A Universal Approach to Optimize the Folding and Stability of Prefusion-Closed HIV-1 Envelope Trimers.

The heavily glycosylated native-like envelope (Env) trimer of HIV-1 is expected to have low immunogenicity, whereas misfolded forms are often highly immunogenic. High-quality correctly folded Envs may therefore be critical for developing a vaccine that induces broadly neutralizing antibodies. Moreover, the high variability of Env may require immunizations with multiple Envs. Here, we report a universal strategy that provides for correctly folded Env trimers of high quality and yield through a repair-and-stabilize approach. In the repair stage, we utilized a consensus strategy that substituted rare strain-specific residues with more prevalent ones. The stabilization stage involved structure-based design and experimental assessment confirmed by crystallographic feedback. Regions important for the refolding of Env were targeted for stabilization. Notably, the α9-helix and an intersubunit ß sheet proved to be critical for trimer stability. Our approach provides a means to produce prefusion-closed Env trimers from diverse HIV-1 strains, a substantial advance for vaccine development.

A highly stable prefusion RSV F vaccine derived from structural analysis of the fusion mechanism.

Respiratory syncytial virus (RSV) causes acute lower respiratory tract infections and is the leading cause of infant hospitalizations. Recently, a promising vaccine antigen based on the RSV fusion protein (RSV F) stabilized in the native prefusion conformation has been described. Here we report alternative strategies to arrest RSV F in the prefusion conformation based on the prevention of hinge movements in the first refolding region and the elimination of proteolytic exposure of the fusion peptide. A limited number of unique mutations are identified that stabilize the prefusion conformation of RSV F and dramatically increase expression levels. This highly stable prefusion RSV F elicits neutralizing antibodies in cotton rats and induces complete protection against viral challenge. Moreover, the structural and biochemical analysis of the prefusion variants suggests a function for p27, the excised segment that precedes the fusion peptide in the polypeptide chain.

Crystal Structure of a Hidden Protein, YcaC, a Putative Cysteine Hydrolase from Pseudomonas aeruginosa, with and without an Acrylamide Adduct.

As part of the ongoing effort to functionally and structurally characterize virulence factors in the opportunistic pathogen Pseudomonas aeruginosa, we determined the crystal structure of YcaC co-purified with the target protein at resolutions of 2.34 and 2.56 Å without a priori knowledge of the protein identity or experimental phases. The three-dimensional structure of YcaC adopts a well-known cysteine hydrolase fold with the putative active site residues conserved. The active site cysteine is covalently bound to propionamide in one crystal form, whereas the second form contains an S-mercaptocysteine. The precise biological function of YcaC is unknown; however, related prokaryotic proteins have functions in antibacterial resistance, siderophore production and NADH biosynthesis. Here, we show that YcaC is exceptionally well conserved across both bacterial and fungal species despite being non-ubiquitous. This suggests that whilst YcaC may not be part of an integral pathway, the function could confer a significant evolutionary advantage to microbial life.

Structure and thermodynamics of effector molecule binding to the nitrogen signal transduction PII protein GlnZ from Azospirillum brasilense.

The trimeric PII signal transduction proteins regulate the function of a variety of target proteins predominantly involved in nitrogen metabolism. ATP, ADP and 2-oxoglutarate (2-OG) are key effector molecules influencing PII binding to targets. Studies of PII proteins have established that the 20-residue T-loop plays a central role in effector sensing and target binding. However, the specific effects of effector binding on T-loop conformation have remained poorly documented. We present eight crystal structures of the Azospirillum brasilense PII protein GlnZ, six of which are cocrystallized and liganded with ADP or ATP. We find that interaction with the diphosphate moiety of bound ADP constrains the N-terminal part of the T-loop in a characteristic way that is maintained in ADP-promoted complexes with target proteins. In contrast, the interactions with the triphosphate moiety in ATP complexes are much more variable and no single predominant interaction mode is apparent except for the ternary MgATP/2-OG complex. These conclusions can be extended to most investigated PII proteins of the GlnB/GlnK subfamily. Unlike reported for other PII proteins, microcalorimetry reveals no cooperativity between the three binding sites of GlnZ trimers for any of the three effectors under carefully controlled experimental conditions.

