Biochemical evidence for conformational variants in the anti-viral and pro-metastatic protein IFITM1.

Interferon induced transmembrane proteins (IFITMs) play a dual role in the restriction of RNA viruses and in cancer progression, yet the mechanism of their action remains unknown. Currently, there is no data about the basic biochemical features or biophysical properties of the IFITM1 protein. In this work, we report on description and biochemical characterization of three conformational variants/oligomeric species of recombinant IFITM1 protein derived from an Escherichia coli expression system. The protein was extracted from the membrane fraction, affinity purified, and separated by size exclusion chromatography where two distinct oligomeric species were observed in addition to the expected monomer. These species remained stable upon re-chromatography and were designated as "dimer" and "oligomer" according to their estimated molecular weight. The dimer was found to be less stable compared to the oligomer using circular dichroism thermal denaturation and incubation with a reducing agent. A two-site ELISA and HDX mass spectrometry suggested the existence of structural motif within the N-terminal part of IFITM1 which might be significant in oligomer formation. Together, these data show the unusual propensity of recombinant IFITM1 to naturally assemble into very stable oligomeric species whose study might shed light on IFITM1 anti-viral and pro-oncogenic functions in cells.

Biochemical and Biophysical Characterization of the dsDNA Packaging Motor from the Lactococcus lactis Bacteriophage Asccphi28.

Double-stranded DNA viruses package their genomes into pre-assembled protein procapsids. This process is driven by macromolecular motors that transiently assemble at a unique vertex of the procapsid and utilize homomeric ring ATPases to couple genome encapsidation to ATP hydrolysis. Here, we describe the biochemical and biophysical characterization of the packaging ATPase from Lactococcus lactis phage asccφ28. Size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), small angle X-ray scattering (SAXS), and negative stain transmission electron microscopy (TEM) indicate that the ~45 kDa protein formed a 443 kDa cylindrical assembly with a maximum dimension of ~155 Å and radius of gyration of ~54 Å. Together with the dimensions of the crystallographic asymmetric unit from preliminary X-ray diffraction experiments, these results indicate that gp11 forms a decameric D5-symmetric complex consisting of two pentameric rings related by 2-fold symmetry. Additional kinetic analysis shows that recombinantly expressed gp11 has ATPase activity comparable to that of functional ATPase rings assembled on procapsids in other genome packaging systems. Hence, gp11 forms rings in solution that likely reflect the fully assembled ATPases in active virus-bound motor complexes. Whereas ATPase functionality in other double-stranded DNA (dsDNA) phage packaging systems requires assembly on viral capsids, the ability to form functional rings in solution imparts gp11 with significant advantages for high-resolution structural studies and rigorous biophysical/biochemical analysis.

The N-terminal domain of the Escherichia coli PriA helicase contains both the DNA- and nucleotide-binding sites. Energetics of domain--DNA interactions and allosteric effect of the nucleotide cofactors.

Functional interactions of the Escherichia coli PriA helicase 181N-terminal domain with the DNA and nucleotide cofactors have been quantitatively examined. The isolated 181N-terminal domain forms a stable dimer in solution, most probably reflecting the involvement of the domain in specific cooperative interactions of the intact PriA protein--double-stranded DNA (dsDNA) complex. Only one monomer of the domain dimer binds the DNA; i.e., the dimer has one effective DNA-binding site. Although the total site size of the dimer--single-stranded DNA (ssDNA) complex is ~13 nucleotides, the DNA-binding subsite engages in direct interactions with approximately five nucleotides. A small number of interacting nucleotides indicates that the DNA-binding subsites of the PriA helicase, i.e., the strong subsite on the helicase domain and the weak subsite on the N-terminal domain, are spatially separated in the intact enzyme. Contrary to current views, the subsite has an only slight preference for the 3'-end OH group of the ssDNA and lacks any significant base specificity, although it has a significant dsDNA affinity. Unlike the intact helicase, the DNA-binding subsite of the isolated domain is in an open conformation, indicating the presence of the direct helicase domain--N-terminal domain interactions. The discovery that the 181N-terminal domain possesses a nucleotide-binding site places the allosteric, weak nucleotide-binding site of the intact PriA on the N-terminal domain. The specific effect of ADP on the domain DNA-binding subsite indicates that in the intact helicase, the bound ADP not only opens the DNA-binding subsite but also increases its intrinsic DNA affinity.

