Significance Events that occur between entry of the HIV-1 capsid into the cytoplasm of the target cell and the delivery of the viral genetic material into the nucleus constitute some of the less well understood processes in the viral life cycle. We demonstrated that PF74, a small-molecule inhibitor of HIV-1, and the host proteins CPSF6 and NUP153 bind to a preformed pocket within the CA protein hexamers that exist within the assembled capsid. Our results suggest that key features of the CA hexameric lattice remain intact upon docking at the nuclear pore. In addition, low molecular weight ligands that better mimic virus–host, protein–protein interactions at the intersubunit interfaces within the assembled viral capsid may offer novel avenues for therapeutic intervention.

The crystal structure has been determined at 3.0 A resolution for an unphosphorylated STAT1 (1-683) complexed with a phosphopeptide derived from the alpha chain of interferon gamma (IFNgamma) receptor. Two dimer interfaces are seen, one between the N domains (NDs) (amino acid residues 1-123) and the other between the core fragments (CFs) (residues 132-683). Analyses of the wild-type (wt) and mutant STAT1 proteins by static light scattering, analytical ultracentrifugation, and coimmunoprecipitation suggest that STAT1 is predominantly dimeric prior to activation, and the dimer is mediated by the ND interactions. The connecting region between the ND and the CF is flexible and allows two interconvertable orientations of the CFs, termed "antiparallel" or "parallel," as determined by SH2 domain orientations. Functional implications of these dimer conformations are discussed. Also revealed in this structure is the detailed interaction between STAT1 SH2 domain and its docking site on IFNgamma receptor.

Hsp70s mediate protein folding, translocation, and macromolecular complex remodeling reactions. Their activities are regulated by proteins that exchange ADP for ATP from the nucleotide-binding domain (NBD) of the Hsp70. These nucleotide exchange factors (NEFs) include the Hsp110s, which are themselves members of the Hsp70 family. We report the structure of an Hsp110:Hsc70 nucleotide exchange complex. The complex is characterized by extensive protein:protein interactions and symmetric bridging interactions between the nucleotides bound in each partner protein's NBD. An electropositive pore allows nucleotides to enter and exit the complex. The role of nucleotides in complex formation and dissociation, and the effects of the protein:protein interactions on nucleotide exchange, can be understood in terms of the coupled effects of the nucleotides and protein:protein interactions on the open-closed isomerization of the NBDs. The symmetrical interactions in the complex may model other Hsp70 family heterodimers in which two Hsp70s reciprocally act as NEFs.

The dissociation of apo-and metal-bound human copper-zinc superoxide dismutase (SOD1) dimers induced by the chaotrope guanidine hydrochloride (GdnHCl) or the reductant Tris(2-carboxyethyl)phosphine (TCEP) has been analyzed using analytical ultracentrifugation. Global fitting of sedimentation equilibrium data under native solution conditions (without GdnHCl or TCEP) demonstrate that both the apo-and metal-bound forms of SOD1 are stable dimers. Sedimentation velocity experiments show that apo-SOD1 dimers dissociate cooperatively over the range 0.5-1.0 M GdnHCl. In contrast, metal-bound SOD1 dimers possess a more compact shape and dissociate at significantly higher GdnHCl concentrations (2.0 -3.0 M). Reduction of the intrasubunit disulfide bond within each SOD1 subunit by 5-10 mM TCEP promotes dissociation of apo-SOD1 dimers, whereas the metal-bound enzyme remains a stable dimer under these conditions. The Cys-57 3 Ser mutant of SOD1, a protein incapable of forming the intrasubunit disulfide bond, sediments as a monomer in the absence of metal ions and as a dimer when metals are bound. Taken together, these data indicate that the stability imparted to the human SOD1 dimer by metal binding and the formation of the intrasubunit disulfide bond are mediated by independent molecular mechanisms. By combining the sedimentation data with previous crystallographic results, a molecular explanation is provided for the existence of different SOD1 macromolecular shapes and multiple SOD1 dimeric species with different stabilities.

No abstract

We report a model-independent analysis approach for fitting sedimentation velocity data which permits simultaneous determination of shape and molecular weight distributions for mono- and polydisperse solutions of macromolecules. Our approach allows for heterogeneity in the frictional domain, providing a more faithful description of the experimental data for cases where frictional ratios are not identical for all components. Because of increased accuracy in the frictional properties of each component, our method also provides more reliable molecular weight distributions in the general case. The method is based on a fine grained two-dimensional grid search over s and f/f (0), where the grid is a linear combination of whole boundary models represented by finite element solutions of the Lamm equation with sedimentation and diffusion parameters corresponding to the grid points. A Monte Carlo approach is used to characterize confidence limits for the determined solutes. Computational algorithms addressing the very large memory needs for a fine grained search are discussed. The method is suitable for globally fitting multi-speed experiments, and constraints based on prior knowledge about the experimental system can be imposed. Time- and radially invariant noise can be eliminated. Serial and parallel implementations of the method are presented. We demonstrate with simulated and experimental data of known composition that our method provides superior accuracy and lower variance fits to experimental data compared to other methods in use today, and show that it can be used to identify modes of aggregation and slow polymerization.

Bacteriophage T4 AsiA is a versatile transcription factor capable of inhibiting host gene expression as an 'anti-sigma' factor while simultaneously promoting gene-specific expression of T4 middle genes in conjunction with T4 MotA. To accomplish this task, AsiA engages conserved region 4 of Eschericia coli sigma70, blocking recognition of most host promoters by sequestering the DNA-binding surface at the AsiA/sigma70 interface. The three-dimensional structure of an AsiA/region 4 complex reveals that the C-terminal alpha helix of region 4 is unstructured, while four other helices adopt a completely different conformation relative to the canonical structure of unbound region 4. That AsiA induces, rather than merely stabilizes, this rearrangement can be realized by comparison to the homologous structures of region 4 solved in a variety of contexts, including the structure of Thermotoga maritima sigmaA region 4 described herein. AsiA simultaneously occupies the surface of region 4 that ordinarily contacts core RNA polymerase (RNAP), suggesting that an AsiA-bound sigma70 may also undergo conformational changes in the context of the RNAP holoenzyme.

Intensely- and broadly-absorbing nanoparticles (IBANs)of silver protected by arylthiolates were recently synthesized and showed unique optical properties, yet question of their dispersity and their molecular formulas remained. Here IBANs are identified as a superatom complex with a molecular formula of Ag44(SR)304− and an electron count of 18.This molecular character is shared by IBANs protected by 4-fluorothiophenol or 2-naphthalenethiol. The molecular formula and purity is determined by mass spectrometry and confirmed by sedimentation velocity-analytical ultracentrifugation. The data also give preliminary indications of a unique structure and environment for Ag44(SR)304−.