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Proline-rich domains (PRDs) are among the most prevalent signaling modules of eukaryotes but often unexplored by biophysical techniques as their heterologous recombinant expression poses significant difficulties. Using a "divide-and-conquer" approach, we present a detailed investigation of a PRD (166 residues; ∼30% prolines) belonging to a human protein ALIX, a versatile adaptor protein involved in essential cellular processes including ESCRT-mediated membrane remodeling, cell adhesion, and apoptosis. In solution, the N-terminal fragment of ALIX-PRD is dynamically disordered. It contains three tandem sequentially similar proline-rich motifs that compete for a single binding site on its signaling partner, TSG101-UEV, as evidenced by heteronuclear NMR spectroscopy. Global fitting of relaxation dispersion data, measured as a function of TSG101-UEV concentration, allowed precise quantitation of these interactions. In contrast to the soluble N-terminal portion, the C-terminal tyrosine-rich fragment of ALIX-PRD forms amyloid fibrils and viscous gels validated using dye-binding assays with amyloid-specific probes, congo red and thioflavin T (ThT), and visualized by transmission electron microscopy. Remarkably, fibrils dissolve at low temperatures (2 to 6 °C) or upon hyperphosphorylation with Src kinase. Aggregation kinetics monitored by ThT fluorescence shows that charge repulsion dictates phosphorylation-mediated fibril dissolution and that the hydrophobic effect drives fibril formation. These data illuminate the mechanistic interplay between interactions of ALIX-PRD with TSG101-UEV and polymerization of ALIX-PRD and its central role in regulating ALIX function. This study also demonstrates the broad functional repertoires of PRDs and uncovers the impact of posttranslational modifications in the modulation of reversible amyloids.

Viral capsids are dynamic assemblies that undergo controlled conformational transitions to perform various biological functions. The replication-derived four-molecule RNA progeny of Brome mosaic virus (BMV) is packaged by a single capsid protein (CP) into three types of morphologically indistinguishable icosahedral virions with T=3 quasisymmetry. Type 1 (B1V) and type 2 (B2V) virions package genomic RNA1 and RNA2, respectively, while type 3 (B3+4V) virions copackage genomic RNA3 (B3) and its subgenomic RNA4 (sgB4). In this study, the application of a robust Agrobacterium-mediated transient expression system allowed us to assemble each virion type separately in planta Experimental approaches analyzing the morphology, size, and electrophoretic mobility failed to distinguish between the virion types. Thermal denaturation analysis and protease-based peptide mass mapping experiments were used to analyze stability and the conformational dynamics of the individual virions, respectively. The crystallographic structure of the BMV capsid shows four trypsin cleavage sites (K65, R103, K111, and K165 on the CP subunits) exposed on the exterior of the capsid. Irrespective of the digestion time, while retaining their capsid structural integrity, B1V and B2V released a single peptide encompassing amino acids 2 to 8 of the N-proximal arginine-rich RNA binding motif. In contrast, B3+4V capsids were unstable with trypsin, releasing several peptides in addition to the peptides encompassing four predicted sites exposed on the capsid exterior. These results, demonstrating qualitatively different dynamics for the three types of BMV virions, suggest that the different RNA genes they contain may have different translational timing and efficiency and may even impart different structures to their capsids.IMPORTANCE The majority of viruses contain RNA genomes protected by a shell of capsid proteins. Although crystallographic studies show that viral capsids are static structures, accumulating evidence suggests that, in solution, virions are highly dynamic assemblies. The three genomic RNAs (RNA1, -2, and -3) and a single subgenomic RNA (RNA4) of Brome mosaic virus (BMV), an RNA virus pathogenic to plants, are distributed among three physically homogeneous virions. This study examines the thermal stability by differential scanning fluorimetry (DSF) and capsid dynamics by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analyses following trypsin digestion of the three virions assembled separately in vivo using the Agrobacterium-mediated transient expression approach. The results provide compelling evidence that virions packaging genomic RNA1 and -2 are distinct from those copackaging RNA3 and -4 in their stability and dynamics, suggesting that RNA-dependent capsid dynamics play an important biological role in the viral life cycle.

Chlamydia species are bacterial pathogens that affect over 140 million individuals worldwide. Ocular infection by Chlamydia trachomatis is the leading cause of preventable blindness, and urogenital tract infection by Chlamydia causes sexually transmitted disease. As obligate intracellular organisms, Chlamydia species have evolved mechanisms to evade the host immune system, including the degradation of the transcription factors regulatory factor X5 and upstream stimulation factor 1, which are required for the expression of major histocompatibility complex molecules I and II by CPAF and cleavage of p65 of the NF-[kappa]B pathway by the encoded CT441 protein. Here, we report the characterization of CT441 as a tail-specific protease. CT441 contains a PDZ domain of protein-protein interactions and a Ser/Lys dyad catalytic unit. Mutation at either Ser455 or Lys481 in the active site ablated CT441 activity of p65 cleavage. In addition, we found that the production of CT441 Tsp, which was detected at the middle and late stages of an infection, correlated with p65 cleavage activity. In addition to high homology, human and mouse p65 proteins also contain an identical C-terminal tail of 22 amino acid (aa) residues. However, only human p65 was susceptible to cleavage. Using molecular biology approaches, we mapped the p65 cleavage site(s) to a region that differs from that of mouse p65 by 6 aa residues. Additionally, the substitution of T352 with a proline inhibited p65 cleavage. Together, the study demonstrates that CT441 is a tail-specific protease that is capable of interfering with the NF-[kappa]B pathway of host antimicrobial and inflammatory responses.

Research describes the crystal and solution structure of an heat shock locus gene products U and V complex of influenza virus. Data reveal that HsIU ATPase and HsIV protease of the complex bear 80% amino acid sequence homology to the Escherichia coli counterparts.

Molecular simulations have provided a means of analyzing self-assembly of viral proteins into icosahedral capsids. The studies have shown the complex processes that govern viral capsid assembly and have suggested some features of the assembly process that can be used for controlling the assembly of icosahedral capsids and nonicosahedral capsules.

The procedure for phase extension (PX) involves gradually extending the initial phases from low resolution (e.g. ∼8 Å) to the high-resolution limit of a diffraction data set. Structural redundancy present in the viral capsids that display icosahedral symmetry results in a high degree of non-crystallographic symmetry (NCS), which in turn translates into higher phasing power and is critical for improving and extending phases to higher resolution. Greater completeness of the diffraction data and determination of a molecular replacement solution, which entails accurately identifying the virus particle orientation(s) and position(s), are important for the smooth progression of the PX procedure. In addition, proper definition of a molecular mask (envelope) around the NCS-asymmetric unit has been found to be important for the success of density modification procedures, such as density averaging and solvent flattening. Regardless of the degree of NCS, the PX method appears to work well in all space groups, provided an accurate molecular mask is used along with reasonable initial phases. However, in the cases with space group P1, in addition to requiring a molecular mask, starting the PX at a higher resolution (e.g. 6 Å) overcame the previously reported problems due to Babinet phases and phase flipping errors. [ABSTRACT FROM PUBLISHER]