Andrea Carfi

Crystal Structure of the Ebola Virus Membrane Fusion Subunit, GP2, from the Envelope Glycoprotein Ectodomain

We have determined the structure of GP2 from the Ebola virus membrane fusion glycoprotein by X-ray crystallography. The molecule contains a central triple-stranded coiled coil followed by a disulfide-bonded loop homologous to an immunosuppressive sequence in retroviral glycoproteins, which reverses the chain direction and connects to an alpha helix packed antiparallel to the core helices. The structure suggests that fusion peptides near the N termini form disulfide-bonded loops at one end of the molecule and that the C-terminal membrane anchors are at the same end. In this conformation, GP2 could both bridge two membranes and facilitate their apposition to initiate membrane fusion. We also find a heptad irregularity like that in low-pH-induced influenza HA2 and a solvent ion trapped in a coiled coil like that in retroviral TMs.

Unraveling the hidden catalytic activity of vertebrate class IIa histone deacetylases

Previous findings have suggested that class IIa histone deacetylases (HDACs) (HDAC4, -5, -7, and -9) are inactive on acetylated substrates, thus differing from class I and IIb enzymes. Here, we present evidence supporting this view and demonstrate that class IIa HDACs are very inefficient enzymes on standard substrates. We identified HDAC inhibitors unable to bind recombinant human HDAC4 while showing inhibition in a typical HDAC4 enzymatic assay, suggesting that the observed activity rather reflects the involvement of endogenous copurified class I HDACs. Moreover, an HDAC4 catalytic domain purified from bacteria was 1,000-fold less active than class I HDACs on standard substrates. A catalytic Tyr is conserved in all HDACs except for vertebrate class IIa enzymes where it is replaced by His. Given the high structural conservation of HDAC active sites, we predicted the class IIa His-Nε2 to be too far away to functionally substitute the class I Tyr-OH in catalysis. Consistently, a Tyr-to-His mutation in class I HDACs severely reduced their activity. More importantly, a His-976-Tyr mutation in HDAC4 produced an enzyme with a catalytic efficiency 1,000-fold higher than WT, and this “gain of function phenotype” could be extended to HDAC5 and -7. We also identified trifluoroacetyl-lysine as a class IIa-specific substrate in vitro. Hence, vertebrate class IIa HDACs may have evolved to maintain low basal activities on acetyl-lysines and to efficiently process restricted sets of specific, still undiscovered natural substrates.

Herpes simplex virus glycoprotein D bound to the human receptor HveA.

Herpes simplex virus (HSV) infection requires binding of the viral envelope glycoprotein D (gD) to cell surface receptors. We report the X-ray structures of a soluble, truncated ectodomain of gD both alone and in complex with the ectodomain of its cellular receptor HveA. Two bound anions suggest possible binding sites for another gD receptor, a 3-O-sulfonated heparan sulfate. Unexpectedly, the structures reveal a V-like immunoglobulin (Ig) fold at the core of gD that is closely related to cellular adhesion molecules and flanked by large N- and C-terminal extensions. The receptor binding segment of gD, an N-terminal hairpin, appears conformationally flexible, suggesting that a conformational change accompanying binding might be part of the viral entry mechanism.

Structure of Unliganded Hsv Gd Reveals a Mechanism for Receptor-Mediated Activation of Virus Entry.

Herpes simplex virus (HSV) entry into cells requires binding of the envelope glycoprotein D (gD) to one of several cell surface receptors. The 50 C‐terminal residues of the gD ectodomain are essential for virus entry, but not for receptor binding. We have determined the structure of an unliganded gD molecule that includes these C‐terminal residues. The structure reveals that the C‐terminus is anchored near the N‐terminal region and masks receptor‐binding sites. Locking the C‐terminus in the position observed in the crystals by an intramolecular disulfide bond abolished receptor binding and virus entry, demonstrating that this region of gD moves upon receptor binding. Similarly, a point mutant that would destabilize the C‐terminus structure was nonfunctional for entry, despite increased affinity for receptors. We propose that a controlled displacement of the gD C‐terminus upon receptor binding is an essential feature of HSV entry, ensuring the timely activation of membrane fusion.

