During HIV-1 entry, the highly conserved gp41 N-trimer pocket region becomes transiently exposed and vulnerable to inhibition. Using mirror-image phage display and structure-assisted design, we have discovered protease-resistant D-amino acid peptides (Dpeptides) that bind the N-trimer pocket with high affinity and potently inhibit viral entry. We also report high-resolution crystal structures of two of these D-peptides in complex with a pocket mimic that suggest sources of their high potency. A trimeric version of one of these peptides is the most potent pocket-specific entry inhibitor yet reported by three orders of magnitude (IC 50 ‫؍‬ 250 pM). These results are the first demonstration that D-peptides can form specific and high-affinity interactions with natural protein targets and strengthen their promise as therapeutic agents. The D-peptides described here address limitations associated with current L-peptide entry inhibitors and are promising leads for the prevention and treatment of HIV/AIDS. microbicide ͉ phage display ͉ protein design

Ubiquitin (Ub) functions in many different biological pathways, where it typically interacts with proteins that contain modular Ub recognition domains. One such recognition domain is the Npl4 zinc finger (NZF), a compact zinc-binding module found in many proteins that function in Ub-dependent processes. We now report the solution structure of the NZF domain from Npl4 in complex with Ub. The structure reveals that three key NZF residues (13TF14/M25) surrounding the zinc coordination site bind the hydrophobic ‘Ile44' surface of Ub. Mutations in the 13TF14/M25 motif inhibit Ub binding, and naturally occurring NZF domains that lack the motif do not bind Ub. However, substitution of the 13TF14/M25 motif into the nonbinding NZF domain from RanBP2 creates Ub-binding activity, demonstrating the versatility of the NZF scaffold. Finally, NZF mutations that inhibit Ub binding by the NZF domain of Vps36/ESCRT-II also inhibit sorting of ubiquitylated proteins into the yeast vacuole. Thus, the NZF is a versatile protein recognition domain that is used to bind ubiquitylated proteins during vacuolar protein sorting, and probably many other biological processes

The HIV gp41 N-trimer pocket region is an ideal viral target because it is extracellular, highly conserved, and essential for viral entry. Here, we report on the design of a pocket-specific D-peptide, PIE12-trimer, that is extraordinarily elusive to resistance and characterize its inhibitory and structural properties. D-Peptides (peptides composed of D-amino acids) are promising therapeutic agents due to their insensitivity to protease degradation. PIE12-trimer was designed using structure-guided mirror-image phage display and linker optimization and is the first D-peptide HIV entry inhibitor with the breadth and potency required for clinical use. PIE12-trimer has an ultrahigh affinity for the gp41 pocket, providing it with a reserve of binding energy (resistance capacitor) that yields a dramatically improved resistance profile compared to those of other fusion inhibitors. These results demonstrate that the gp41 pocket is an ideal drug target and establish PIE12-trimer as a leading anti-HIV antiviral candidate.

Paramyxovirus entry into cells requires the fusion protein (F) and a receptor binding protein (hemagglutinin-neuraminidase [HN], H, or G

Recent iPrEx clinical trial results provided evidence that systemic preexposure prophylaxis (PrEP) with emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF

