Structural basis of an epitope tagging system derived from Haloarcula marismortui bacteriorhodopsin I D94N and its monoclonal antibody GD-26.

Specific antibody interactions with short peptides have made epitope tagging systems a vital tool employed in virtually all fields of biological research. Here, we present a novel epitope tagging system comprised of a monoclonal antibody named GD-26, which recognises the TD peptide (GTGATPADD) derived from Haloarcula marismortui bacteriorhodopsin I (HmBRI) D94N mutant. The crystal structure of the antigen-binding fragment (Fab) of GD-26 complexed with the TD peptide was determined to a resolution of 1.45 Å. The TD peptide was found to adopt a 310 helix conformation within the binding cleft, providing a characteristic peptide structure for recognition by GD-26 Fab. Based on the structure information, polar and nonpolar forces collectively contribute to the strong binding. Attempts to engineer the TD peptide show that the proline residue is crucial for the formation of the 310 helix in order to fit into the binding cleft. Isothermal calorimetry (ITC) reported a dissociation constant KD of 12 ± 2.8 nm, indicating a strong interaction between the TD peptide and GD-26 Fab. High specificity of GD-26 IgG to the TD peptide was demonstrated by western blotting, ELISA and immunofluorescence as only TD-tagged proteins were detected, suggesting the effectiveness of the GD-26/TD peptide tagging system. In addition to already-existing epitope tags such as the FLAG tag and the ALFA tag adopting either extended or α-helix conformations, the unique 310 helix conformation of the TD peptide together with the corresponding monoclonal antibody GD-26 offers a novel tagging option for research.

Biochemical and molecular dynamics studies of archaeal polyisoprenyl pyrophosphate phosphatase from Saccharolobus solfataricus.

The undecaprenyl pyrophosphate phosphatase (UppP) is an integral membrane pyrophosphatase. In bacteria, UppP catalyzes the dephosphorylation of undecaprenyl pyrophosphate (C55-pp) to undecaprenyl phosphate (C55-P) in the periplasmic space, which is an essential step for the isoprenyl lipid carrier to reenter the peptidoglycan synthesis cycle. Besides bacteria, the UppP homologs are widely distributed in archaea genome. However, all archaea lack peptidoglycan structure in their cell wall components, and the major archaeal lipid carriers are dolichol phosphate (Dol-p) and dolichol pyrophosphate (Dol-pp), so the functions of the UppP homolog in archaea remain unclear. Here, we purified a recombinant polyisoprenyl pyrophosphatase of a thermoacidophilic archaeon, Saccharolobus solfataricus (SsUppP), and characterized its enzymatic properties. Two isoprenyl pyrophosphate, farnesyl pyrophosphate (Fpp) and geranylgeranyl pyrophosphate (Ggpp), were used as the surrogate substrates, simulating the bacterial and archaeal lipid carriers. SsUppP dephosphorylated Fpp and Ggpp at 37 °C, but retained the phosphatase activity at high temperatures. The optimal condition for the enzymatic activity was found to be at pH 7 and 70 °C. The thermostability of SsUppP was also supported by molecular dynamics simulation studies. Our results indicated that the archaeal SsUppP can dephosphorylate isoprenyl pyrophosphates at the natural environment of high temperature, and the possibility to catalyze the dephosphorylation of archaeal lipid carriers.

Transient oligomerization of the SARS-CoV N protein--implication for virus ribonucleoprotein packaging.

The nucleocapsid (N) phosphoprotein of the severe acute respiratory syndrome coronavirus (SARS-CoV) packages the viral genome into a helical ribonucleocapsid and plays a fundamental role during viral self-assembly. The N protein consists of two structural domains interspersed between intrinsically disordered regions and dimerizes through the C-terminal structural domain (CTD). A key activity of the protein is the ability to oligomerize during capsid formation by utilizing the dimer as a building block, but the structural and mechanistic bases of this activity are not well understood. By disulfide trapping technique we measured the amount of transient oligomers of N protein mutants with strategically located cysteine residues and showed that CTD acts as a primary transient oligomerization domain in solution. The data is consistent with the helical oligomer packing model of N protein observed in crystal. A systematic study of the oligomerization behavior revealed that altering the intermolecular electrostatic repulsion through changes in solution salt concentration or phosphorylation-mimicking mutations affects oligomerization propensity. We propose a biophysical mechanism where electrostatic repulsion acts as a switch to regulate N protein oligomerization.

Molecular mechanism of oxidation-induced TDP-43 RRM1 aggregation and loss of function.

Cysteine oxidation of the two RNA recognition motifs (RRM1 and RRM2) of TDP-43, a multi-domain protein involved in neurodegenerative diseases, results in loss of function and accumulation of insoluble aggregates under both in vitro and in vivo conditions. However, the molecular mechanisms linking cysteine oxidation to protein aggregation and functional aberration remain unknown. We report that oxidation of cysteines in RRM1, but not in other domains, induced conformational changes which subsequently resulted in protein aggregation and loss of nucleic acid-binding activity. Thus, oxidation-induced conformational change of RRM1 plays a key role in TDP-43 aggregation and disease progression.

The N-terminus of TDP-43 promotes its oligomerization and enhances DNA binding affinity.

TDP-43 is a DNA/RNA-binding protein associated with different neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-U). Here, the structural and physical properties of the N-terminus on TDP-43 have been carefully characterized through a combination of nuclear magnetic resonance (NMR), circular dichroism (CD) and fluorescence anisotropy studies. We demonstrate for the first time the importance of the N-terminus in promoting TDP-43 oligomerization and enhancing its DNA-binding affinity. An unidentified structural domain in the N-terminus is also disclosed. Our findings provide insights into the N-terminal domain function of TDP-43.