Molecular Insights into Quorum Sensing in the Human Pathogen Pseudomonas aeruginosa from the Structure of the Virulence Regulator LasR Bound to Its Autoinducer

Many Gram-negative bacteria communicate via molecules called autoinducers to coordinate the activities of their populations. Such communication is termed quorum sensing and can regulate pathogenic virulence factor production and antimicrobial resistance. The quorum sensing system of Pseudomonas aeruginosa is currently the most intensively researched, because this bacterium is an opportunistic human pathogen annually responsible for the death of thousands of cystic fibrosis sufferers and many other immunocompromised individuals. Quorum sensing inhibitors can attenuate the pathogenicity of P. aeruginosa. Here we present the crystal structure of the P. aeruginosa LasR ligand-binding domain bound to its autoinducer 3-oxo-C12-acylhomoserine lactone. The structure is a symmetrical dimer, with each monomer exhibiting an α-β-α fold similar to the TraR and SdiA quorum sensing proteins of Agrobacterium tumefaciens and Escherichia coli. The structure was determined up to 1.8-Å resolution and reveals the atomic interactions between LasR and its autoinducer. The monomer structures of LasR, TraR, and SdiA are comparable but display differences in their quaternary organization. Inspection of their binding sites shows some unexpected variations resulting in quite different conformations of their bound autoinducers. We modeled interactions between LasR and various quorum sensing inhibitors, yielding insight into their possible mechanisms of action. The structure also provides a platform for the optimization, or de novo design, of quorum sensing inhibitors.

Structural and Functional Analysis of the Human HDAC4 Catalytic Domain Reveals a Regulatory Structural Zinc-binding Domain

Histone deacetylases (HDACs) regulate chromatin status and gene expression, and their inhibition is of significant therapeutic interest. To date, no biological substrate for class IIa HDACs has been identified, and only low activity on acetylated lysines has been demonstrated. Here, we describe inhibitor-bound and inhibitor-free structures of the histone deacetylase-4 catalytic domain (HDAC4cd) and of an HDAC4cd active site mutant with enhanced enzymatic activity toward acetylated lysines. The structures presented, coupled with activity data, provide the molecular basis for the intrinsically low enzymatic activity of class IIa HDACs toward acetylated lysines and reveal active site features that may guide the design of class-specific inhibitors. In addition, these structures reveal a conformationally flexible structural zinc-binding domain conserved in all class IIa enzymes. Importantly, either the mutation of residues coordinating the structural zinc ion or the binding of a class IIa selective inhibitor prevented the association of HDAC4 with the N-CoR·HDAC3 repressor complex. Together, these data suggest a key role of the structural zinc-binding domain in the regulation of class IIa HDAC functions.

Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers

Respiratory syncytial virus (RSV), the main cause of infant bronchiolitis, remains a major unmet vaccine need despite more than 40 years of vaccine research. Vaccine candidates based on a chief RSV neutralization antigen, the fusion (F) glycoprotein, have foundered due to problems with stability, purity, reproducibility, and potency. Crystal structures of related parainfluenza F glycoproteins have revealed a large conformational change between the prefusion and postfusion states, suggesting that postfusion F antigens might not efficiently elicit neutralizing antibodies. We have generated a homogeneous, stable, and reproducible postfusion RSV F immunogen that elicits high titers of neutralizing antibodies in immunized animals. The 3.2-Å X-ray crystal structure of this substantially complete RSV F reveals important differences from homology-based structural models. Specifically, the RSV F crystal structure demonstrates the exposure of key neutralizing antibody binding sites on the surface of the postfusion RSV F trimer. This unanticipated structural feature explains the engineered RSV F antigens efficiency as an immunogen. This work illustrates how structural-based antigen design can guide the rational optimization of candidate vaccine antigens.

Substrate binding to histone deacetylases as shown by the crystal structure of the HDAC8-substrate complex.