The Paramyxoviridae family of enveloped viruses enters cells through the concerted action of two viral glycoproteins. The receptor-binding protein, hemagglutinin-neuraminidase (HN), H, or G, binds its cellular receptor and activates the fusion protein, F, which, through an extensive refolding event, brings viral and cellular membranes together, mediating virus-cell fusion. However, the underlying mechanism of F activation on receptor engagement remains unclear. Current hypotheses propose conformational changes in HN, H, or G propagating from the receptor-binding site in the HN, H, or G globular head to the F-interacting stalk region. We provide evidence that the receptor-binding globular head domain of the paramyxovirus parainfluenza virus 5 HN protein is entirely dispensable for F activation. Considering together the crystal structures of HN from different paramyxoviruses, varying energy requirements for fusion activation, F activation involving the parainfluenza virus 5 HN stalk domain, and properties of a chimeric paramyxovirus HN protein, we propose a simple model for the activation of paramyxovirus fusion. Paramyxovirus-mediated fusion depends on the concerted actions of two glycoproteins, an attachment protein [hemagglutinin-neuraminidase (HN), H, or G] and its cognate fusion (F) protein, the latter initially folding into a metastable form. The attachment protein is thought to trigger the fusion protein in a receptor-dependent manner (1-5). This triggering of the metastable F (6) by the receptor-binding protein couples receptor binding of HN, H, or G to lowering the activation energy barrier of F so that F refolds into a highly stable postfusion form (7). In the process, F undergoes a series of structural rearrangements involving several intermediates and brings about membrane merger (8).HN proteins bind sialic acid as their receptor and also have neuraminidase (receptor-destroying) activity. The PIV5 HN protein comprises 565 residues and has a short N-terminal cytoplasmic tail followed by a single transmembrane domain and a large ectodomain (residues 37-565). The protein consists of a stalk region (residues 1-117) that supports a large globular head domain (residues 118-565) containing the receptor-binding and neuraminidase-active site. X-ray crystal structures of the globular head domain of PIV5, NDV, Nipah virus, Hendra virus, measles virus, and human parainfluenza virus 3 (hPIV3) attachment proteins have been obtained (9-16). The PIV5 HN globular head structure (16) reveals a neuraminidase-like fold with a six-bladed β-propeller structure that is a common feature of the other paramyxovirus HN/H/G head domains, regardless of receptor specificity. The sialic acid-binding site is placed centrally within the β-propeller. PIV5 HN exists as a pair of disulfide-linked dimers with the disulfide bond at cysteine 111 (16,17). These dimers are associated noncovalently to form a dimer-of-dimer oligomer (16,18). The PIV5 HN dimer-of-dimer structure showed the dimers arranged at an approximately 90°angle to each oth...

Paramyxoviruses cause a wide variety of human and animal diseases. They infect host cells using the coordinated action of two surface glycoproteins, the receptor binding protein (HN, H, or G) and the fusion protein (F). HN binds sialic acid on host cells (hemagglutinin activity) and hydrolyzes these receptors during viral egress (neuraminidase activity, NA). Additionally, receptor binding is thought to induce a conformational change in HN that subsequently triggers major refolding in homotypic F, resulting in fusion of virus and target cell membranes. HN is an oligomeric type II transmembrane protein with a short cytoplasmic domain and a large ectodomain comprising a long helical stalk and large globular head domain containing the enzymatic functions (NA domain). Extensive biochemical characterization has revealed that HN-stalk residues determine F specificity and activation. However, the F/HN interaction and the mechanisms whereby receptor binding regulates F activation are poorly defined. Recently, a structure of Newcastle disease virus (NDV) HN ectodomain revealed the heads (NA domains) in a “4-heads-down” conformation whereby two of the heads form a symmetrical interaction with two sides of the stalk. The interface includes stalk residues implicated in triggering F, and the heads sterically shield these residues from interaction with F (at least on two sides). Here we report the x-ray crystal structure of parainfluenza virus 5 (PIV5) HN ectodomain in a “2-heads-up/2-heads-down” conformation where two heads (covalent dimers) are in the “down position,” forming a similar interface as observed in the NDV HN ectodomain structure, and two heads are in an “up position.” The structure supports a model in which the heads of HN transition from down to up upon receptor binding thereby releasing steric constraints and facilitating the interaction between critical HN-stalk residues and F.

Linear peptides derived from the HIV gp41 C-terminus (C-peptides), such as the 36-residue Fuzeon, are potent HIV fusion inhibitors. These molecules bind to the N-peptide region of gp41 and inhibit an intramolecular protein-protein interaction that powers fusion of the viral and host cell membranes. The N-peptide region contains a surface pocket that is occupied in the post-fusion state by three alpha-helical residues found near the gp41 C-terminus: Trp628, Trp631, and Ile635-the WWI epitope. Here, we describe a set of beta3-decapeptides (betaWWI-1-4) in which the WWI epitope is presented on one face of a short 14-helix stabilized by macrodipole neutralization and side chain-side chain salt bridges. betaWWI-1-4 bind in vitro to IZN17, a validated gp41 model, and inhibit syncytia formation in cell culture. Molecules lacking a complete WWI functional epitope neither bind IZN17 nor inhibit syncytia formation. These results provide evidence that short beta-peptide 14-helices can inhibit an intramolecular protein-protein interaction in vivo. Molecules related to betaWWI-1-4 could represent starting points for the development of highly potent inhibitors or antigens effective against HIV or other viruses, including SARS, Ebola, HRSV, and influenza, that employ common fusion mechanisms.