Stephen C. Harrison

X-ray structure of a protein-conducting channel

A conserved heterotrimeric membrane protein complex, the Sec61 or SecY complex, forms a protein-conducting channel, allowing polypeptides to be transferred across or integrated into membranes. We report the crystal structure of the complex from Methanococcus jannaschii at a resolution of 3.2 Å. The structure suggests that one copy of the heterotrimer serves as a functional translocation channel. The α-subunit has two linked halves, transmembrane segments 1–5 and 6–10, clamped together by the γ-subunit. A cytoplasmic funnel leading into the channel is plugged by a short helix. Plug displacement can open the channel into an ‘hourglass’ with a ring of hydrophobic residues at its constriction. This ring may form a seal around the translocating polypeptide, hindering the permeation of other molecules. The structure also suggests mechanisms for signal-sequence recognition and for the lateral exit of transmembrane segments of nascent membrane proteins into lipid, and indicates binding sites for partners that provide the driving force for translocation.

Structure of the dengue virus envelope protein after membrane fusion.

Dengue virus enters a host cell when the viral envelope glycoprotein, E, binds to a receptor and responds by conformational rearrangement to the reduced pH of an endosome. The conformational change induces fusion of viral and host-cell membranes. A three-dimensional structure of the soluble E ectodomain (sE) in its trimeric, postfusion state reveals striking differences from the dimeric, prefusion form. The elongated trimer bears three ‘fusion loops’ at one end, to insert into the host-cell membrane. Their structure allows us to model directly how these fusion loops interact with a lipid bilayer. The protein folds back on itself, directing its carboxy terminus towards the fusion loops. We propose a fusion mechanism driven by essentially irreversible conformational changes in E and facilitated by fusion-loop insertion into the outer bilayer leaflet. Specific features of the folded-back structure suggest strategies for inhibiting flavivirus entry.

A ligand-binding pocket in the dengue virus envelope glycoprotein.

Dengue virus is an emerging global health threat. Its major envelope glycoprotein, E, mediates viral attachment and entry by membrane fusion. A crystal structure of the soluble ectodomain of E from dengue virus type 2 reveals a hydrophobic pocket lined by residues that influence the pH threshold for fusion. The pocket, which accepts a hydrophobic ligand, opens and closes through a conformational shift in a β-hairpin at the interface between two domains. These features point to a structural pathway for the fusion-activating transition and suggest a strategy for finding small-molecule inhibitors of dengue and other flaviviruses.

The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted α Helices: Crystal structure of the protein-DNA complex

The yeast transcriptional activator GCN4 is 1 of over 30 identified eukaryotic proteins containing the basic region leucine zipper (bZIP) DNA-binding motif. We have determined the crystal structure of the GCN4 bZIP element complexed with DNA at 2.9 A resolution. The bZIP dimer is a pair of continuous alpha helices that form a parallel coiled coil over their carboxy-terminal 30 residues and gradually diverge toward their amino termini to pass through the major groove of the DNA-binding site. The coiled-coil dimerization interface is oriented almost perpendicular to the DNA axis, giving the complex the appearance of the letter T. There are no kinks or sharp bends in either bZIP monomer. Numerous contacts to DNA bases and phosphate oxygens are made by basic region residues that are conserved in the bZIP protein family. The details of the bZIP dimer interaction with DNA can explain recognition of the AP-1 site by the GCN4 protein.

Crystal Structures of c-Src Reveal Features of Its Autoinhibitory Mechanism

Src family kinases are maintained in an assembled, inactive conformation by intramolecular interactions of their SH2 and SH3 domains. Full catalytic activity requires release of these restraints as well as phosphorylation of Tyr-416 in the activation loop. In previous structures of inactive Src kinases, Tyr-416 and flanking residues are disordered. We report here four additional c-Src structures in which this segment adopts an ordered but inhibitory conformation. The ordered activation loop forms an alpha helix that stabilizes the inactive conformation of the kinase domain, blocks the peptide substrate-binding site, and prevents Tyr-416 phosphorylation. Disassembly of the regulatory domains, induced by SH2 or SH3 ligands, or by dephosphorylation of Tyr-527, could lead to exposure and phosphorylation of Tyr-416.

Structure of an IκBα/NF-κB Complex

An aqueous pharmaceutical preparation is provided containing chloride ion and stably containing chlorhexidine gluconate, characterized in that said preparation further contains at least one pharmaceutically acceptable polycarboxylic acid of the formula I: HOOC-(CRi 1 -CRi 2)n -COOH (I) wherein n is an integer 1, 2 or 3; i is a positive integer not more than n; R11 and R12, when n=1, independently from each other, are H, -OH or -COOH; each of R11, R12, R21 and R22, when n=2, is H, -OH or -COOH, but not all of them are H simultaneously; and each of R11, R12, R21, R22, R31 and R32, when n=3, is H, -OH or -COOH, but two adjacent carbons in no case contain four hydrogen atoms in total; or a pharmaceutically acceptable salt thereof.