Histone deacetylases (HDACs)—an enzyme family that deacetylates histones and non‐histone proteins—are implicated in human diseases such as cancer, and the first‐generation of HDAC inhibitors are now in clinical trials. Here, we report the 2.0 Å resolution crystal structure of a catalytically inactive HDAC8 active‐site mutant, Tyr306Phe, bound to an acetylated peptidic substrate. The structure clarifies the role of active‐site residues in the deacetylation reaction and substrate recognition. Notably, the structure shows the unexpected role of a conserved residue at the active‐site rim, Asp 101, in positioning the substrate by directly interacting with the peptidic backbone and imposing a constrained cis‐conformation. A similar interaction is observed in a new hydroxamate inhibitor–HDAC8 structure that we also solved. The crucial role of Asp 101 in substrate and inhibitor recognition was confirmed by activity and binding assays of wild‐type HDAC8 and Asp101Ala, Tyr306Phe and Asp101Ala/Tyr306Phe mutants.

Effects of pH and Low Density Lipoprotein (LDL) on PCSK9-dependent LDL Receptor Regulation

Mutations within PCSK9 (proprotein convertase subtilisin/kexin type 9) are associated with dominant forms of familial hyper- and hypocholesterolemia. Although PCSK9 controls low density lipoprotein (LDL) receptor (LDLR) levels post-transcriptionally, several questions concerning its mode of action remain unanswered. We show that purified PCSK9 protein added to the medium of human endothelial kidney 293, HepG2, and Chinese hamster ovary cell lines decreases cellular LDL uptake in a dose-dependent manner. Using this cell-based assay of PCSK9 activity, we found that the relative potencies of several PCSK9 missense mutants (S127R and D374Y, associated with hypercholesterolemia, and R46L, associated with hypocholesterolemia) correlate with LDL cholesterol levels in humans carrying such mutations. Notably, we found that in vitro wild-type PCSK9 binds LDLR with an ∼150-fold higher affinity at an acidic endosomal pH (KD = 4.19 nm) compared with a neutral pH (KD = 628 nm). We also demonstrate that wild-type PCSK9 and mutants S127R and R46L are internalized by cells to similar levels, whereas D374Y is more efficiently internalized, consistent with their affinities for LDLR at neutral pH. Finally, we show that LDL diminishes PCSK9 binding to LDLR in vitro and partially inhibits the effects of secreted PCSK9 on LDLR degradation in cell culture. Together, the results of our biochemical and cell-based experiments suggest a model in which secreted PCSK9 binds to LDLR and directs the trafficking of LDLR to the lysosomes for degradation.

A PCSK9-binding antibody that structurally mimics the EGF(A) domain of LDL-receptor reduces LDL cholesterol in vivo

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) regulates LDL cholesterol levels by inhibiting LDL receptor (LDLr)-mediated cellular LDL uptake. We have identified a fragment antigen-binding (Fab) 1D05 which binds PCSK9 with nanomolar affinity. The fully human antibody 1D05-IgG2 completely blocks the inhibitory effects of wild-type PCSK9 and two gain-of-function human PCSK9 mutants, S127R and D374Y. The crystal structure of 1D05-Fab bound to PCSK9 reveals that 1D05-Fab binds to an epitope on the PCSK9 catalytic domain which includes the entire LDLr EGF(A) binding site. Notably, the 1D05-Fab CDR-H3 and CDR-H2 loops structurally mimic the EGF(A) domain of LDLr. In a transgenic mouse model (CETP/LDLr-hemi), in which plasma lipid and PCSK9 profiles are comparable to those of humans, 1D05-IgG2 reduces plasma LDL cholesterol to 40% and raises hepatic LDLr protein levels approximately fivefold. Similarly, in healthy rhesus monkeys, 1D05-IgG2 effectively reduced LDL cholesterol 20%–50% for over 2 weeks, despite its relatively short terminal half-life (t1/2 = 3.2 days). Importantly, the decrease in circulating LDL cholesterol corresponds closely to the reduction in free PCSK9 levels. Together these results clearly demonstrate that the LDL-lowering effect of the neutralizing anti-PCSK9 1D05-IgG2 antibody is mediated by reducing the amount of PCSK9 that can bind to the LDLr.