domain, which comprises the critical adhesion domain.

Crystal structure of hypothetical protein HP0062 (O24902_HELPY) from Helicobacter pylori at 1.65 A resolution.

The HP0062 gene encodes a small acidic protein of 86 amino acids with a theoretical pI of 4.6. The crystal structure of hypothetical protein HP0062 from Helicobacter pylori has been determined at 1.65 A by molecular-replacement method. The crystallographic asymmetric unit contains dimer, in which HP0062 monomer folds into a helix-hairpin-helix structure. The two protomers are primarily held together by extensive hydrophobic interactions in an antiparallel arrangement, forming a four helix bundle. Aromatic residues located at a or g position in the heptad leucine zipper are not major contributor required for HP0062 dimerization but important for the thermostability of this protein.

NMR solution structure of HP0827 (O25501_HELPY) from Helicobacter pylori: model of the possible RNA-binding site.

The HP0827 protein is an 82-residue protein identified as a putative ss-DNA-binding protein 12RNP2 Precursor from Helicobacter pylori. Here, we have determined 3D structure of HP0827 using Nuclear Magnetic Resonance. It has a ferredoxin-like fold, beta1-alpha1-beta2-beta3-alpha2-beta4 (alpha; alpha-helix and beta; beta-sheet) and ribonucleoprotein (RNP) motifs which are thought to be important in RNA binding. By using structural homologues search and analyzing electrostatic potential of surface, we could compared HP0827 with other RNA-binding proteins (sex-lethal, T-cell restricted intracellular antigen-1, U1A) to predict RNA-binding sites of HP0827. We could predict that beta sheets of HP0827, especially beta1 and beta3, are primary region for RNA binding. Consequently, similar to other RNA-binding proteins, RNP motifs (Y5, F45, F47), positively charged and hydrophobic regions (K32, R37, K40, K41, K43, R70, R73) are proposed as a putative RNA-binding sites. In addition, differences in amino acids composition of RNP motifs, N, C-terminal residues, loop-region fold and the orientation of alpha1-helix with other RNA recognition motif proteins could give specific biological functions to HP0827. Finally, the study on natural RNA target is also important to completely understand the biological function of HP0827.