The Escherichia coli PriA helicase-double-stranded DNA complex: location of the strong DNA-binding subsite on the helicase domain of the protein and the affinity control by the two nucleotide-binding sites of the enzyme.

The Escherichia coli PriA helicase complex with the double-stranded DNA (dsDNA), the location of the strong DNA-binding subsite, and the effect of the nucleotide cofactors, bound to the strong and weak nucleotide-binding site of the enzyme on the dsDNA affinity, have been analyzed using the fluorescence titration, analytical ultracentrifugation, and photo-cross-linking techniques. The total site size of the PriA-dsDNA complex is only 5±1 bp, that is, dramatically lower than 20±3 nucleotides occluded in the enzyme-single-stranded DNA (ssDNA) complex. The helicase associates with the dsDNA using its strong ssDNA-binding subsite in an orientation very different from the complex with the ssDNA. The strong DNA-binding subsite of the enzyme is located on the helicase domain of the PriA protein. The dsDNA intrinsic affinity is considerably higher than the ssDNA affinity and the binding process is accompanied by a significant positive cooperativity. Association of cofactors with strong and weak nucleotide-binding sites of the protein profoundly affects the intrinsic affinity and the cooperativity, without affecting the stoichiometry. ATP analog binding to either site diminishes the intrinsic affinity but preserves the cooperativity. ADP binding to the strong site leads to a dramatic increase of the cooperativity and only slightly affects the affinity, while saturation of both sites with ADP strongly increases the affinity and eliminates the cooperativity. Thus, the coordinated action of both nucleotide-binding sites on the PriA-dsDNA interactions depends on the structure of the phosphate group. The significance of these results for the enzyme activities in recognizing primosome assembly sites or the ssDNA gaps is discussed.

The Escherichia coli PriA helicase specifically recognizes gapped DNA substrates: effect of the two nucleotide-binding sites of the enzyme on the recognition process.

Energetics and specificity of interactions between the Escherichia coli PriA helicase and the gapped DNAs have been studied, using the quantitative fluorescence titration and analytical ultracentrifugation methods. The gap complex has a surprisingly low minimum total site size, corresponding to approximately 7 nucleotides of the single-stranded DNA (ssDNA), as compared with the site size of approximately 20 nucleotides of the enzyme-ssDNA complex. The dramatic difference in stoichiometries indicates that the enzyme predominantly engages the strong DNA-binding subsite in interactions with the gap and assumes a very different orientation in the gap complex, as compared with the complex with the ssDNA. The helicase binds the ssDNA gaps with 4-5 nucleotides with the highest affinity, which is approximately 3 and approximately 2 orders of magnitude larger than the affinities for the ssDNA and double-stranded DNA, respectively. In the gap complex, the protein does not engage in cooperative interactions with the enzyme predominantly associated with the surrounding dsDNA. Binding of nucleoside triphosphate to the strong and weak nucleotide-binding sites of the helicase eliminates the selectivity of the enzyme for the size of the gap, whereas saturation of both sites with ADP leads to amplified affinity for the ssDNA gap containing 5 nucleotides and engagement of an additional protein area in interactions with the nucleic acid.

Mechanisms of interactions of the nucleotide cofactor with the RepA protein of plasmid RSF1010. Binding dynamics studied using the fluorescence stopped-flow method.