Lipid-protein interactions in double-layered two-dimensional AQP0 crystals.

Lens-specific aquaporin-0 (AQP0) functions as a specific water pore and forms the thin junctions between fibre cells. Here we describe a 1.9 Å resolution structure of junctional AQP0, determined by electron crystallography of double-layered two-dimensional crystals. Comparison of junctional and non-junctional AQP0 structures shows that junction formation depends on a conformational switch in an extracellular loop, which may result from cleavage of the cytoplasmic amino and carboxy termini. In the centre of the water pathway, the closed pore in junctional AQP0 retains only three water molecules, which are too widely spaced to form hydrogen bonds with each other. Packing interactions between AQP0 tetramers in the crystalline array are mediated by lipid molecules, which assume preferred conformations. We were therefore able to build an atomic model for the lipid bilayer surrounding the AQP0 tetramers, and we describe lipid–protein interactions.Membrane organizationAquaporin-0 (AQP0) is the most abundant protein in the membranes of fibre cells in the lens of the mammalian eye, acting both as a water-conducting channel and as an adhesion molecule at cell junctions. The structure of AQP0 unbound (shown on the cover), and together with the lipids that surround it in the membrane, has now been determined at high resolution by electron microscopy — high enough to resolve single water molecules. The structure gives us the first close look at how a membrane protein is embedded in a lipid bilayer. When junctions form between lens fibre cells, the associated lipids, already partly immobilized by interaction with AQP0, mediate the lattice contacts. AQP0 mutations are known to cause cataracts; these mutations may be interfering with AQP0s interaction with the lipids, preventing the integration of AQP0 into a bilayerLens-specific aquaporin-0 (AQP0) functions as a specific water pore and forms the thin junctions between fibre cells. Here we describe a 1.9 Å resolution structure of junctional AQP0, determined by electron crystallography of double-layered two-dimensional crystals. Comparison of junctional and non-junctional AQP0 structures shows that junction formation depends on a conformational switch in an extracellular loop, which may result from cleavage of the cytoplasmic amino and carboxy termini. In the centre of the water pathway, the closed pore in junctional AQP0 retains only three water molecules, which are too widely spaced to form hydrogen bonds with each other. Packing interactions between AQP0 tetramers in the crystalline array are mediated by lipid molecules, which assume preferred conformations. We were therefore able to build an atomic model for the lipid bilayer surrounding the AQP0 tetramers, and we describe lipid–protein interactions.

Structure of an unliganded simian immunodeficiency virus gp120 core

Envelope glycoproteins of human and simian immunodeficiency virus (HIV and SIV) undergo a series of conformational changes when they interact with receptor (CD4) and co-receptor on the surface of a potential host cell, leading ultimately to fusion of viral and cellular membranes. Structures of fragments of gp120 and gp41 from the envelope protein are known, in conformations corresponding to their post-attachment and postfusion states, respectively. We report the crystal structure, at 4 Å resolution, of a fully glycosylated SIV gp120 core, in a conformation representing its prefusion state, before interaction with CD4. Parts of the protein have a markedly different organization than they do in the CD4-bound state. Comparison of the unliganded and CD4-bound structures leads to a model for events that accompany receptor engagement of an envelope glycoprotein trimer. The two conformations of gp120 also present distinct antigenic surfaces. We identify the binding site for a compound that inhibits viral entry.

Structure of PAK1 in an Autoinhibited Conformation Reveals a Multistage Activation Switch

The p21-activated kinases (PAKs), stimulated by binding with GTP-liganded forms of Cdc42 or Rac, modulate cytoskeletal actin assembly and activate MAP-kinase pathways. The 2.3 A resolution crystal structure of a complex between the N-terminal autoregulatory fragment and the C-terminal kinase domain of PAK1 shows that GTPase binding will trigger a series of conformational changes, beginning with disruption of a PAK1 dimer and ending with rearrangement of the kinase active site into a catalytically competent state. An inhibitory switch (IS) domain, which overlaps the GTPase binding region of PAK1, positions a polypeptide segment across the kinase cleft. GTPase binding will refold part of the IS domain and unfold the rest. A related switch has been seen in the Wiskott-Aldrich syndrome protein (WASP).

Structure of the DNA-binding domains from NFAT, Fos and Jun bound specifically to DNA.

The nuclear factor of activated T cells (NFAT) and the AP-1 heterodimer, Fos–Jun, cooperatively bind a composite DNA site and synergistically activate the expression of many immune-response genes. A 2.7-Å-resolution crystal structure of the DNA-binding domains of NFAT, Fos and Jun, in a quaternary complex with a DNA fragment containing the distal antigen-receptor response element from the interleukin-2 gene promoter, shows an extended interface between NFAT and AP-1, facilitated by the bending of Fos and DNA. The tight association of the three proteins on DNA creates a continuous groove for the recognition of 15 base pairs.