High-level over-expression, purification, and crystallization of a novel phospholipase C/sphingomyelinase from Pseudomonas aeruginosa.

The hemolytic phospholipase C/sphingomyelinase PlcH from the opportunistic pathogen Pseudomonas aeruginosa represents the founding member of a growing family of virulence factors identified in a wide range of bacterial and fungal pathogens. In P. aeruginosa PlcH is co-expressed with a 17 kDa chaperone (PlcR2) and secreted as a fully folded heterodimer (PlcHR2) of approximately 95 kDa, by the twin arginine translocase (TAT) via the cytoplasmic membrane and through the outer membrane, by the Xcp (TypeII) secretory system. PlcHR2 has been shown to be an important virulence factor in model P. aeruginosa infections and is selectively cytotoxic, at picomolar concentrations to mammalian endothelial cells. Here we report how the various challenges starting from protein overexpression in the native organism P. aeruginosa, the use of detergents in the crystallization and data collection using the most advanced µ-focus synchrotron beam lines were overcome. Native diffraction data of this heterodimeric protein complex were collected up to a resolution of 4Å, whereas needle-shaped crystals of l-selenomethionine substituted PlcHR2 with a maximum diameter of 10 micron were used to collect data sets with a maximum resolution of 2.75Å.

The crystal structure of non-modified and bipyridine-modified PNA duplexes.

Peptide nucleic acid (PNA) is a synthetic analogue of DNA that commonly has an N-aminoethyl glycine backbone. The crystal structures of two PNA duplexes, one containing eight standard nucleobase pairs (GGCATGCC)(2), and the other containing the same nucleobase pairs and a central pair of bipyridine ligands, have been solved with a resolution of 1.22 and 1.10 Å, respectively. The non-modified PNA duplex adopts a P-type helical structure similar to that of previously characterized PNAs. The atomic-level resolution of the structures allowed us to observe for the first time specific modes of interaction between the terminal lysines of the PNA and the backbone and the nucleobases situated in the vicinity of the lysines, which are considered an important factor in the induction of a preferred handedness in PNA duplexes. Our results support the notion that whereas PNA typically adopts a P-type helical structure, its flexibility is relatively high. For example, the base-pair rise in the bipyridine-containing PNA is the largest measured to date in a PNA homoduplex. The two bipyridines bulge out of the duplex and are aligned parallel to the major groove of the PNA. In addition, two bipyridines from adjacent PNA duplexes form a π-stacked pair that relates the duplexes within the crystal. The bulging out of the bipyridines causes bending of the PNA duplex, which is in contrast to the structure previously reported for biphenyl-modified DNA duplexes in solution, where the biphenyls are π stacked with adjacent nucleobase pairs and adopt an intrahelical geometry. This difference shows that relatively small perturbations can significantly impact the relative position of nucleobase analogues in nucleic acid duplexes.

A new P(II) protein structure identifies the 2-oxoglutarate binding site.

P(II) proteins of bacteria, archaea, and plants regulate many facets of nitrogen metabolism. They do so by interacting with their target proteins, which can be enzymes, transcription factors, or membrane proteins. A key feature of the ability of P(II) proteins to sense cellular nitrogen status and to interact accordingly with their targets is their binding of the key metabolic intermediate 2-oxoglutarate (2-OG). However, the binding site of this ligand within P(II) proteins has been controversial. We have now solved the X-ray structure, at 1.4 A resolution, of the Azospirillum brasilense P(II) protein GlnZ complexed with MgATP and 2-OG. This structure is in excellent agreement with previous biochemical data on 2-OG binding to a variety of P(II) proteins and shows that 2-oxoglutarate binds within the cleft formed between neighboring subunits of the homotrimer. The 2-oxo acid moiety of bound 2-OG ligates the bound Mg(2+) together with three phosphate oxygens of ATP and the side chain of the T-loop residue Gln39. Our structure is in stark contrast to an earlier structure of the Methanococcus jannaschii GlnK1 protein in which the authors reported 2-OG binding to the T-loop of that P(II) protein. In the light of our new structure, three families of T-loop conformations, each associated with a distinct effector binding mode and characterized by a different interaction partner of the ammonium group of the conserved residue Lys58, emerge as a common structural basis for effector signal output by P(II) proteins.