The dynamics of the nucleotide binding to a single, noninteracting nucleotide-binding site of the hexameric helicase RepA protein of plasmid RSF1010 has been examined, using the fluorescence stopped-flow method. The experiments have been performed with fluorescent analogues of ATP and ADP, TNP-ATP and TNP-ADP, respectively. In the presence of Mg(2+), the association of the cofactors proceeds as a sequential three-step process [Formula: see text] The sequential nature of the mechanism indicates the lack of significant conformational equilibria of the helicase prior to nucleotide binding. The major conformational change of the RepA helicase-nucleotide complex occurs in the formation of (H-N)(2), which is characterized by a very high value of the partial equilibrium constant and large positive changes in the apparent enthalpy and entropy. Strong stabilizing interactions between subunits of the RepA hexamer contribute to the observed dynamics and energetics of the internal transitions of the formed complexes. Magnesium cations mediate the efficient and fast conformational transitions of the protein, in a manner independent of the structure of the cofactor phosphate group. The ssDNA bound to the enzyme preferentially selects a single intermediate of the RepA-ATP analogue complex, (H-N)(2), while the DNA has no effect on the intermediates of the RepA-ADP complex. Allosteric interactions between the nucleotide- and DNA-binding site are established in the initial stages of formation of the complex. Moreover, in the presence of the single-stranded DNA, all the transitions in the nucleotide binding to the helicase become sensitive to the structure of the phosphate group of the cofactor.

Zinc binding in pestivirus N(pro) is required for interferon regulatory factor 3 interaction and degradation.

Pestiviruses, such as bovine viral diarrhea virus and classical swine fever virus (CSFV), use the viral protein N(pro) to subvert host cell antiviral responses. N(pro) is the first protein encoded by the single large open reading frame of the pestivirus positive-sense RNA genome and has an autoproteolytic activity, cleaving itself off from the polyprotein. N(pro) also targets interferon regulatory factor 3 (IRF3), a transcription factor for alpha/beta interferon genes, and promotes its proteasomal degradation, a process that is independent of the proteolytic activity of N(pro). We determined that N(pro) contains a novel metal-binding TRASH motif consisting of Cys-X(21)-Cys-X(3)-Cys (where X is any amino acid) at its C-terminus. We also found that N(pro) coordinates a single zinc atom as determined by graphite furnace-atomic absorption spectrophotometry and inductively coupled plasma-mass spectrometry. Mutational and biochemical analyses show that the cysteine residues in the TRASH motif are required for zinc binding and protein stability. Individual substitutions of the cysteines in the TRASH motif of CSFV N(pro) abolished the interaction of N(pro) with IRF3 and resulted in the loss of virus-mediated IRF3 degradation in CSFV-infected cells. Thus, the zinc-binding ability of N(pro) in pestiviruses appears to be essential for the virus-mediated degradation of IRF3.

Dynamics of the ssDNA recognition by the RepA hexameric helicase of plasmid RSF1010: analyses using fluorescence stopped-flow intensity and anisotropy methods.

The kinetic mechanism of the single-stranded DNA (ssDNA) recognition by the RepA hexameric replicative helicase of the plasmid RSF1010 and the nature of formed intermediates, in the presence of the ATP nonhydrolyzable analog, beta,gamma-imidoadenosine-5'-triphosphate (AMP-PNP), have been examined, using the fluorescence intensity and anisotropy stopped-flow and analytical ultracentrifugation methods. Association of the RepA hexamer with the ssDNA oligomers that engage the total DNA-binding site and exclusively the strong DNA-binding subsite is a minimum four-step mechanism [formula: see text]. Extreme stability of the RepA hexamer precludes any disintegration of its structure, and the sequential character of the mechanism indicates that the enzyme exists in a predominantly single conformation prior to the association with the nucleic acid. Moreover, the hexameric helicase possesses a DNA-binding site located outside its cross channel. The reaction steps have dramatically different dynamics, with rate constants differing by 2-3 orders of magnitude. Such behavior indicates a very diverse nature of the observed transitions, which comprises binding steps and large conformational transitions of the helicase, including local opening of the hexameric structure. Steady-state fluorescence anisotropies of intermediates indicate that the entry of the DNA into the cross channel is initiated from the 5' end of the bound nucleic acid. The global structure of the tertiary complex RepA-ssDNA-AMP-PNP is very different from the structure of the binary complex RepA-AMP-PNP, indicating that, in equilibrium, the RepA hexamer-ssDNA-AMP-PNP complex exists as a mixture of partially open states.